PILRalpha / CD99 pathway inhibitor and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a PILR alpha / CD99 pathway inhibitor and application thereof in immunotherapy. Specifically, the invention provides application and a method of a PILR alpha / CD99 pathway inhibitor for treating tumors and / or regulating immune response. In addition, the invention also provides a PILR alpha functional epitope and an antibody targeting the PILR alpha functional epitope, and the antibody inhibits the immunosuppression function of the PILR alpha by blocking the interaction between the PILR alpha and CD99, recovers the immune cell activity and enhances the immune response.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a PILRα / CD99 pathway inhibitor and its application in immunotherapy. Background Art

[0002] The molecular mechanisms by which tumor cells escape immune surveillance are highly heterogeneous, among which the upregulation of the PD-1 / PD-L1 immune checkpoint has been the most extensively studied. Although the blockade of the PD-1 / PD-L1 immune checkpoint using specific monoclonal antibodies (mAbs) has achieved success in clinical applications, the response rate is limited, with only 25% of solid tumor patients and 40 - 60% of certain lymphoma patients showing a positive response to anti-PD-1 / PD-L1 antibody therapy. In addition, strong evidence indicates that in addition to the upregulation of PD-1 / PD-L1, many other molecular or cellular mechanisms may also promote immune evasion in the tumor microenvironment. These mechanisms include, but are not limited to, insufficient infiltration of immune cells and the accumulation of regulatory T cells, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells. In addition, the upregulation of other immunosuppressive molecules, cytokines, and metabolites, as well as the downregulation of immune-stimulatory molecules, also promote immune evasion. In the field of tumor immunotherapy, it is crucial to identify and explore immune checkpoints that can regulate immune responses, especially novel immune checkpoints that regulate T cell function.

[0003] T cells have become the focus of attention in tumor immunotherapy. Dong et al. in CD8 +CRISPR / Cas9 library screening was performed in T cells, and a batch of genes including Dhx37 that can regulate the effector functions of T cells were identified (Dong M B, Wang G C, Chow R D, et al. Systematic Immunotherapy Target Discovery Using Genome-Scale In Vivo CRISPR Screens in CD8 T Cells. Cell. 2019, 178(5):1189-1204). Wang et al. developed a system for high-throughput screening of T cell activity using genomic arrays to identify immune regulatory molecules expressed on the surface of T cells (Wang J, Sun J W, Liu L N, et al. Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy. Nat Med. 2019, 25(4):656-666). Tumor-infiltrating lymphocytes (TILs), including T cells, can secrete interferon γ (IFNγ) and other cytokines after recognizing tumor antigens, inducing the expression of immune inhibitory molecules (such as PD-L1) in tumor cells. These immune inhibitory molecules can also be targets for immunotherapy.

[0004] Paired immunoglobulin-like receptor 2 (PILR) contains two molecules, PILRα and PILRβ, and is a member of the paired receptor family. In the cytoplasmic tail, PILRα has two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which transmit inhibitory signals by recruiting phosphatases SHP-1 and SHP-2; while PILRβ has one tyrosine-based activation motif (ITAM), which transmits activation signals by binding to the transmembrane adaptor protein DAP-12. PILRα is a transmembrane protein that is widely expressed on myeloid cells including macrophages, dendritic cells, and granulocytes. PILRα can regulate granulocyte recruitment in inflammatory responses by modulating integrins. At the same time, PILRα knockout mice showed an increase in inflammatory cytokines and were more susceptible to autoimmune arthritis. In addition, PILRα can serve as a co-receptor for gB in HSV-1 infection; the G78R mutation of PILRα can prevent microglial cell infection during HSV-1 recurrence by reducing inhibitory signals in microglial cells, thus reducing the risk of Alzheimer's disease. Although the roles of PILRα in HSV-1 infection and Alzheimer's disease have been reported, its functions and roles in cancer have not been explored. Summary of the Invention

[0005] The present invention for the first time discovers that PILRα has a negative regulatory effect on the activation, proliferation and effector functions of T cells, and identifies the functional epitopes through which PILRα exerts its regulatory effect. Based on this, the present invention for the first time provides inhibitors (such as antibodies) targeting the functional epitopes of PILRα, which can help improve the tumor microenvironment and enhance anti-tumor immunity by blocking the interaction between PILRα and CD99, thereby exerting a tumor therapeutic effect. Thus, the following inventions are provided.

[0006] Application of PILRα / CD99 Pathway Inhibitors

[0007] In one aspect, the present invention provides the use of a PILRα / CD99 pathway inhibitor for treating tumors, or for use in the preparation of a medicament for treating tumors. A method for treating tumors is also provided, the method comprising: administering to a subject (such as a human) in need thereof an effective amount of a PILRα / CD99 pathway inhibitor. In certain embodiments, the treatment of tumors is anti-tumor immunotherapy. In certain embodiments, the anti-tumor immunotherapy includes enhancing the anti-tumor immune response and / or reducing tumor immunosuppression in the subject.

[0008] In another aspect, the present invention provides the use of a PILRα / CD99 pathway inhibitor for regulating the immune response, or for use in the preparation of a medicament for regulating the immune response. A method for regulating the immune response is also provided, the method comprising: administering to a subject (such as a human) in need thereof an effective amount of a PILRα / CD99 pathway inhibitor. In certain embodiments, the immune response includes a T cell immune response. In certain embodiments, the regulation of the immune response includes enhancing or stimulating the T cell immune response and / or enhancing T cell activity in the subject (such as a human). In certain embodiments, the subject has a tumor.

[0009] In any of the above aspects, the PILRα / CD99 pathway inhibitor enhances the T cell immune response and / or promotes T cell function, for example, as measured by the activation and / or proliferation of T cells (such as CD8+ T cells). In certain embodiments, the activation may include the activation of nuclear factor of activated T cells NFAT or an increase in cytokines (such as IFN-γ, IL-2, TNF-α, etc.).

[0010] I. PILRα / CD99 Pathway Inhibitors

[0011] In any of the above aspects, the "PILRα / CD99 pathway inhibitor" refers to any inhibitor that can inhibit the biological activity of PILRα, block the binding or interaction between PILRα and CD99, or inhibit the intracellular signal transduction induced by the binding or interaction between PILRα and CD99. The binding or interaction between PILRα and CD99 results in the downregulation of the ZAP70 / NFAT / IL2 / JAK / STAT signaling pathway activated by CD99. Thus, by relieving the inhibition of PILRα, the ZAP70 / NFAT / IL2 / JAK / STAT signaling pathway activated by CD99 can be upregulated to activate T cells.

[0012] In certain embodiments, the PILRα / CD99 pathway inhibitor includes a PILRα inhibitor. "PILRα inhibitor" includes, but is not limited to, any molecule that can block, reduce, or otherwise limit the interaction or activity of PILRα.

[0013] The PILRα inhibitors described herein can exert their inhibitory effects through any mechanism, such as inhibiting the expression of PILRα at the RNA or protein level (e.g., reducing or inhibiting gene transcription, and / or reducing or inhibiting the translation of the mRNA product of the gene); or, for example, inhibiting or blocking the interaction between PILRα and its receptor protein (e.g., CD99); or, for example, by inhibiting or blocking the biological activity of PILRα; or, for example, by degrading PILRα or disrupting its stability.

[0014] In certain embodiments, the inhibitor inhibits the biological function of PILRα by directly binding to PILRα or blocks its interaction with the receptor protein (e.g., CD99). Methods for assaying such inhibitors are within the capabilities of those skilled in the art. Assays for biological function can include, for example, measuring whether the ZAP70 / NFAT / IL2 / JAK / STAT signaling pathway activated by CD99 is upregulated (compared to in the absence of the inhibitor), or detecting whether T cells are activated and / or proliferating (compared to in the absence of the inhibitor). Assays for interaction can be determined by techniques such as BLI, SPR, ELISA, etc.

[0015] In certain embodiments, the inhibitor inhibits the expression level of the PILRα gene. In such embodiments, the determination of the expression level can be carried out at the nucleic acid level or the protein level. Methods for assaying expression at the nucleic acid level include, but are not limited to, Northern blotting, PCR, RT-PCR, or real-time RT-PCR. Methods for assaying expression at the protein level include, but are not limited to, Western blotting or polyacrylamide gel electrophoresis combined with protein staining techniques such as Coomassie brilliant blue or silver staining, mass spectrometry, ELISA, etc.

[0016] In certain embodiments, the inhibitor is selected from antibodies, small molecule compounds, aptamers, RNA interference agents (e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA), or microRNA (miRNA)), or antisense oligonucleotides, gene editing systems (e.g., CRISPR-Cas systems).

[0017] A) Affinity Inhibitors

[0018] In some embodiments, the inhibitor is selected from affinity molecules that have binding specificity for PILRα (e.g., human PILRα).

[0019] In certain embodiments, the affinity molecule specifically binds to the Stalk domain of PILRα. In certain embodiments, the affinity molecule does not bind, or binds minimally or undetectably, to a PILRα protein lacking the Stalk domain (e.g., the polypeptide shown in SEQ ID NO:31).

[0020] In certain embodiments, the affinity molecule specifically binds to a functional epitope in the Stalk domain of PILRα, and the functional epitope contains at least one O-glycosylation site. In certain embodiments, the O-glycosylation site is selected from Ser and / or Thr residues. In certain embodiments, the O-glycosylation site is selected from S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176, or any combination thereof, and the amino acid positions are determined according to SEQ ID NO:25. In certain embodiments, the functional epitope contains one or more selected from S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176, and the amino acid positions are determined according to SEQ ID NO:25.

[0021] Antibodies

[0022] In certain embodiments, the affinity molecule is an antibody or an antigen-binding fragment thereof.

[0023] In certain embodiments, the antibody or an antigen-binding fragment thereof specifically binds to PILRα (e.g., human PILRα). In certain embodiments, the antibody or an antigen-binding fragment thereof specifically binds to the functional epitope in the Stalk domain of PILRα described herein. In certain embodiments, the antibody or an antigen-binding fragment thereof inhibits or blocks the interaction between PILRα and CD99.

[0024] In certain embodiments, the antibody can be a murine antibody, a simian antibody, a rabbit antibody, a camelid antibody, a human antibody, a chimeric antibody, or a humanized antibody.

[0025] In certain embodiments, the antigen-binding fragment is selected from ScFv, Fab, Fab’, (Fab’) 2 , Fv fragment, disulfide-linked Fv (dsFv).

[0026] Antibodies that specifically bind to PILRα (e.g., human PILRα) can be prepared by a variety of conventional techniques, such as hybridoma technology, recombinant DNA technology, or phage antibody library technology.

[0027] For example, monoclonal antibodies can be prepared as follows. First, a mouse or other suitable host animal is immunized with an immunogen (optionally with an adjuvant). After the animal is immunized, lymphocytes that secrete antibodies specifically binding to the immunogen will be generated in vivo. The lymphocytes of interest are collected and fused with myeloma cells using a suitable fusogen, such as PEG, to obtain hybridoma cells. The hybridoma cells prepared above can be inoculated into a suitable culture medium for growth, and the culture medium preferably contains one or more substances that can inhibit the growth of unfused, parental myeloma cells. For example, for parental myeloma cells lacking hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), adding substances such as hypoxanthine, aminopterin, and thymidine (HAT medium) to the culture medium will inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells should have characteristics such as a high fusion rate, stable antibody secretion ability, and sensitivity to HAT culture medium. Among them, murine myeloma is preferred, such as MOP-21 or MC-11 mouse tumor-derived strains, and SP-2 / 0 or X63-Ag8-653 cell lines. The culture medium for growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as enzyme-linked immunosorbent assay (ELISA). After determining the specificity, affinity, and reactivity of the antibodies produced by the hybridoma, the target cell line can be subcloned by standard limiting dilution methods. Using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, etc., the monoclonal antibodies secreted by the subcloned cells can be separated from cell culture supernatants, ascites, or sera.

[0028] Monoclonal antibodies can also be obtained by genetic engineering recombination techniques. Using nucleic acid primers that specifically bind to the heavy and light chain genes of the monoclonal antibody for PCR amplification, DNA molecules encoding the heavy and light chain genes of the monoclonal antibody can be isolated from hybridoma cells. Insert the obtained DNA molecules into an expression vector, then transfect host cells, and culture them under appropriate conditions to obtain the target antibody expressed recombinantly.

[0029] Antibodies can be purified by well-known techniques, such as affinity chromatography using Protein A or Protein G. Subsequently or as an alternative, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column, and the immunospecific antibody can be purified by immunoaffinity chromatography.

[0030] After obtaining a monoclonal antibody that specifically binds to PILRα, the inhibitory activity of the monoclonal antibody against PILRα can be further determined using the methods described above.

[0031] After obtaining the monoclonal antibody prepared as above, its chimeric antibody or humanized antibody can be further prepared. For example, DNA encoding the heavy and light chains is obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.

[0032] To prepare a chimeric antibody, methods known in the art can be used to link the murine immunoglobulin variable region to the human immunoglobulin constant region. For example, the DNA encoding VH can be operably linked to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but usually IgG1 or IgG4 constant regions are preferred. For example, the DNA encoding VL can be operably linked to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and Fab light chain gene). The sequences of human light chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a κ or λ constant region, but usually the κ constant region is preferred.

[0033] To prepare a humanized antibody, methods known in the art can be used to insert the murine CDR regions into the human framework sequence. Alternatively, transgenic animals can also be utilized, which are capable of not producing endogenous immunoglobulins after immunization and can generate a complete human antibody library.

[0034] Exemplary Antibodies

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from antibodies having the CDRs and / or variable region sequences of antibody C21, antibodies having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to its CDR or variable region sequences, and antibodies having only one or more (such as at most 20, at most 15, at most 10, or at most 5) conservative substitutions compared to its CDR or variable region sequences.

[0036] In certain embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 4, 5, and 6, respectively. In certain embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 4, 5, and 6, respectively.

[0037] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 7 or a sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 8 or a sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence as shown in SEQ ID NO: 7 and a VL having the sequence as shown in SEQ ID NO: 8.

[0038] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from antibodies having the CDRs and / or variable region sequences of antibody C29, antibodies having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to its CDR or variable region sequences, and antibodies having no more than one or more (e.g., at most 20, at most 15, at most 10, or at most 5) conservative substitutions compared to its CDR or variable region sequences.

[0039] In certain embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 9, 10, and 11, respectively, and light chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 12, 13, and 14, respectively. In certain embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 9, 10, and 11, respectively, and light chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 12, 13, and 14, respectively.

[0040] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 15 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 16 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having the sequence as shown in SEQ ID NO: 15 and a VL having the sequence as shown in SEQ ID NO: 16.

[0041] Small Molecules

[0042] In certain embodiments, the affinity molecule is selected from small molecule compounds. The term "small molecule compound" or "small molecule" refers to an organic non-protein compound.

[0043] In certain embodiments, the molecular weight of the small molecule compound is no greater than 1500 Da. In certain embodiments, the molecular weight of the small molecule compound is at most 900 Da.

[0044] In certain embodiments, the small molecule compound specifically binds to PILRα (such as human PILRα). In certain embodiments, the small molecule compound specifically binds to a functional epitope in the PILRα Stalk domain described herein. In certain embodiments, the small molecule compound inhibits or blocks the interaction between PILRα and CD99.

[0045] The small molecule compound can be obtained by screening existing small molecule compound libraries (e.g., Chem Bridge, Chem Div, Inter BioScreen, Life Chemicals, Specs or Vitas-m), and measuring the binding activity and inhibitory activity of the compound against PILRα using the methods described above.

[0046] Aptamers

[0047] In certain embodiments, the affinity molecule is selected from aptamers. The term "aptamer" refers to an oligonucleotide that specifically and with high affinity binds to a target molecule. Under defined conditions, an aptamer can fold into a specific three-dimensional structure.

[0048] In certain embodiments, the aptamer specifically binds to PILRα (such as human PILRα). In certain embodiments, the aptamer specifically binds to a functional epitope in the PILRα Stalk domain described herein. In certain embodiments, the aptamer inhibits or blocks the interaction between PILRα and CD99.

[0049] A variety of SELEX-based aptamer selection procedures have been reported (see, e.g., Ali MH, Elsherbiny ME, Emara M. Updates on Aptamer Research. Int J Mol Sci. 2019;20(10):2511.), which enables the selection of aptamers for almost all conceivable targets (see, e.g., Stoltenburg R, Nikolaus N, Strehlitz B. Capture-SELEX: Selection of DNA Aptamers for Aminoglycoside Antibiotics. J Anal Methods Chem. 2012;2012:415697.), and the binding activity and inhibitory activity of the aptamer against PILRα are measured using the methods described above to obtain the aptamer.

[0050] B) Expression Inhibitors

[0051] In other embodiments, the inhibitor is selected from reagents that inhibit the expression level of the PILRα gene.

[0052] RNA Interference Agents

[0053] In certain embodiments, the inhibitor comprises an RNA interfering agent.

[0054] As used herein, the term "RNA interfering agent" refers to any agent that inhibits the expression of a target gene through the RNA interference (RNAi) mechanism. "RNA interference (RNAi)" is an evolutionarily conserved process in which the expression or introduction of RNA having a sequence identical or highly similar to the target gene results in sequence-specific degradation of the messenger RNA (mRNA) transcribed from the target gene or specific post-transcriptional gene silencing (PTGS), thereby inhibiting the expression of the target gene.

[0055] Thus, those skilled in the art will appreciate that a target sequence can be selected based on the sequence of the PILRα-encoding gene or the mRNA transcribed therefrom to design small interfering RNA (siRNA) or small RNA (microRNA, miRNA) molecules. The siRNA or miRNA molecules can interfere with the transcription, translation, or post-transcriptional and post-translational modifications of the gene, thereby affecting protein expression.

[0056] As used herein, the term "siRNA" refers to Small interfering RNA, which is a small RNA molecule composed of approximately 21-25 nucleotides and is processed by Dicer (an enzyme specific for double-stranded RNA in the RNAaseⅢ family). siRNA is a major member of siRISC and triggers the silencing of the target mRNA complementary thereto.

[0057] As used herein, the term "microRNA (miRNA)" refers to a class of naturally occurring non-coding RNA molecules approximately 21-25 nucleotides in length. Based on sequence complementarity with the target mRNA, they can cause degradation of the target mRNA or inhibit its translation through specific base pairing with the target mRNA, thereby regulating the post-transcriptional expression of the gene. miRNAs can be referenced in the miRBase database (http: / / microrna.sanger.ac.uk / ).

[0058] In addition, in order to prolong the inhibitory effect of siRNA on target gene expression, a pair of specific oligonucleotide sequences can be designed, annealed and cloned into a vector. The transcription product of this recombinant vector is short hairpin RNA (shRNA), which can self-fold and pair into a stem-loop structure with a stem length of 19-21 bases. The 19-21 bases are derived from a specific sequence of the target gene mRNA. The precursor of this stem-loop structure is quickly cleaved in the cell to form functional siRNA. The siRNA formed by the cleavage of the shRNA expressed by this vector has the characteristics of stable expression level and long duration, thus causing long-term inhibition of target gene expression.

[0059] In certain embodiments, the RNA interfering agent is selected from small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA).

[0060] Antisense Oligonucleotides

[0061] In certain embodiments, the inhibitor includes antisense oligonucleotides.

[0062] As used herein, the term "antisense oligonucleotide" refers to a molecule that is complementary to a sense nucleic acid, such as complementary to the coding strand of the PILRα gene or to the mRNA sequence of the PILRα gene. Thus, the antisense oligonucleotide can form hydrogen bonds with the sense nucleic acid (i.e., anneal thereto). The antisense oligonucleotide can be complementary to the entire coding strand of the nucleic acid sequence encoding PILRα, or only to a portion thereof, such as all or part of the protein coding region (or open reading frame). The antisense oligonucleotide can also be antisense to all or part of the non-coding region of the coding strand of the nucleic acid sequence encoding PILRα. The length of the antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30 or more nucleotides.

[0063] The above antisense oligonucleotides can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, they can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides, which are designed to increase the biological stability of the molecule or the physical stability of the duplex formed between the antisense and sense nucleic acids. For example, phosphorothioate derivatives and acridine-substituted nucleotides can be used. Alternatively, the above antisense oligonucleotides can be produced biologically using an expression vector that contains the target nucleic acid cloned therein, and the RNA transcribed from the inserted nucleic acid will have an antisense orientation to the target nucleic acid.

[0064] After obtaining the RNA interfering agent or antisense oligonucleotide, the inhibitory activity of the RNA interfering agent or antisense oligonucleotide on the mRNA or protein expression level of PILRα can be further determined using the method described above.

[0065] Gene Editing Systems

[0066] In certain embodiments, the inhibitor is a gene editing system. Those skilled in the art are familiar with using gene editing systems to knockdown or knockout target genes (such as PILRα).

[0067] The gene editing system that can be used in the present invention can be any site-specific (sequence-specific) genome editing system known now. In certain embodiments, the genome editing system includes at least one site-specific nuclease, such as an RNA-guided nuclease (such as a Cas nuclease), a zinc finger nuclease, a megabase meganuclease, a TALE-nuclease, a recombinase, a transposase, and any combination thereof. In certain embodiments, the gene editing system is a CRISPR / Cas system, which includes a Cas effector protein (including but not limited to Cas9, Cas12a (Cpf1), Cas12b (C2c1), Cas13a (C2c2), C2c3, Cas13b) and the corresponding guide RNA (gRNA), which includes a guide sequence complementary to the target sequence in the target locus. In certain embodiments, the genome editing system is present on one or more vectors.

[0068] II. Administration

[0069] The inhibitors described herein can be administered by any suitable method known in the art. Preferred routes of administration include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration. Parenteral administration refers to a mode of administration that is typically by injection rather than enteral and topical administration, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, it can be administered via non-parenteral routes, such as local, epidermal or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or topical.

[0070] The inhibitors described herein can be formulated into dosage forms compatible with their intended route of administration. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by incorporating the required dose of the inhibitor described herein in a suitable solvent, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) simultaneously, followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for ease of storage and use.

[0071] The inhibitors described herein can be formulated in unit dosage form for easy administration. Unit dosage form refers to physically discrete units suitable as a single dose for a subject to be treated; each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0072] The inhibitors described herein are administered to a subject in need thereof, and the subject includes any individual capable of generating an immune response. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0073] III. Indications

[0074] The inhibitors described herein can be used for anti-tumor immunotherapy, improving the ability of the immune system to recognize and kill tumors, and overcoming the immunosuppressive tumor microenvironment. The "tumor" and "cancer" described herein are used interchangeably and include any form of disease characterized by uncontrolled growth of abnormal cells in the body.

[0075] In certain embodiments, the tumor includes a solid tumor or a hematological tumor.

[0076] In certain embodiments, the tumor is selected from breast cancer, cholangiocarcinoma, esophageal cancer, glioblastoma, head and neck cancer, kidney cancer (such as clear cell renal cell carcinoma (KIRC) or papillary renal cell carcinoma (KIRP)), sarcoma, thyroid cancer, and gastric cancer.

[0077] In certain embodiments, the tumor expresses PILRα or is PILRα-positive. The PILRα positivity can be determined at the nucleic acid level or the protein level. Methods for determining expression at the nucleic acid level include but are not limited to RT-PCR or real-time RT-PCR. Methods for determining expression at the protein level include but are not limited to immunological assays, such as enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance, etc.

[0078] The inhibitors described herein can be used to modulate the immune response, particularly to enhance or stimulate T cell immune responses and / or enhance T cell activity, and thus for the treatment of any disease that would benefit from enhanced immune responses.

[0079] In certain embodiments, the enhancement of the immune response can be determined by EC50 or maximal activity levels in assays measuring activation of nuclear factor of activated T cells NFAT activation, cytokine (such as IFN-γ, IL-2, TNF-α, etc.) release, T cell proliferation, etc. In certain embodiments, the inhibitors described herein enhance the immune response by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or more.

[0080] Affinity Molecule Antibodies that Bind to PILRα

[0081] The present invention unexpectedly found that the PILRα stalk domain (especially the O-glycosylation therein) plays a key role in PILRα-mediated inhibition of T cell activity, and thus the present invention provides a functional epitope that inhibits PILRα activity and an affinity molecule that targets this functional epitope.

[0082] In another aspect, the present invention provides an affinity molecule that specifically binds to the stalk domain of PILRα. Herein, the stalk domain refers to the segment of PILRα corresponding to aa 151-197 of the reference sequence SEQ ID NO:25. An exemplary sequence of the stalk domain can be referred to SEQ ID NO:33.

[0083] In certain embodiments, the affinity molecule does not bind, or binds substantially not, or undetectably binds to a PILRα protein lacking the stalk domain (such as the polypeptide shown in SEQ ID NO:31).

[0084] In certain embodiments, the affinity molecule specifically binds to a functional epitope in the stalk domain of PILRα, and the functional epitope contains at least one O-glycosylation site.

[0085] In certain embodiments, the O-glycosylation site is selected from Ser and / or Thr residues.

[0086] In certain embodiments, the O-glycosylation site is selected from S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176 or any combination thereof, and the amino acid positions are determined according to SEQ ID NO:25.

[0087] In certain embodiments, the functional epitope comprises one or more of the following amino acid residues: S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176, wherein the amino acid positions are determined according to SEQ ID NO:25.

[0088] In certain embodiments, the affinity molecule is an antibody or an antigen-binding fragment thereof, a small molecule compound, or an aptamer.

[0089] In certain embodiments, the affinity molecule is an antibody or an antigen-binding fragment thereof.

[0090] Competitive Antibodies

[0091] In another aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to PILRα, and the antibody or the antigen-binding fragment thereof is selected from:

[0092] (i) an antibody having the CDR and / or variable region sequences of antibody C21 or C29;

[0093] (ii) an antibody having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the CDR or variable region sequences described in (i); or

[0094] (iii) an antibody having only one or more (e.g., at most 20, at most 15, at most 10, or at most 5) conservative substitutions compared to the CDR or variable region sequences described in (i).

[0095] In certain embodiments, the antibody or the antigen-binding fragment thereof specifically binds to the Stalk domain of PILRα. In certain embodiments, the antibody or the antigen-binding fragment thereof does not bind, or binds substantially not at all, or undetectably binds to the PILRα protein lacking the Stalk domain (e.g., the polypeptide shown in SEQ ID NO:31).

[0096] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to a functional epitope in the Stalk domain of PILRα, and the functional epitope comprises at least one O-glycosylation site. In certain embodiments, the O-glycosylation site is selected from Ser and / or Thr residues. In certain embodiments, the O-glycosylation site is selected from S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176 or any combination thereof, and the amino acid positions are determined according to SEQ ID NO:25. In certain embodiments, the functional epitope comprises one or more of the following amino acid residues: S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176, and the amino acid positions are determined according to SEQ ID NO:25.

[0097] C21

[0098] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from antibodies having the CDR and / or variable region sequences of antibody C21, antibodies having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to its CDR or variable region sequences, and antibodies having only one or more (such as, at most 20, at most 15, at most 10, or at most 5) conservative substitutions compared to its CDR or variable region sequences.

[0099] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: heavy chain CDR1, CDR2, and CDR3 respectively comprising SEQ ID NOs: 1, 2, and 3, and light chain CDR1, CDR2, and CDR3 respectively comprising SEQ ID NOs: 4, 5, and 6. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: heavy chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 1, 2, and 3 respectively, and light chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 4, 5, and 6 respectively.

[0100] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:7 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:8 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH having the sequence as shown in SEQ ID NO:7 and a VL having the sequence as shown in SEQ ID NO:8.

[0101] C29

[0102] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from antibodies having the CDRs and / or variable region sequences of antibody C29, antibodies having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to its CDR or variable region sequences, and antibodies having only one or more (e.g., at most 20, at most 15, at most 10, or at most 5) conservative substitutions compared to its CDR or variable region sequences.

[0103] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: heavy chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs:9, 10, and 11, respectively, and light chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs:12, 13, and 14, respectively. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: heavy chain CDR1, CDR2, and CDR3 having the sequences as shown in SEQ ID NOs:9, 10, and 11, respectively, and light chain CDR1, CDR2, and CDR3 having the sequences as shown in SEQ ID NOs:12, 13, and 14, respectively.

[0104] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:15 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:16 or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH having the sequence as shown in SEQ ID NO:15 and a VL having the sequence as shown in SEQ ID NO:16.

[0105] In certain embodiments, the antibody or antigen-binding fragment thereof in any of the above embodiments may further comprise a heavy chain constant region (CH) and a light chain constant region (CL). The constant region sequences may be derived from mammalian (e.g., murine or human) immunoglobulins.

[0106] In certain embodiments, the heavy chain constant region is selected from IgG, IgM, IgE, IgD or IgA.

[0107] In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is a murine IgG1, IgG2, IgG3 or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region. In certain embodiments, preferably the heavy chain constant region is a human IgG1 or IgG4 heavy chain constant region.

[0108] In certain embodiments, the light chain constant region is selected from κ or λ.

[0109] In certain embodiments, the light chain constant region is a κ light chain constant region. In certain embodiments, the light chain constant region is a murine κ light chain constant region. In certain embodiments, the light chain constant region is a human κ light chain constant region.

[0110] In certain embodiments, the heavy chain constant region may comprise one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibodies of the present invention: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function, etc. Functional alterations can be generated by replacing at least one amino acid residue in the antibody constant region with a different residue or by chemical modification. For example, altering the affinity of the antibody for effector ligands (such as FcR or complement C1q) can change effector function (e.g., reducing or enhancing it). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

[0111] In certain embodiments, the antigen-binding fragment in any of the above embodiments may be selected from Fab, Fab’, (Fab’) 2 , Fv, disulfide-linked Fv, scFv, diabody.

[0112] In certain embodiments, the antibody in any of the above embodiments is a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0113] Antibody Derivatives

[0114] In another aspect, the present invention provides an antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof described in the previous aspect for binding to PILRα and / or binds to the same epitope on PILRα.

[0115] In certain embodiments, the antibody or antigen-binding fragment thereof competes with the specific antibody C21 or C29 described herein or an antibody comprising its CDR and / or variable region sequence for binding to PILRα and / or binds to the same epitope on PILRα.

[0116] In certain embodiments, the antibody or antigen-binding fragment thereof inhibits the binding of the specific antibody C21 or C29 described herein or an antibody comprising its CDR and / or variable region sequence to PILRα by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0117] In certain embodiments, the antibody or antigen-binding fragment thereof does not bind, or binds substantially not at all, or undetectably binds to a PILRα protein lacking a Stalk domain (such as the polypeptide shown in SEQ ID NO:31).

[0118] Competitive antibodies can be obtained by using methods known in the art. For example, mice can be immunized with PILRα to generate hybridomas, and the resulting monoclonal antibodies can be screened for their ability to compete with the antibodies described herein for binding to PILRα. Mice can also be immunized with a smaller fragment of PILRα that contains the epitope to which the antibodies described herein bind.

[0119] The ability to compete for binding to PILRα can be determined based on conventional quantitative or qualitative antibody binding assays, such as ELISA, RIA, surface plasmon resonance, flow cytometry, etc. For example, a standard ELISA assay or a competitive ELISA assay can be used, in which the PILRα protein (e.g., human PILRα) is immobilized on a plate, different concentrations of unlabeled first antibody are added, the plate is washed, a labeled second antibody is added, the plate is washed again, and the amount of bound label is measured. If increasing concentrations of the unlabeled (first) antibody (also referred to as the "blocking antibody") inhibit the binding of the labeled (second) antibody, the first antibody is said to inhibit the binding of the second antibody to the target on the plate, or to compete for binding with the second antibody. Additionally or alternatively, SPR analysis can be used to evaluate the ability of an antibody to compete. Testing the ability of an antibody to inhibit the binding of an antibody or its antigen-binding fragment described in the third or fourth aspect to PILRα demonstrates that the test antibody is capable of competing with that antibody for binding to PILRα.

[0120] Techniques for determining that a test antibody binds to the "same epitope on PILRα" as the antibodies described herein include conventional epitope mapping methods, such as site-directed mutagenesis (where loss of binding due to mutations within the antigen sequence is often considered an indication of an epitope component), competitive binding experiments, crystal structure analysis (such as x-ray analysis of crystals of the antigen:antibody complex), etc.

[0121] Preparation

[0122] The antibodies or their antigen-binding fragments of any of the above aspects can be derivatized, for example, by being linked to another molecule (such as another polypeptide or protein or small molecule). Generally, derivatization of the antibody or its antigen-binding fragment does not adversely affect its binding to PILRα (especially human PILRα). Accordingly, the present invention also intends to include such derivatized forms. For example, the antibodies or their antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating the binding of the antibody or antigen-binding fragment of the present invention to another molecule (e.g., avidin or a polyhistidine tag).

[0123] In another aspect, the present invention provides a multispecific antibody comprising the antibody or antigen-binding fragment thereof described in any of the above aspects.

[0124] In certain embodiments, the multispecific antibody specifically binds to PILRα and additionally specifically binds to other targets.

[0125] In certain embodiments, the multispecific antibody further comprises at least one second antibody having a second binding specificity for a second target.

[0126] In certain embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0127] In another aspect, the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof described in any of the above aspects and a conjugate moiety.

[0128] In certain embodiments, the conjugate moiety is selected from therapeutic agents. In certain embodiments, the conjugate is an antibody-drug conjugate (ADC). In certain embodiments, the therapeutic agent is a cytotoxic agent.

[0129] In certain embodiments, the conjugate moiety is selected from detectable labels. A detectable label can be any substance detectable by means such as fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrochemistry, optics, or chemistry. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes, chemiluminescent substances (such as acridinium ester compounds), or biotin. In certain embodiments, such labels are suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable label as described above can be linked to the antibody or antigen-binding fragment of the present invention through linkers of different lengths to reduce potential steric hindrance.

[0130] In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof described in any of the above aspects as an extracellular antigen-binding domain. A CAR typically comprises an extracellular antigen-binding domain (e.g., scFv), an optional spacer domain, a transmembrane domain, and an intracellular signaling domain. The present invention also provides a host cell containing or expressing the chimeric antigen receptor, such as an immune cell (e.g., T lymphocyte, NK cell).

[0131] Application

[0132] The antibodies, multispecific antibodies or chimeric antigen receptors of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant techniques. For example, DNA molecules encoding them are obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector, and then the host cells are transfected. Then, the transfected host cells are cultured under specific conditions, and the antibodies, multispecific antibodies or chimeric antigen receptors of the present invention are expressed.

[0133] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules. Additionally, these antigen-binding fragments can also be directly produced by recombinant host cells. For example, Fab’ fragments can be directly obtained from host cells; Fab’ fragments can be chemically conjugated to form F(ab’) 2 fragments. Additionally, Fv, Fab or F(ab’) 2 fragments can also be directly isolated from the culture broth of recombinant host cells. Those of ordinary skill in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0134] In another aspect, the present invention provides an isolated nucleic acid molecule comprising: (i) a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof, in any aspect of the present invention; (ii) a nucleotide sequence of a multispecific antibody of the present invention or at least one of its polypeptide chains; or (iii) a nucleotide sequence encoding a chimeric antigen receptor of the present invention.

[0135] In certain embodiments, the isolated nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof, of the present invention. In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain variable region or a heavy chain of an antibody or an antigen-binding fragment thereof, of the present invention, and a second nucleotide sequence encoding a light chain variable region or a light chain of an antibody or an antigen-binding fragment thereof, of the present invention. The first nucleotide sequence and the second nucleotide sequence are on the same or different isolated nucleic acid molecules.

[0136] In certain embodiments, the isolated nucleic acid molecule encodes a multispecific antibody of the present invention, or at least one of its polypeptide chains. In certain embodiments, the isolated nucleic acid molecule comprises nucleotide sequences encoding each polypeptide chain of a multispecific antibody of the present invention. The nucleotide sequences encoding each polypeptide chain are on the same or different isolated nucleic acid molecules.

[0137] In certain embodiments, the isolated nucleic acid molecule encodes a CAR of the present invention.

[0138] In another aspect, the present invention provides a vector (such as a cloning vector or an expression vector) that comprises the isolated nucleic acid molecule as described above.

[0139] In certain embodiments, the vector comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof of the present invention, or the variable region of the heavy chain and / or the variable region of the light chain thereof. In certain embodiments, the vector comprises a first nucleotide sequence encoding the variable region of the heavy chain or the heavy chain of an antibody or an antigen-binding fragment thereof of the present invention, and a second nucleotide sequence encoding the variable region of the light chain or the light chain of an antibody or an antigen-binding fragment thereof of the present invention. The first nucleotide sequence and the second nucleotide sequence are on the same or different vectors. In certain embodiments, the vector is, for example, a plasmid, a cosmid, a phage, etc.

[0140] In certain embodiments, the vector comprises a nucleotide sequence encoding a multispecific antibody of the present invention, or at least one polypeptide chain thereof. In certain embodiments, the vector comprises nucleotide sequences encoding each polypeptide chain of a multispecific antibody of the present invention. The nucleotide sequences encoding each polypeptide chain are on the same or different vectors. In certain embodiments, the vector is, for example, a plasmid, a cosmid, a phage, etc.

[0141] In certain embodiments, the vector comprises a nucleotide sequence encoding a CAR of the present invention. In certain embodiments, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector or a retroviral vector.

[0142] In another aspect, the present invention provides a host cell that comprises the isolated nucleic acid molecule or vector as described above.

[0143] In certain embodiments, the host cell comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof of the present invention, or the variable region of the heavy chain and / or the variable region of the light chain thereof, or an isolated nucleic acid molecule or vector comprising the nucleotide sequence. In certain embodiments, the host cell comprises a prokaryotic cell such as an Escherichia coli cell, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, for example, mouse cells, human cells, etc.).

[0144] In certain embodiments, the host cell comprises a nucleotide sequence encoding a multispecific antibody of the present invention, or at least one polypeptide chain thereof, or an isolated nucleic acid molecule or vector comprising the nucleotide sequence. In certain embodiments, the host cell comprises a prokaryotic cell such as an Escherichia coli cell, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, for example, mouse cells, human cells, etc.).

[0145] In certain embodiments, the host cell comprises a nucleotide sequence encoding a CAR of the present invention or an isolated nucleic acid molecule or vector comprising the nucleotide sequence. In certain embodiments, the host cell comprises an immune cell (e.g., a human immune cell), such as a T lymphocyte, NK cell, monocyte, macrophage, or dendritic cell, and any combination thereof, such as a T cell and / or an NK cell.

[0146] In another aspect, provided is a method for preparing an antibody or an antigen-binding fragment thereof, a multispecific antibody, or a chimeric antigen receptor of the present invention, which comprises culturing the host cell as described above under conditions permitting protein expression, and recovering the antibody or an antigen-binding fragment thereof, the multispecific antibody, or the chimeric antigen receptor from the cultured host cell culture.

[0147] Drug Screening

[0148] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof described in any of the above aspects, or an affinity molecule, a multispecific antibody, a conjugate, a chimeric antigen receptor, or an immune cell expressing a chimeric antigen receptor, an isolated nucleic acid molecule, a vector, or a host cell described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0149] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In certain embodiments, the additional pharmaceutically active agent is a drug having anti-tumor activity, such as an alkylating agent, a mitosis inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a specific tumor cell-targeting antibody, or an immune checkpoint inhibitor.

[0150] In certain embodiments, the additional pharmaceutically active agent is an immune checkpoint inhibitor, such as a PD-1 / PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, or any combination thereof.

[0151] In certain embodiments, the additional pharmaceutically active agent is a PD-1 / PD-L1 inhibitor, such as a PD-1 or PD-L1 antibody.

[0152] The pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections, inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by incorporating the required dose of the active ingredient in a suitable solvent, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) at the same time, followed by filtration sterilization. In addition, the sterile injection solution can be prepared as a sterile freeze-dried powder (for example, by vacuum drying or lyophilization) for ease of storage and use.

[0153] The pharmaceutical composition of the present invention can be administered by any suitable method known in the art. Preferred routes of administration include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration. Parenteral administration refers to a mode of administration that is usually by injection rather than enteral and topical administration, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, it can be administered via non-parenteral routes, such as local, epidermal or mucosal administration routes, for example, intranasal, oral, vaginal, rectal, sublingual or topical.

[0154] In another aspect, the present invention provides the use of the antibody or its antigen-binding fragment described in any of the above aspects, or the affinity molecule, multispecific antibody, conjugate, chimeric antigen receptor or immune cell expressing a chimeric antigen receptor, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition described herein for the treatment of tumors, or the use in the preparation of a drug for the treatment of tumors. In addition, a method for treating tumors is also provided, the method comprising: administering to a subject in need thereof an effective amount of the antibody or its antigen-binding fragment described in any of the above aspects, or the affinity molecule, multispecific antibody, conjugate, chimeric antigen receptor or immune cell expressing a chimeric antigen receptor, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition described herein.

[0155] In certain embodiments, the treatment comprises administering the antibody or its antigen-binding fragment described in any aspect herein or a pharmaceutical composition comprising the same.

[0156] In certain embodiments, the treatment comprises administering the affinity molecule described herein or a pharmaceutical composition comprising the same.

[0157] In certain embodiments, the treatment comprises administering a multispecific antibody or a pharmaceutical composition comprising the same as described herein.

[0158] In certain embodiments, the treatment comprises administering a conjugate or a pharmaceutical composition comprising the same as described herein.

[0159] In certain embodiments, the treatment comprises administering a chimeric antigen receptor or an immune cell expressing a chimeric antigen receptor or a pharmaceutical composition comprising the same as described herein.

[0160] In certain embodiments, the treatment is anti-tumor immunotherapy. In certain embodiments, the anti-tumor immunotherapy comprises enhancing an anti-tumor immune response and / or reducing tumor immunosuppression in a subject.

[0161] In certain embodiments, the tumor comprises a solid tumor or a hematological tumor. In certain embodiments, the tumor is selected from breast cancer, cholangiocarcinoma, esophageal cancer, glioblastoma, head and neck cancer, kidney cancer (such as clear cell renal cell carcinoma KIRC or papillary renal cell carcinoma KIRP), sarcoma, thyroid cancer, and gastric cancer.

[0162] In certain embodiments, the tumor expresses PILRα or is PILRα-positive. The PILRα positivity can be determined at the nucleic acid level or the protein level. Methods for determining expression at the nucleic acid level include, but are not limited to, RT-PCR or real-time RT-PCR. Methods for determining expression at the protein level include, but are not limited to, immunological detection.

[0163] In another aspect, the present invention provides the use of the antibody or its antigen-binding fragment, or the affinity molecule, multispecific antibody, conjugate, chimeric antigen receptor or immune cell expressing a chimeric antigen receptor, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition described in any of the above aspects for modulating an immune response, or for use in the preparation of a medicament for modulating an immune response. In addition, a method for modulating an immune response is provided, the method comprising: administering to a subject in need thereof an effective amount of the antibody or its antigen-binding fragment, or the affinity molecule, multispecific antibody, conjugate, chimeric antigen receptor or immune cell expressing a chimeric antigen receptor, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition described in any of the above aspects.

[0164] In certain embodiments, the modulating the immune response comprises enhancing or stimulating a T cell immune response and / or enhancing T cell activity in a subject (such as a human), thereby for the treatment of any disease that would benefit from enhanced immune response. In certain embodiments, the subject has a tumor.

[0165] In certain embodiments, said modulating an immune response comprises administering an antibody or an antigen-binding fragment thereof or a pharmaceutical composition comprising the same as described in any aspect herein.

[0166] In certain embodiments, said modulating an immune response comprises administering an affinity molecule or a pharmaceutical composition comprising the same as described herein.

[0167] In certain embodiments, said modulating an immune response comprises administering a multispecific antibody or a pharmaceutical composition comprising the same as described herein.

[0168] In certain embodiments, said modulating an immune response comprises administering a conjugate or a pharmaceutical composition comprising the same as described herein.

[0169] In certain embodiments, said modulating an immune response comprises administering a chimeric antigen receptor or an immune cell expressing a chimeric antigen receptor or a pharmaceutical composition comprising the same.

[0170] Term Definition

[0171] In another aspect, the present invention provides a method for screening a drug for treating a tumor or modulating an immune response, wherein the method comprises screening for a PILRα / CD99 pathway inhibitor (such as a PILRα inhibitor). The screening step is carried out in vitro.

[0172] In another aspect, the present invention provides a method for screening a drug for treating a tumor or modulating an immune response, wherein the method comprises: screening for a reagent (such as an antibody, a small molecule or an aptamer) that can bind to the stalk domain of PILRα. The screening step is carried out in vitro.

[0173] In certain embodiments, the method comprises: screening a reagent that can bind to the stalk domain of PILRα as a test reagent, detecting whether the test reagent can inhibit or block the interaction between PILRα and CD99, and selecting a test reagent that can inhibit or block the interaction between PILRα and CD99 as a candidate drug. The above screening can be determined by conventional binding assays in the art, such as by BLI, SPR, or ELISA assays.

[0174] In another aspect, the present invention provides a method for screening a drug for treating a tumor or modulating an immune response, wherein the method comprises: screening for a reagent (such as an antibody, a small molecule or an aptamer) that competes with an antibody or an antigen-binding fragment thereof as described in any aspect herein for binding to PILRα and / or binds to the same epitope on PILRα. The screening step is carried out in vitro.

[0175] In certain embodiments, the method includes: screening a reagent that competes with the antibody or antigen-binding fragment thereof described in any aspect herein for binding to PILRα and / or binds to the same epitope on PILRα as a test reagent, detecting whether the test reagent can inhibit or block the interaction between PILRα and CD99, and selecting a test reagent that can inhibit or block the interaction between PILRα and CD99 as a candidate drug. The above screening can be determined by conventional competitive binding assays or epitope mapping methods in the art.

[0176] Figure 1

[0177] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand the present invention, the definitions and explanations of related terms are provided below.

[0178] As used herein, the term "PILRα" refers to paired immunoglobulin-like receptor α, which consists of an immunoglobulin-like V-type (IgV) domain, a stalk domain, a transmembrane (TM) domain, and a cytoplasmic carboxyl-terminal domain (CTD). The IgV domain and the stalk domain together form the extracellular domain (ECD). PILRα is preferably human, and can also be a homologous gene from other species (e.g., non-human mammals, fish, reptiles or birds, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, primates, zebrafish, etc.). The sequence of PILRα is well known to those skilled in the art and can be found in various public databases, such as GenBank: NM_013439. An exemplary amino acid sequence of human PILRα can be found in SEQ ID NO:25.

[0179] In this text, when referring to the amino acid sequence of PILRα, the sequence shown in SEQ ID NO:25 is used for description. For example, the expression "the Xth amino acid residue of PILRα" refers to the Xth amino acid residue of the polypeptide shown in SEQ ID NO:25. However, those skilled in the art understand that in the amino acid sequence of PILRα, mutations or variations can occur naturally or be introduced artificially without affecting its biological function. Therefore, in the present invention, the term "PILRα" and its similar expressions shall include all such sequences, including, for example, the sequence shown in SEQ ID NO:25 and its natural or artificial variants (such as variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity). And when describing the residue position or sequence fragment of PILRα, it includes not only the residue position or sequence fragment of SEQ ID NO:25, but also the corresponding residue position or corresponding sequence fragment in its natural or artificial variants. For example, the expression "the Xth amino acid residue of PILRα" includes the Xth amino acid residue of SEQ ID NO:25 and the corresponding position in its variants (natural or artificial). According to the present invention, the expression "corresponding position" or "corresponding fragment" means the residues or fragments at equivalent positions in the sequences being compared when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage identity. The best alignment of the sequences for comparison can be performed by a computerized implementation of known algorithms or by visual inspection. Existing sequence alignment and multiple sequence alignment algorithms include BLAST, ClustalW / ClustalW2 / Clustal Omega, etc.

[0180] As used herein, the term "CD99", also known as MIC2 or single-chain type 1 glycoprotein, is a glycosylated protein located on the cell membrane and is typically expressed on the cell membranes of lymphocytes, thymic cortical cells, and ovarian granulosa cells. CD99 is preferably of human origin and can also be a homologous gene from other species (e.g., non-human mammals, fish, reptiles, or birds, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, primates, zebrafish, etc.). The sequence of CD99 is well-known to those skilled in the art and can be found in various public databases, such as GenBank: NM_002414.

[0181] As used herein, the term "specifically binds" or "is specific for" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and the antigen it targets. The binding affinity between two molecules can be described by the KD value. The KD value is the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also referred to as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also referred to as kon), or kd / ka expressed as a molar concentration (M). The smaller the KD value, the tighter the binding between the two molecules and the higher the affinity. In certain embodiments, an antibody that specifically binds an antigen (or an antibody that is specific for an antigen) refers to an antibody that binds the antigen with an affinity (KD) of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less. The KD value can be determined by methods well known in the art, such as by measuring it in a BIACORE instrument using surface plasmon resonance (SPR).

[0182] As used herein, the term "epitope" in its broadest sense refers to the site on an antigen that is specifically bound by an affinity molecule (such as an immunoglobulin or antibody, small molecule, or aptamer). Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational". In a linear epitope, all the points of interaction between the protein and the interacting molecule (such as an antibody) are linearly present along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction exist across protein amino acid residues that are separated from each other.

[0183] As used herein, the term "antibody" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant and can include various antibody structures, provided they exhibit the required antigen-binding activity. Typically, an antibody can be an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotypes of the antibody are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be further subdivided into regions of high variability (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site.

[0184] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0185] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined by the IMGT, Kabat, or Chothia numbering systems. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined by the Kabat numbering system.

[0186] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0187] The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0188] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". Antigen-binding fragments of an antibody can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of a full-length antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab’, F(ab’) 2 , Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), probody, and such polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0189] As used herein, the term "full-length antibody" means an antibody composed of two "full-length heavy chains" and two "full-length light chains". Wherein, a "full-length heavy chain" refers to a polypeptide chain that, in the N-terminal to C-terminal direction, is composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is of the IgE isotype, optionally further includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention can be from a single species, such as human; or can be chimeric antibodies or humanized antibodies. The full-length antibodies of the present invention comprise two antigen-binding sites formed by VH and VL pairs respectively, and these two antigen-binding sites specifically recognize / bind the same antigen.

[0190] As used herein, the term "Fd fragment" means an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" means an antibody fragment composed of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment composed of VL, VH, CL, and CH1 domains; the term "F(ab’) 2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region; the term "Fab’ fragment" means a reduced-linked F(ab’) 2The fragment obtained after the disulfide bond of the two heavy chain fragments in the fragment consists of a complete light chain and the Fd fragment of the heavy chain (composed of the VH and CH1 domains).

[0191] As used herein, the term "Fv fragment" refers to an antibody fragment composed of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a single variable region (such as the Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind an antigen, although its affinity may be lower than that of the complete binding site.

[0192] As used herein, the term "Fc fragment" refers to an antibody fragment formed by the disulfide bond of the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various different functions but does not participate in antigen binding.

[0193] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains, wherein the VL and VH are linked by a linker. Such scFv molecules can have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH. Suitable prior art linkers consist of a repeated GGGGS amino acid sequence or variants thereof. In some cases, a disulfide bond may also exist between the VH and VL of the scFv.

[0194] As used herein, the term "diabody" refers to an antibody in which the VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains on the other chain and generating two antigen-binding sites.

[0195] Each of the above antibody fragments retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen.

[0196] Antigen-binding fragments of an antibody (such as the above antibody fragments) can be obtained from a given antibody (such as the antibodies provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as for the full-length antibody.

[0197] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only the full-length antibody but also the antigen-binding fragments of the antibody.

[0198] As used herein, the terms "monoclonal antibody", "single antibody", and "mAb" have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, relative to monoclonal antibodies, generally contain at least two or more different antibodies, and these different antibodies generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.

[0199] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0200] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0201] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. "Percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared × 100. For example, if 6 of 10 positions in two sequences match, then the two sequences have 60% identity. For instance, the DNA sequences CTGACT and CAGGTT have 50% identity (3 of a total of 6 positions match). Generally, comparison is made when the two sequences are aligned to yield maximum identity. Such alignment can be achieved by using, e.g., the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). The algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), incorporated into the ALIGN program (version 2.0), can also be used, with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com) can be used, with a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences.

[0202] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue with another amino acid residue having a similar side chain, such as a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having a similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art.

[0203] The notations of the twenty conventional amino acids involved in this article follow the conventional usage. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present invention, amino acids are generally represented by the single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0204] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are well known in the art and include, but are not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining reagents, absorption delaying reagents, preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining reagents include, but are not limited to, sugars, NaCl and its analogs. Absorption delaying reagents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Stabilizers have the meaning commonly understood by those skilled in the art, and can stabilize the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (such as 0.9% (w / v) NaCl), glucose solutions (such as 5% glucose), solutions containing surfactants (such as 0.01% polysorbate 20), pH buffer solutions (such as phosphate buffer solutions), Ringer's solution and any combination thereof.

[0205] As used herein, the term "subject" includes any individual capable of generating an immune response. In certain embodiments, the subject refers to a mammal. In certain embodiments, the subject refers to a human. In certain embodiments, the subject (such as a human) has a tumor or any other disease that would benefit from enhanced immune response, or is at risk of having the above diseases.

[0206] As used herein, the term "treatment" refers to a method of administering for the purpose of obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission of symptoms (whether partial or total), whether detectable or not. In addition, "treatment" can also refer to an extension of survival as compared to the expected survival period if not treated. For "anti-tumor effects", it includes, but is not limited to, for example, reduction in tumor volume, reduction in the number of cancer cells, reduction in the number of metastatic foci, increase in life expectancy, reduction in cancer cell proliferation, reduction in cancer cell survival, or improvement of various physiological symptoms associated with the cancer condition.

[0207] As used herein, the term "effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of a particular condition. The effective amount herein can vary depending on factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit the desired response in an individual. The effective amount is also the amount at which the beneficial effects of treatment outweigh any toxic or adverse effects of the treatment. For prophylactic use, beneficial or desired outcomes include outcomes such as elimination or reduction of risk, alleviation of severity, or delay in the onset of a disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and the intermediate pathological phenotypes presented during disease formation. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reduction of one or more symptoms resulting from the disease, improvement in the quality of life of subjects suffering from the disease, reduction in the dosage of other medications required to treat the disease, enhancement of the effect of another medication (such as via targeting), delay in the progression of the disease, and / or extension of survival. In the case of cancer or a tumor, an effective amount of a drug can have an effect in reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., slowing to some extent and desirably stopping) the invasion of cancer cells into peripheral organs; inhibiting (i.e., slowing to some extent and desirably stopping) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating one or more symptoms associated with the condition to some extent. The effective amount can be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly effect a prophylactic or therapeutic treatment.

[0208] As used herein, the terms "cancer" and "tumor" are used interchangeably and refer to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer includes solid tumors as well as hematological malignancies such as lymphoma, leukemia, myeloma or lymphoid malignancies, and cancers of the spleen and lymph nodes.

[0209] Beneficial effects

[0210] The present invention for the first time discovers that PILRα has a negative regulatory effect on the activation, proliferation and effector functions of T cells, and the T cell surface antigen CD99 serves as a ligand for PILRα to mediate its immunosuppressive function. Thus, the present invention for the first time provides an immunotherapy based on an inhibitor of the PILRα / CD99 signaling pathway. In addition, the present invention for the first time identifies a functional epitope through which PILRα exerts its regulatory effect and provides a monoclonal antibody targeting the functional epitope of PILRα. Such antibodies can effectively regulate the immune response and inhibit tumor growth by blocking the interaction between PILRα and CD99.

[0211] The embodiments of the present invention will be described in detail below in conjunction with the drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and not to limit the scope of the present invention. According to the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art. Brief description of the drawings

[0212] Figure 2This is the experimental result graph of Example 1 of the present invention. This graph shows that PILRα was identified as a T cell activity inhibitory molecule by high-throughput screening. (A) Schematic diagram of screening for co-inhibitory or co-stimulatory cell surface molecules against T cells in tumor cells. RNA sequencing analysis was performed on U87 cells co-cultured with EGFR CAR-T, and genes upregulated by CAR-T (n = 600, FC ≥ 2, P ≤ 0.05), genes encoding membrane proteins among them (n = 148), and genes overexpressed in GBM cells and associated with poor prognosis (n = 32) were shown. Expression vectors (pTT5) of these 32 genes were transfected into aAPC-293T cells expressing transmembrane form of anti-human CD3 single-chain antibody (OKT3) and transmembrane signaling molecules (FCER1G, CD3Z, DAP10, and DAP12) respectively. Jurkat T cells carrying NFAT-GFP reporter gene were added to aAPC-293T cells, and GFP fluorescence was recorded. Genes with changed GFP reporter signals (n = 5, FC ≥ 1, P ≤ 0.05) were further verified for their effects on human primary T cells. (B) GO enrichment analysis was performed on CAR-T-induced genes encoding membrane proteins (n = 148). (C, D) aAPC-293T cells were transfected with control vector or pTT5-PD-L1, PILRα, CD70, or ICOSLG, co-cultured with Jurkat T cells carrying NFAT-GFP (C) and NFAT-Lucia reporter genes (D) for 8 h, and then imaging and fluorescence activity measurements were performed (mean ± standard error, **P < 0.01; *P < 0.05).

[0213] Figure 3This is the experimental result graph of Example 2 of the present invention. This graph shows that the mucin stalk domain of PILRα is crucial for its immunosuppressive function. (A) Schematic diagrams of the domains of PILRα (full length, FL), PILRβ (full length, FL), as well as PILRα (ECD, extracellular domain region), PILRβ (ECD, extracellular domain region), PILRα isoform 1 (iso1), PILRα isoform 2 (iso2), and the fusion protein of the IgV domain of PILRα (IgV) with the Fc of human IgG1 (hIg). (B) Jurkat T cells carrying the NFAT-Lucia reporter gene were added to a plate pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and then treated or not treated with purified PILRα protein [PILRα(ECD)-hIg, 20 μg / mL] for 12 h. Finally, luciferase activity was measured (mean ± s.e.m, ***P < 0.001; n.s., not significant). (C) Jurkat T cells carrying the NFAT-Lucia reporter gene as described in (B) were treated with the indicated concentrations of PILRα(ECD)-hIg for 12 h, and then luciferase activity was measured. (D, E, F) CFSE-labeled human primary T cells were added to a plate pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and then cultured for 72 h with or without the addition of purified PILRα protein (20 μg / mL). Finally, the divided T cells (D), CD25 expression (E) were detected by flow cytometry, and the cytokine release levels including IFNγ, granzyme B, and IL2 in the supernatant were measured by ELISA (F) (mean ± s.e.m, **P < 0.01; ***P < 0.001; n.s., not significant). (G, H) CFSE-labeled human primary T cells were added to a plate pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and then cultured for 72 h with or without the addition of hIg, PILRα(ECD)-hIg, or PILRα(IgV)-hIg (20 μg / mL). Finally, the divided T cells (G) were detected by flow cytometry, and the cytokine release amounts including IFNγ, granzyme B, and IL2 in the supernatant were measured by ELISA (H) (mean ± s.e.m, ***P < 0.001; n.s., not significant). (I) Immunoblot analysis of purified PILRα(ECD)-hIg and PILRα(IgV)-hIg proteins using biotin-conjugated peanut agglutinin (PNA) and streptavidin-horseradish peroxidase (streptavidin-HRP). (J) The purified PILRα(ECD)-hIg protein was treated or not treated with a combination of O-glycosidase and neuraminidase for 4 h, and then IB analysis was performed using an anti-PILRα antibody.(K) Human primary T cells were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and then co-cultured for 72 h with or without O-glycosidase / neuraminidase-treated or untreated PILRα protein (PILRα(ECD)-hIg, 20 μg / mL). Finally, flow cytometry was performed to detect CD25 expression. (L) Schematic diagrams of wild-type (WT) PILRα(ECD) and a PILRα mutant [T&S(M)] in which all identified O-glycosylated Ser (S) and Thr (T) residues were replaced with alanine (Ala). (M) Immunoblot analysis of purified PILRα(ECD)-hIg (WT) and T&S(M) proteins with the indicated antibodies. (N-P) CFSE-labeled human primary T cells were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and then cultured for 72 h with or without purified PILRα(ECD) (WT) or T&S(M) (20 μg / mL). Subsequently, dividing T cells (N), CD25 expression (O) were detected by flow cytometry, and the levels of cytokine release including IFNγ, granzyme B, and IL2 in the supernatant were determined by ELISA (P) (mean ± standard error, ***P < 0.001; n.s., not significant).

[0214] Figure 4 This is the experimental result figure of Example 3 of the present invention. This figure shows that the binding of PILRα to the T cell surface protein CD99 inhibits T cell activity. (A) Flow cytometric analysis of human primary T cells stained with FITC-conjugated anti-CD99 antibody. (B) Purified His-tagged CD99 protein was immobilized on the surface using a Ni 2+ -NTA biosensor for surface plasmon resonance analysis, and PILRα(ECD) protein at the indicated concentrations was added. K D , equilibrium dissociation constant. (C) Purified PILRα(ECD) protein at the indicated concentrations was added to plates coated with His-tagged CD99 protein, and then ELISA was performed (mean ± standard error). (D) HEK293T cells were co-transfected to express Flag-tagged PILRα(WT) or T&S(M) and Myc-tagged CD99, and then immunoprecipitation was performed using anti-Flag M2 affinity gel, and immunoblot analysis was performed with the indicated antibodies. (E) Using Ni 2+- The -NTA biosensor immobilized purified PILRα(ECD) on the surface for surface plasmon resonance analysis, and anti-PILRα antibody C21 was added at the indicated concentrations. (F) PILRα(ECD), PILRα(IgV), or PILRαT&S(M) proteins at the indicated concentrations were added to the C21-coated plates, followed by ELISA assay (mean ± standard error). (G) Purified PILRα(ECD) (2 μg / mL) was incubated with a plate coated with His-tagged CD99 for 1 h, and then isotype control (CTL) or C21 was added at the indicated concentrations for ELISA assay (mean ± standard error). (H) HEK293T cells expressing PILRα(WT), PILRαT&S mutant (M), or PILRα(IgV) were stained with C21, followed by flow cytometry analysis. (I, J, K) CFSE-labeled human primary T cells were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3) and incubated for 72 h in the presence or absence of PILRα(ECD) (20 μg / mL) and isotype control CTL or C21 (50 μg / mL). Subsequently, dividing T cells (I) and CD25 expression (J) were detected by flow cytometry, and cytokine release including IFNγ, granzyme B, and IL2 was measured by ELISA (K) (mean ± standard error, ***P < 0.0001). (L) Wild-type (CD99 WT ) or CD99 knockdown (CD99 KD ) human primary T cells were stained with FITC anti-CD99 antibody and analyzed by flow cytometry. (M, N, O) CFSE-labeled CD99 WT and CD99 KD human primary T cells were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3) and incubated for 72 h in the presence or absence of purified PILRα(ECD) (20 μg / mL). Subsequently, dividing T cells (M) and CD25 expression (N) were detected by flow cytometry, and IFNγ release levels were measured by ELISA (O) (mean ± standard error, ***P < 0.0001; n.s., not significant). (P, Q) CFSE-labeled human primary T cells were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3) and co-incubated with isotype control antibody CTL or C21 antibody (50 μg / mL) for 72 h in the presence or absence of purified PILRα(ECD) (20 μg / mL). Subsequently, dividing T cells (P) were detected by flow cytometry, and IFNγ release was measured by ELISA (Q) (mean ± standard error, ***P < 0.001; n.s., not significant).

[0215] Figure 5 This is the experimental result graph of Example 4 of the present invention. This graph shows that PILRα inhibits CD99-activated ZAP70 / NFAT / IL2 / JAK / STAT signal transduction in T cells. (A, D) Human primary T cells were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and then cultured for 48 h with or without purified PILRα (ECD) (20 μg / mL) for RNA-seq (A) and immunoblot analysis (D). The genes induced and inhibited by PILRα (FPKM ≥ 0.5, FC ≥ 1.5) are shown as pie charts in the figure. (B) GO analysis was performed on the genes inhibited by PILRα, as described in (A). (C) KEGG pathway analysis was performed on the genes inhibited by PILRα, as described in (A). (E, F) Human primary T cells were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and in the presence or absence of purified PILRα (ECD) (20 μg / mL) protein, with or without inhibitors against NF-κB (NFκBi, 20 μM), NFAT (NFATi, 25 μM), c-Jun (JUNi, 250 nM), and c-Fos (FOSi, 10 μM), and cultured for 48 h for ELISA determination of IFNγ (E) and IL2 (F) release levels (mean ± s.e.m, **P < 0.01; ***P < 0.001; n.s., not significant). (G) Human primary T cells were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and in the presence or absence of purified PILRα (ECD) (20 μg / mL), with or without cyclosporin A (1 μM) and cultured for 72 h for ELISA determination of IFNγ release (mean ± s.e.m, ***P < 0.001; n.s., not significant). (H) Human primary T cells were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and in the presence or absence of purified PILRα (ECD) (20 μg / mL), with or without NFATi (25 μM) and cultured for 72 h for flow cytometry detection of CD25 expression. (I) The human primary T cells of CD99 WT and CD99 KD were added to plates pre-coated with 1 μg / mL anti-CD3 antibody (αCD3), and then treated for 48 h with or without purified PILRα (ECD) (20 μg / mL), followed by immunoblot analysis.

[0216] Figure 6 This is the experimental result graph of Example 5 of the present invention. This graph shows that PILRα inhibits anti-tumor T cell immune responses in humanized mouse xenograft and murine syngeneic transplantation models. (A) Control (CTL), PILRα knockdown (PILRαKD ), overexpression of PILRα (PILRα OE ), or overexpression of the PILRα T&S mutant (PILRα M ) in U87-luc cells were subjected to immunoblot analysis. (B, C) CTL, PILRα KD , PILRα OE , or PILRα M U87-luc cells were injected intracranially into 6-week-old female NCG mice (3.0×10 5 cells per mouse, n = 5), and then on day 7 after inoculation (day 0), PBMC isolated from the peripheral blood of healthy volunteers were injected intraperitoneally (1.0×10 7 cells per mouse, n = 5). Tumor growth curves (B) and bioluminescence-based tumor images (C) were shown (mean ± s.e.m, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant). (D) IHC analysis of the tumor tissues in (C) was performed using anti-CD8 antibody. Scale bar, 100 μm. (E) Quantification of CD8-positive (CD8 + ) staining in (D) was carried out (mean ± s.e.m; *P < 0.05; **P < 0.01; ***P < 0.001). (F) Flow cytometry analysis of the tumor tissues in (C) was performed, showing the percentages of CD25 + , IFNγ + , granzyme B + , and IL2 + cells among all CD3 + CD8 + cells (mean ± s.e.m, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant). (G) Control, PILRα knockdown (PILRα KD ), overexpression of PILRα (PILRα OE ), or overexpression of the PILRα T&S mutant (PILRα M ) in MDA-MB-231 cells were subjected to immunoblot analysis. (H, I, J) 6-week-old female NCG mice were subcutaneously injected with CTL, PILRα KD , PILRα OE , or PILRα M MDA-MB-231 cells (3.0×10 6 cells per mouse, n = 5), and then on day 15 after inoculation (day 0), PBMC isolated from the peripheral blood of healthy volunteers were injected intraperitoneally (1.0×10 7cells, n = 5). Tumor growth curves (H), tumors dissected on day 42 (I), and tumor weights (J) are shown (mean ± s.e.m, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant). (K) IHC analysis of the tumor tissues described in (I) was performed using anti-CD8 antibody. Scale bar, 100 μm. (L) Quantification of the CD8-positive (CD8 + ) staining shown in (K) was performed (n = 5; mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001). (M) Flow cytometry analysis of the tumor tissues described in (I) was performed, showing the percentages of CD25 + , IFNγ + , granzyme B + , and IL2 + in all CD3 + CD8 + cells (n = 5, mean ± s.e.m, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant). (N) Immunoblot analysis of CTL, Pilrα-knockdown (Pilrα KD ), or Pilrα-overexpressing (Pilrα OE ) Hepa1-6 cells was performed. (O, P, Q) CTL, Pilrα KD , or Pilrα OE Hepa1-6 cells (5.0 × 10 6 cells per mouse, n = 5) were subcutaneously injected into 6-week-old female C57BL / 6 mice. Tumor growth curves (O), tumor weights (P), and survival rates (Q) are shown (mean ± s.e.m, **P < 0.01; ***P < 0.001). (R) Flow cytometry analysis of the spleen (left) and tumor (right) tissues described in (O) was performed. The numbers of CD3 4 T cells, CD3 + CD8 + T cells, CD3 + CD4 + T cells, CD19 + B cells, NK1.1 + NK cells, CD11b + myeloid cells, and CD11b + F4 / 80 + macrophages in 1 × 10 + CD45 +Number of macrophages (n = 5, mean ± s.e.m, *P < 0.05; **P < 0.01; n.s., not significant). Flow cytometry analysis of spleen (left) and tumor (right) tissues of the mice described in (S) to (O) showed all CD3 + CD8 + percentage of CD25 + 、IFNγ + 、granzyme B + and IL2 + cells (n = 3, mean ± s.e.m, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant). (T, U) Three days before subcutaneous inoculation of CTL or Pilrα KD Hepa1-6 cells (5.0 × 10 6 cells per mouse, n = 5), 6-week-old female C57BL / 6 mice were injected with isotype CTL or anti-CD3 antibody (10 mg / kg) respectively, and then injected twice a week. Tumor growth curve (T) and mouse survival rate (U) were shown (mean ± s.e.m, **P < 0.01; ***P < 0.001; n.s., not significant).

[0217] Figure 7 is one of the experimental result graphs of Example 6 of the present invention. This figure shows that the anti-PILRα monoclonal antibody significantly inhibits the growth of tumors in the humanized mouse xenograft model. (A) U87-luc cells (3.0 × 10 5 cells per mouse, n = 5) were injected intracranially (i.c.) into 6-week-old female NCG mice. Seven days later, PBMC isolated from the peripheral blood of healthy volunteers (1.0 × 10 7 cells per mouse) were injected intraperitoneally. Within 24 hours after injection, antibodies including isotype CTL, C21 and C126 (10 mg / kg) were injected intraperitoneally as shown in the figure. (B-D) Tumor growth curve (B), tumor (C) and mouse survival rate (D) shown according to bioluminescence (mean ± s.e.m, *P < 0.05; **P < 0.01; n.s., not significant). (E) MDA-MB-231 cells (3.0 × 10 6 cells per mouse, n = 5) were inoculated subcutaneously (s.c.). 15 days after inoculation, PBMC isolated from the peripheral blood of healthy volunteers (1.0 × 10 7After injecting (3.0×10 cells per mouse, n = 5) U87-luc cells intracranially (i.c.) into 6-week-old female NCG mice, 7 days later, PBMC isolated from the peripheral blood of healthy volunteers was intraperitoneally injected (1.0×10 cells per mouse). Within 24 h after the injection, antibodies including isotype CTL, C21, and C126 (10 mg / kg) were intraperitoneally injected again. (F-I) Tumor growth curves (F), tumors on day 28 (G), tumor weights (H), and survival rates of mice (I) shown according to bioluminescence (mean ± s.e.m, *P < 0.05; **P < 0.01; n.s., not significant).

[0218] Figure 6 This is the second experimental result figure of Example 6 of the present invention. This figure shows that the anti-PILRα antibody can enhance T cell infiltration and is relatively safe in a humanized mouse xenograft model. (A, F) IHC analysis of tumor tissue sections using antibodies against CD8, CD25, IFNγ, granzyme B, and IL2 was performed as shown in Figure 6 B (A) and Figure 6 F (F). Scale bar, 100 μm. (B, G) Quantification of positive staining for CD8, CD25, IFNγ, granzyme B, and IL2 in (A)(B) and (F)(G) (n = 5, mean ± s.e.m, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant). (C, H) Body weights of the mice as described in Figure 6 B (C) and Figure 6 F (H). (D, I) Images of organs such as the heart, liver, spleen, lung, and kidney collected from the mice as shown in Figure 6 B (D) and Figure 8 F (I). (E, J) HE staining was performed on the organ tissue sections shown in (D)(E) and (I)(J). Scale bar, 100 μm.

[0219] Figure 9 This is the first experimental result figure of Example 7 of the present invention. This figure shows that the anti-PILRα monoclonal antibody enhances the antitumor effect of anti-PD-1 immunotherapy on tumors in a humanized mouse xenograft model. (A) U87-luc cells (3.0×10 5 cells per mouse, n = 5) were injected intracranially (i.c.) into 6-week-old female NCG mice, and 7 days later, PBMC isolated from the peripheral blood of healthy volunteers was intraperitoneally injected (1.0×10 7Individual cells), within 24 h after injection, intraperitoneal injection of antibodies including isotype CTL, C21, and PD-1 (10 mg / kg) was performed as shown in the figure. (B-D) Tumor growth curves (B), tumors (C), and survival rates of mice (D) as shown by bioluminescence (mean ± s.e.m, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant). (E) IHC analysis of tumor tissue samples (as described in (C)) using anti-CD8, CD25, IFNγ, granzyme B, and IL2 antibodies. Scale bar, 100 μm. (F) Quantification of CD8, CD25, IFNγ, granzyme B, and IL2 positive staining in (E) (mean ± s.e.m, n = 5, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant).

[0220] Figure 7 This is the second experimental result figure of Example 7 of the present invention. This figure shows that the treatment with anti-PILRα antibody combined with anti-PD-1 antibody is relatively safe in a humanized mouse xenograft model. (A) As shown in Figure 10 Panel B, images of organs such as the heart, liver, spleen, lung, and kidney collected from mice are shown. (B) HE staining was performed on the organ tissue sections shown in (A). Scale bar, 100 μm.

[0221] Figure 11 This is the experimental result figure of Example 9 of the present invention. This figure shows that PILRα is highly expressed in various cancers and is associated with poor prognosis of cancer patients. (A) According to the UALCAN database, the levels of PILRα mRNA (TPM, transcripts per million reads) in normal (blue) and tumor (red) samples are shown. TNBC, triple-negative breast cancer; CHOL, cholangiocarcinoma; ESCA, esophageal cancer; GBM, glioblastoma; HNSC, head and neck squamous cell carcinoma; KIRC, renal cell carcinoma; KIRP, renal papillary cell carcinoma; SARC, sarcoma; THCA, thyroid cancer; STAD, gastric cancer. (B) Kaplan-Meier plot of median baseline overall survival according to PILRα levels in patients with TNBC, ESCA, GBM, KIRC, and STAD. (C) Through TIMER analysis, the correlation between PILRα expression and immune cell infiltration in patients with TNBC, ESCA, GBM, KIRC, and STAD is shown.

[0222] Figure 1 This is the experimental result figure of Example 6 of the present invention, in which the binding of anti-PILRα antibodies C29 and C126 to PILRα (ECD) and PILRα lacking the stalk domain (IgV) was detected.

[0223] Description of Sequence Information

[0224] The information of the sequences involved in the present invention is provided in Table 1.

[0225] Table 1: Sequence Information

[0226]

[0227]

[0228]

[0229] Examples

[0230] The present invention will now be described with reference to the following examples which are intended to illustrate the invention (but not to limit the invention).

[0231] Those skilled in the art will appreciate that the examples describe the invention by way of illustration and are not intended to limit the scope claimed in the present application. The experimental methods in the examples are all conventional methods unless otherwise specified. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained commercially.

[0232] Example 1: Identification of PILRα as an Inhibitory Molecule of T Cell Activity by High-Throughput Screening

[0233] Previous studies have shown that in triple-negative breast cancer (TNBC) and glioblastoma (GBM) cells after receiving chimeric antigen receptor T (CAR-T) cell therapy targeting epidermal growth factor receptor (EGFR), a large number of immunosuppressive molecules are upregulated by IFNγ signaling. Long-term exposure to IFNγ can lead to tumor adaptive immune tolerance. Therefore, the tumor immune response induced by CAR-T cells can mimic T cells in the tumor microenvironment (TME), thereby rapidly identifying key regulatory molecules of T cell anti-tumor immunity and providing potential targets for cancer immunotherapy.

[0234] By performing RNA sequencing (RNA-seq) analysis on the glioblastoma cell line U87 (ATCC) co-cultured with EGFR-targeted CAR-T, it was found that CAR-T upregulated 600 genes (FC≥2, P≤0.05)( Figure 1 A). Among these genes, this example focused on genes encoding membrane proteins because these genes are usually related to anti-tumor immunity. The search results identified 148 genes encoding membrane proteins( Figure 1 A). Gene ontology (GO) analysis showed that the most abundant gene group among these 148 genes was related to T cell co-stimulation(Figure 1 B). Additionally, according to The Cancer Genome Atlas (TCGA) analysis, 32 genes were found to be significantly overexpressed in GBM clinical samples and were associated with poor prognosis ( Figure 1 A).

[0235] To identify potential membrane proteins involved in the regulation of T cell activity, in this example, an artificial antigen-presenting - 293T cell line (aAPC-293T) was used. The construction of this cell line was to engineer the expression of a membrane-localized anti-human CD3 single-chain antibody fragment (scFv, SEQ ID NO:35) on HEK293T cells (ATCC) to stimulate the T cell receptor (TCR), and at the same time express several transmembrane signal transduction molecules (DAP10 (Genbank: NM_014266), DAP12 (Genbank: NM_003332), FCER1G (Genbank: NM_004106) and CD3Z (Genbank: NM_198053)) to enhance antigen presentation. To evaluate the effects of these 32 membrane proteins on T cell activity, in this example, Jurkat T cells (ATCC) stably expressing a nuclear factor of activated T cells (NFAT) response element-driven green fluorescent protein (NFAT-GFP) (construct plasmid: System Biosciences, TR451PA-P) were constructed as reporter cells. Subsequently, aAPC-293T cells were co-cultured with Jurkat T reporter cells, thereby enabling the determination of T cell activity ( Figure 1 A). Compared with the control transfected with empty vector, aAPC-293T cells expressing 5 transmembrane proteins showed a significant decrease in GFP expression ( Figure 1 A). Among these five proteins, PD-L1 (Genbank: NM_014143) has been reported to inhibit T cell activity. Among them, PILRα is a newly identified T cell inhibitory molecule, and this finding was Figure 1 verified in C. Two stimulatory molecules CD70 (Genbank: NM_001252) and ICOSLG (Genbank: NM_015259) were included as controls ( Figure 1 C). The inhibitory effect of PILRα on T cells was further confirmed using the NFAT-Lucia-Jurkat T reporter cell line (InvivoGen, jktl-nfat) ( Figure 2 D).

[0236] Example 2: The mucin stalk domain of PILRα is crucial for its immunosuppressive function

[0237] PILRα is a paired receptor family protein consisting of an immunoglobulin-like V-type (IgV) domain, a stalk domain, a transmembrane (TM) domain, and a cytoplasmic carboxyl-terminal domain (CTD). The IgV domain and the stalk domain together form the extracellular domain (ECD). Among inhibitory and stimulatory molecules, the protein sequence of the IgV domain of PILRα is highly conserved ( Figure 2 A). To investigate the functional role of PILRα, in this example, a fusion protein was generated by binding the ECD of PILRα to the human IgG1 Fc domain (hIg), called PILRα(ECD)-hIg ( Figure 2 A). This example found that PILRα(ECD)-hIg significantly reduced the activity of NFAT-Lucia-Jurkat T reporter cells stimulated with anti-CD3 antibody (αCD3; Biolegend, 317326) ( Figure 2 B), and this inhibitory effect on T cells was concentration-dependent ( Figure 2 C). In addition, PILRα(ECD)-hIg also inhibited the proliferation ( Figure 2 D), activation ( Figure 2 E), and release of IFNγ, granzyme B, and interleukin-2 (IL2) of human primary T cells stimulated with anti-CD3 antibody ( Figure 2 F).

[0238] PILRα and PILRβ have approximately 90% high sequence similarity in the IgV domain, but there are significant differences in the stalk domain. Purified recombinant PILRα(ECD)-hIg can significantly inhibit T cell activity after T cell activation, while PILRβ(ECD)-hIg cannot ( Figure 2 A and Figure 2 E). Similarly, other secreted PILRα, such as PILRα isoform 1 (PILRα-iso1-hIg) lacking the stalk region but having the CTD, and PILRα isoform 2 (PILRα-iso2-hIg) lacking the stalk region and having an undefined region, did not show significant inhibitory effects after T cell activation for their corresponding recombinant proteins ( Figure 2 A and Figure 2 E). In addition, the recombinant protein of PILRα lacking the stalk region [PILRα(IgV)-hIg] also could not inhibit T cell activation ( Figure 2E). These findings indicate that the stalk domain of PILRα plays a crucial role in its function of inhibiting T cells. To confirm this, in this example, PILRα(ECD)-hIg or PILRα(IgV)-hIg proteins were co-incubated with human primary T cells under anti-CD3 antibody stimulation, and it was found that compared with PILRα(ECD)-hIg, under PILRα(IgV)-hIg incubation, the inhibitory effects of PILRα on CD3 + the proliferation of T cells and the cytokines IFNγ, granzyme B, and IL2 completely disappeared ( Figure 2 G and Figure 2 H).

[0239] The stalk domain of PILRα has a tandem repeat of multiple serine (Ser) and threonine (Thr) residues, which is called the ST repeat, and this is a structural feature of the mucin stalk domain. The mucin stalk domain usually has a glycosyl structure, which may cause conformational changes to regulate functional interactions. Using the NetOGlyc 3.1 algorithm (www.cbs.dtu.dk / services / NetOGlyc / ), it was predicted that the PILRα stalk domain (aa 151-197) has enriched O-glycosylation sites at serine (Ser) or threonine (Thr) residues. The O-glycosylation of PILRα-hIg was confirmed by immunoblot analysis using biotinylated peanut agglutinin (Peanut Agglutinin (PNA), Biotinylated; Vector Laboratories, B-1075-5) ( Figure 2 I). However, the binding of PNA was not detected in PILRα(IgV)-hIg, indicating that the presence of the stalk domain is crucial for O-glycosylation ( Figure 2 I). In addition, after treating PILRα with O-glycosidase (NEB, E0540S), the decrease in its molecular weight further confirmed its O-glycosylation ( Figure 2 J). The inhibitory effect of PILRα(ECD)-hIg on T cell activation was weakened in the presence of O-glycosidase, suggesting that O-glycosylation is crucial for PILRα to exert its function ( Figure 2 K).

[0240] To obtain direct evidence of O-glycosylation in the stem domain of PILRα, in this example, purified PILRα-hIg was subjected to liquid chromatography and tandem mass spectrometry (LC-MS / MS) analysis. This analysis revealed the presence of O-glycosylation on several specific Ser and Thr residues in the stem domain, including Ser 162, Ser 171, Ser 187, Ser 189, Thr 164, Thr 165, Thr166, Thr 172, Thr 173, Thr 174, Thr 175, and Thr 176. To evaluate the functional significance of these glycosylation sites, in this example, a PILRα mutant [T&S(M)]( Figure 2 L) was generated by replacing all identified Ser and Thr residues with alanine (Ala). Compared with wild-type (WT) PILRα, T&S(M) had a lower molecular weight and a reduced binding ability to PNA( Figure 2 M). Notably, T&S(M) lost the ability to inhibit αCD3-stimulated T cell proliferation, activation, and cytokine release( Figure 3 N-2P). These findings indicate that the mucin stem domain, particularly the glycosylation of Ser and Thr residues therein, plays a crucial role in PILRα-mediated inhibition of T cell activity.

[0241] Example 3: PILRα Interacts with T Cell Surface Protein CD99 to Inhibit T Cell Activity

[0242] To explore the molecular mechanism of the inhibitory function of PILRα in T cells, in this example, affinity purification combined with mass spectrometry analysis was used to identify potential ligands of PILRα in T cells. Among these proteins, CD99 is highly expressed on the T cell membrane, and CD99 has been reported as a co-stimulatory molecule for T cell proliferation and activation, so CD99 attracted our attention. First, in this example, the expression of CD99 on the surface of human primary T cells was confirmed by flow cytometry( Figure 3 A). Subsequently, in this example, purified PILRα and CD99 proteins (Abcam, ab220556) were used to confirm the direct interaction between PILRα and CD99 by surface plasmon resonance (SPR) (K D = 64 μM) and enzyme-linked immunosorbent assay (ELISA) (IC 50 = 2.146 μg / mL)( Figure 3 B and Figure 3 C). This interaction was further confirmed by co-transfecting FLAG-tagged PILRα and Myc-tagged CD99 on 293T cells (ATCC), and it was found that the binding was dependent on Ser and Thr residues in the stem domain( Figure 3 D).

[0243] Next, this example attempted to determine whether interfering with the interaction between PILRα and CD99 could reduce the inhibitory effect of PILRα on T cell activity. To this end, this example prepared a monoclonal antibody against human PILRα using hybridoma technology, and screened out clone C21 by ELISA assay, which could effectively restore the reporter activity of NFAT-Lucia in Jurkat T reporter cells under the incubation of PILRα(ECD)-hIg protein and promote the secretion of IFNγ in human primary T cells. By SPR (K D = 4.92 nM) and ELISA (IC 50 = 75.6 ng / mL) assays showed that the purified C21 antibody exhibited high binding affinity with PILRα protein ( Figure 3 E and Figure 3 F). Interestingly, C21 could not bind to the PILRα(IgV) protein lacking the stem domain or the T&S(M) protein with Ser / Thr mutations, indicating its specific recognition of the stem domain, especially the Ser and Thr residues of the stem domain ( Figure 3 F). In addition, C21 effectively competed with CD99 for binding to PILRα, with an IC 50 value of 0.43 μg / mL ( Figure 3 G). Flow cytometry analysis showed that C21 exhibited strong binding to 293T cells expressing PILRα, but did not bind to 293T cells expressing PILRα(IgV) or T&S(M) ( Figure 3 H). As expected, C21 effectively reversed the inhibitory effect of PILRα on the proliferation, activation and cytokine release of human primary T cells stimulated by αCD3 ( Figure 3 I-3K).

[0244] To further verify that CD99 is involved in the inhibitory effect of PILRα on T cells, this example used the Cas9 / ribonucleoprotein (RNP; sgRNA: synthesized by GenScript Biotech Corporation; Cas9 protein: Invitrogen, A36499) method to establish human primary T cells with CD99 knockdown (CD99 KD ) ( Figure 3 L). Knockdown of CD99 led to reduced T cell proliferation, activation and cytokine release, which was consistent with previous reports. Importantly, this example observed that knockdown of CD99 alleviated the inhibitory effect of PILRα on T cells ( Figure 3 M-3O). In addition, the ability of the C21 antibody to relieve the inhibitory effect of PILRα on T cells was affected in CD99 KD T cells ( Figure 3 P and Figure 4Q). These findings suggest that the inhibitory effect of PILRα on T cells depends on its interaction with CD99.

[0245] Example 4: PILRα can inhibit the CD99-activated ZAP70 / NFAT / IL2 / JAK / STAT signaling pathway in T cells

[0246] To deeply explore the downstream signaling pathway of PILRα / CD99 in T cells, this example performed transcriptome analysis on T cells incubated with and without PILRα protein. Compared with the control, 202 genes were upregulated and 254 genes were downregulated in T cells in the presence of PILRα ( Figure 4 A). GO analysis of the downregulated genes showed significant enrichment in immune system development, cell activation, and regulation of peptidyl-tyrosine phosphorylation, emphasizing their possible involvement in the PILRα-mediated signaling cascade ( Figure 4 B). The main enriched pathways identified by KEGG pathway analysis were cytokine-cytokine receptor interaction, Janus kinase / signal transducer and activator of transcription (JAK / STAT) signaling, and T cell receptor signaling pathway ( Figure 4 C).

[0247] In T cell receptor (TCR) signaling, the TCR / CD3 complex and co-stimulatory molecules act as the first and second signals, initiating the recruitment and activation of tyrosine kinases. Among these kinases, ZAP70 phosphorylates a transmembrane T cell activation linker protein (LAT), thereby conducting signals through the calcium-calmodulin (Ca 2+ -CaN), nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways. After activation, the Ca 2+ -CaN signaling pathway leads to the nuclear translocation of NFAT; the NF-κB signaling pathway leads to the nuclear translocation of REL and NF-κB transcription factors; the MAPK signaling pathway activates the transcription factors FOS and JUN. The activation of these signaling pathways together induces the expression and secretion of the IL2 cytokine. IL2 binds to the heterotrimeric IL2 receptor (IL2R), initiating the activation of the JAK / STAT signaling pathway, which is crucial for promoting T cell proliferation, differentiation, and function. It has been reported that T cell activation is regulated by CD99, which increases the first signal mediated by TCRζ and provides the second signal as a co-stimulatory molecule, thereby leading to the phosphorylation of the T cell-associated protein kinase ZAP70 and downstream transcription factors including NFAT. It is hypothesized that the interaction between PILRα and CD99 may impair CD99-induced signal transduction. The experimental results of this example confirmed this hypothesis, and the presence of PILRα did significantly reduce the phosphorylation of ZAP70 ( Figure 4D). To determine the specific downstream transcription factors affected by the PILRα / CD99 signaling pathway, in this example, T cells were treated with inhibitors against NF-κB (SC-514; MCE, HY-13802), NFAT (VIVIT peptide; MCE, HY-P1026), JUN (JNK-IN-8; MCE, HY-13319), and FOS (T-5224; MCE, HY-12270), and observed in the presence of PILRα ( Figure 4 E and Figure 4 F). When cells were treated with the NFAT inhibitor (NFATi) in the presence of PILRα, no further inhibition of IL2 and IFNγ secretion was observed, indicating that NFAT may be downstream of PILRα ( Figure 4 E and Figure 4 F). This finding was further verified using another NFAT inhibitor, cyclosporin A (MCE, HY-B0579) ( Figure 4 G). In addition, the inhibition of the JAK / STAT signaling pathway was verified by immunoblot analysis ( Figure 4 D). Consistently, in the presence of NFATi, detection of CD25 expression revealed that PILRα-hIg did not exhibit additional inhibition of T cell activation ( Figure 4 H). These data indicate that PILRα inhibits the activation of the ZAP70 / NFAT / IL2 / JAK / STAT signaling pathway. To investigate whether the function of PILRα is dependent on CD99, in this example, control and CD99 KD -T cells were subjected to immunoblot analysis with or without incubation with PILRα. The results showed that the addition of PILRα to control cells led to a downregulation in the expression of p-ZAP70, p-JAK1, p-JAK3, and p-STAT5. However, in CD99 KD -T cells, the inhibitory effect of PILRα was abolished ( Figure 5 I). These findings indicate that the inhibitory effect of PILRα on T cells is achieved by downregulating the ZAP70 / NFAT / IL2 / JAK / STAT signaling pathway activated by CD99.

[0248] Example 5: PILRα inhibits T cell anti-tumor immune responses in both humanized mouse xenograft and allograft models

[0249] To investigate the role of PILRα in T cell anti-tumor immune responses and tumor growth, this example used triple immunodeficient mice (NOD, CRISPR, Prkdc, Il2rγ, abbreviated as NCG; Jiangsu Jicui Yakang Biopharmaceutical Co., Ltd.), which were reconstituted with human peripheral blood mononuclear cells (PBMC), and were orthotopically injected with U87 glioblastoma cells expressing the firefly luciferase gene (Genbank: XM_031473197.1) in the brain, while knocking down PILRα (PILRα KD ), overexpressing PILRα (PILRα OE ), or overexpressing the T&S(M) mutant of PILRα (PILRα M ) (U87-luc, U87 cells were derived from ATCC) ( Figure 5 A). It should be noted that overexpression of PILRα had no significant effect on the growth of U87 cells. Xenograft tumors derived from U87-luc cells grew rapidly in the brain, invaded blood vessels and spread outside the central nervous system. Compared with the control group (CTL), the growth rate of U87 xenograft tumors with PILRα KD was significantly slower, and there was less tumor metastasis ( Figure 5 B and Figure 5 C). In contrast, the growth and metastasis of U87 xenograft tumors with PILRα OE were significantly increased, while U87 xenograft tumors expressing PILRα M attenuated these effects ( Figure 5 B and Figure 5 C). Consistent with the T cell inhibitory effect of PILRα, immunohistochemistry (IHC) staining showed that there were more infiltrating CD8 KD T cells in U87 xenograft tumors with PILRα + , while the number of infiltrating CD8 OE T cells in U87 xenograft tumors with PILRα + was significantly reduced ( Figure 5 D and Figure 5 E). In addition, this example also evaluated the expression levels of CD25, IFNγ, IL2, and granzyme B in tumor tissues and found that their expression levels increased when PILRα was knocked down and decreased when overexpressed ( Figure 5 F).

[0250] To further investigate the inhibitory effect of PILRα on anti-tumor immune responses, this example used NCG mice reconstituted with human PBMC immunized with xenograft triple-negative breast cancer (TNBC) cells MDA-MB-231. Mice were subcutaneously injected with CTL, PILRα KD , PILRα OE or PILRαM MDA-MB-231 cells (ATCC) of Figure 5 G). Consistent with the observations in U87 tumors, PILRα knockdown significantly inhibited tumor growth, while PILRα overexpression had the opposite effect ( Figure 5 H-5J). Interestingly, overexpression of the stem domain mutant PILRα-T&S(M) did not significantly affect tumor growth ( Figure 5 H-5J). In addition, the number of CD8 KD T cells and the expression levels of CD25, IFNγ, IL2, and granzyme B were significantly increased in the MDA-MB-231 xenograft tumor tissues of + PILRα ( Figure 5 K-5M).

[0251] To further investigate the effects of PILRα on T cell immune responses and tumor growth, in this example, mouse hepatoma cells Hepa1-6 were inoculated to establish an allograft model. First, Pilrα was knocked down (Pilrα KD ) or overexpressed (Pilrα OE ) on Hepa1-6 cells (ATCC), and then subcutaneously injected into syngeneic C57BL / 6 mice (Shanghai Slac Laboratory Animal Co., Ltd.) ( Figure 5 N). Notably, Pilrα knockdown led to inhibited tumor growth, reduced tumor weight, and generally prolonged the survival of mice ( Figure 5 O-5Q). In contrast, Pilrα overexpression showed the opposite effect ( Figure 5 O-5Q). As expected, in mice inoculated with Pilrα KD -Hepa1-6 cells, the numbers of CD3 + and CD8 + T cells in the spleen and tumor were significantly increased ( Figure 5 R). No significant changes were observed in other immune cell populations, including CD4 + T cells, B cells, NK cells, granulocytes, and macrophages ( Figure 5 R). In addition, CD8 KD T cells in the Pilrα + -Hepa1-6 xenografts showed increased expression of CD25, IFNγ, IL2, and granzyme B ( Figure 5 S). In contrast, in mice inoculated with Pilrα OE -Hepa1-6 cells, the numbers of CD3 + and CD8 + T cells in the spleen and tumor were significantly reduced, while CD8+ The expressions of CD25, IFNγ, IL2, and granzyme B in T cells were also significantly decreased ( Figure 5 R and Figure 5 S). To further demonstrate that the reduced growth rate of Pilrα KD -Hepa1-6 xenografts mainly depends on T cells, in this example, an anti-mouse CD3 antibody (Bio XCell, BE0002) was injected to conduct a T cell depletion experiment. Notably, after treatment with the anti-CD3 antibody, the tumor suppression effect caused by Pilrα knockdown was significantly weakened ( Figure 5 T and Preparation of Anti-PILRα Monoclonal Antibody U). Collectively, these data suggest that PILRα can inhibit T cell anti-tumor immune responses and promote tumor growth in both humanized mouse xenograft and murine transplantation models.

[0252] Example 6: Anti-PILRα monoclonal antibodies effectively inhibit tumor growth in humanized mouse xenograft models by blocking the interaction between PILRα and CD99

[0253] Figure 3

[0254] Monoclonal antibodies against human PILRα were prepared using hybridoma technology. After affinity and function screening, functional and effective anti-PILRα monoclonal antibodies C21 and C29, as well as a control antibody C126 that only has binding activity but no function, were obtained. The CDR sequences were determined according to the Kabat numbering system, and the CDRs and variable region sequences of these antibodies are shown in the following table.

[0255] Table 2: CDR and variable region sequences of antibodies

[0256]

[0257] In addition, the epitopes bound by the above monoclonal antibodies were identified as follows. Pilrα

[0258] (ECD)-hIg protein and Pilrα (IgV)-hIg protein lacking the stem domain were coated on ELISA plates, and the binding curves of different concentrations of PILRα-specific antibodies to the proteins were detected by ELISA. The results showed that the functional C21 ( Figure 11 F) and C29 antibodies did not bind to Pilrα lacking the stem domain ( Figure 11 A); in contrast, the non-functional C126 ( In Vivo Antitumor Experiment B) was able to bind to Pilrα lacking the stem domain. The above results further indicate that the stem domain of PILRα is the functional epitope for exerting the inhibitory effect.

[0259] Figure 6

[0260] To evaluate the effect of blocking the interaction between PILRα and CD99 on tumor growth, in this example, U87-luc cells were intracranially injected into NCG mice reconstituted with human PBMC. On the 7th day after injection, intraperitoneal injection of control antibody or anti-PILRα monoclonal antibody C21 or C126 was started ( Figure 6 A). The results showed that compared with control or C126 antibody treatment, C21 antibody treatment significantly inhibited tumor growth and metastasis ( Figure 6 B and Figure 6 C). In addition, the survival time of mice in the C21 treatment group was also significantly prolonged ( Figure 7 D). Importantly, in this example, it was observed that the infiltration of CD8 + T cells and the expression levels of CD25, IFNγ, IL2, and granzyme B in the tumor tissues of the C21 treatment group were significantly higher than those in other groups ( Figure 7 A and Figure 7 B). In C21-treated mice, no significant changes were observed in body weight, morphology, or histopathology of various organs ( Figure 6 C-7E). In addition, in this example, MDA-MB-231 cells were inoculated into the same human PBMC-reconstituted NCG mice, and the effect of C21 antibody was evaluated. When the tumor reached 100-200 mm 3 , control antibody or anti-PILRα antibody clone C21 or C126 was intraperitoneally administered to the mice ( Figure 6 E). Similarly, it was found that compared with control or C126 treatment, C21 treatment effectively reduced the tumor burden and prolonged the survival time of mice ( Figure 7 F-6I). In addition, C21 treatment also led to a significant increase in the infiltration of CD8 + T cells in tumor tissues, and at the same time, the expressions of CD25, IFNγ, IL2, and granzyme B were significantly upregulated ( Figure 7 F and Figure 7 G). Importantly, C21 treatment had no effect on the body weight, morphology, and histopathology of various organs (including the heart, liver, spleen, lungs, and kidneys) ( Figure 8 H-7J).

[0261] Example 7: Anti-PILRα Monoclonal Antibody Enhances the Antitumor Effect of Anti-PD-1 Immunotherapy in Humanized Mouse Xenograft Models

[0262] It was observed that anti-PILRα antibody could enhance CD8 +T cells infiltrate and inhibit tumor growth, leading to the idea in this example to further investigate whether it can enhance the anti-tumor response induced by PD-1 / PD-L1 checkpoint blockade. Brain tumors such as glioblastoma multiforme (GBM) are typically characterized as "cold" tumors with limited response to PD-1 / PD-L1 checkpoint blockade. Therefore, in this example, human PBMCs were injected into NCG mice, U87-luc cells were inoculated, and then treated with anti-PILRα and / or the anti-PD-1 antibody Pembrolizumab ( Figure 8 A). Treatment with C21 alone or the anti-PD-1 antibody significantly reduced tumor growth and metastasis and prolonged the survival of mice compared to the control group ( Figure 8 B-8D). The combined treatment with C21 and the anti-PD-1 antibody had a significantly stronger inhibitory effect on tumor growth and metastasis than monotherapy ( Figure 8 B-8D). In addition, in the tumor tissues of mice in the combined treatment group, the infiltration of CD8 + T cells and the expression of CD25, IL2, granzyme B, and IFNγ showed a stronger effect compared to the treatment with C21 and the anti-PD-1 antibody alone ( Figure 8 E and Figure 9 F). The combined treatment did not cause obvious changes in the morphology or pathology of organs such as the heart, liver, spleen, lung, and kidney ( Figure 9 A and Figure 10 B).

[0263] Example 8: PILRα has an adverse effect on the prognosis of cancer patients

[0264] Analysis of the TCGA database showed that PILRα was upregulated in a wide range of human tumors, including triple-negative breast cancer (TNBC), cholangiocarcinoma (CHOL), esophageal cancer (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), sarcoma (SARC), thyroid cancer (THCA), and stomach adenocarcinoma (STAD) ( Figure 10 A). Notably, the expression level of PILRα was associated with the prognostic survival of patients with TNBC, ESCA, GBM, KIRC, and STAD ( Figure 10 B). In addition, TIMER database analysis showed that PILRα expression was negatively correlated with the infiltration of CD8 + and CD4 + T cells in TNBC, ESCA, GBM, KIRC, and STAD ( ​ C). These findings highlight the potential importance of PILRα as a prognostic marker and its potential role in regulating T cell immune responses in various tumor types.

[0265] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings that have been published, and such changes are within the scope of protection of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of a PILRα / CD99 pathway inhibitor in the preparation of a medicament for treating tumors.

2. Use of a PILRα / CD99 pathway inhibitor in the preparation of a medicament for regulating immune responses (such as T cell immune responses).

3. The use according to claim 1 or 2, wherein, the PILRα / CD99 pathway inhibitor includes a PILRα inhibitor.

4. The use according to claim 3, wherein, the PILRα inhibitor can inhibit or down-regulate the expression of the PILRα gene, inhibit or block the activity of the PILRα protein, inhibit or block the binding of PILRα to CD99, or degrade the PILRα protein.

5. The use according to any one of claims 1-4, wherein, the inhibitor is selected from antibodies, small molecule compounds, aptamers, RNA interference agents (such as siRNA, shRNA or miRNA), or antisense oligonucleotides, gene editing systems (such as CRISPR-Cas).

6. The use according to any one of claims 1-5, wherein, the PILRα inhibitor is selected from affinity molecules that specifically bind to the Stalk domain of PILRα; Preferably, the affinity molecule binds to a functional epitope in the Stalk domain of PILRα, and the functional epitope contains at least one O-glycosylation site; Preferably, the O-glycosylation site is selected from Ser and / or Thr residues; Preferably, the O-glycosylation site is selected from S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176 or any combination thereof; Preferably, the functional epitope contains one or more amino acid residues selected from the following: S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176; Preferably, the affinity molecule is an antibody or its antigen-binding fragment, a small molecule compound or an aptamer.

7. The use according to claim 6, wherein, the PILRα inhibitor is selected from antibodies or their antigen-binding fragments that specifically bind to the Stalk domain of PILRα; Preferably, the antibody or its antigen-binding fragment comprises: (1) Heavy chain CDR1, CDR2 and CDR3 respectively comprising SEQ ID NOs: 1, 2 and 3, and light chain CDR1, CDR2 and CDR3 respectively comprising SEQ ID NOs: 4, 5 and 6; or (2) Heavy chain CDR1, CDR2 and CDR3 respectively comprising SEQ ID NOs: 9, 10 and 11, and light chain CDR1, CDR2 and CDR3 respectively comprising SEQ ID NOs: 12, 13 and 14; Preferably, the antibody or its antigen-binding fragment comprises: (1) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:7 or a sequence having at least 80% identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:8 or a sequence having at least 80% identity thereto; or (2) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:15 or a sequence having at least 80% identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:16 or a sequence having at least 80% identity thereto.

8. The use according to any one of claims 1-7, wherein, the PILRα / CD99 pathway inhibitor is used in combination with an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitor is selected from PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, or any combination thereof; Preferably, the PILRα / CD99 pathway inhibitor is used in combination with a PD-1 / PD-L1 inhibitor (such as a PD-1 or PD-L1 antibody).

9. The use according to any one of claims 1-8, wherein, the tumor includes solid tumors or hematological tumors; Preferably, the tumor is a PILRα-positive tumor; Preferably, the tumor is selected from breast cancer, cholangiocarcinoma, esophageal cancer, glioblastoma, head and neck cancer, kidney cancer, sarcoma, thyroid cancer, and gastric cancer.

10. An affinity molecule that specifically binds to the Stalk domain of PILRα; Preferably, the affinity molecule specifically binds to a functional epitope in the Stalk domain of PILRα, and the functional epitope contains at least one O-glycosylation site; Preferably, the O-glycosylation site is selected from Ser and / or Thr residues; Preferably, the O-glycosylation site is selected from S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176, or any combination thereof; Preferably, the functional epitope contains one or more amino acid residues selected from the following: S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176; Preferably, the affinity molecule is an antibody or an antigen-binding fragment thereof, a small molecule compound, or an aptamer.

11. An antibody or an antigen-binding fragment thereof that specifically binds to PILRα, the antibody or the antigen-binding fragment thereof comprising: (1) Heavy chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs:1, 2, and 3 respectively, and light chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs:4, 5, and 6 respectively; or (2) The heavy chain CDR1, CDR2 and CDR3 respectively comprising SEQ ID NOs: 9, 10 and 11, and the light chain CDR1, CDR2 and CDR3 respectively comprising SEQ ID NOs: 12, 13 and 14.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein, the antibody or antigen-binding fragment thereof comprises: (1) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 7 or a sequence having at least 80% identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 8 or a sequence having at least 80% identity thereto; or (2) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 15 or a sequence having at least 80% identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 16 or a sequence having at least 80% identity thereto.

13. The antibody or antigen-binding fragment thereof according to claim 11 or 12, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and a light chain constant region (CL); Preferably, the CH is selected from IgG, IgM, IgE, IgD or IgA; Preferably, the CH is IgG, such as IgG1, IgG2, IgG3 or IgG4; Preferably, the CL is selected from κ or λ.

14. The antibody or antigen-binding fragment thereof according to any one of claims 11-13, wherein, The antibody or its antigen-binding fragment is selected from Fab, Fab’, (Fab’) 2 , Fv, disulfide-linked Fv, scFv, diabody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.

15. An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof according to any one of claims 11-14 for binding to PILRα and / or binds to the same epitope on PILRα; Preferably, the epitope comprises at least one O-glycosylation site, such as selected from Ser and / or Thr residues; Preferably, the epitope comprises one or more of the following amino acid residues: S162, S171, S187, S189, T164, T165, T166, T172, T173, T174, T175, T176.

16. A multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 11-15; Preferably, the multispecific antibody specifically binds to PILRα and additionally specifically binds to other targets; Preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody.

17. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 11-15 and a conjugate moiety linked to the antibody or antigen-binding fragment thereof; Preferably, the conjugate moiety is selected from a detectable label or a therapeutic agent.

18. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof according to any one of claims 11-15 as an extracellular antigen-binding domain.

19. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 11-15, a nucleotide sequence encoding a multispecific antibody according to claim 16, or a nucleotide sequence encoding a chimeric antigen receptor according to claim 18.

20. A vector comprising the isolated nucleic acid molecule according to claim 19.

21. A host cell comprising the isolated nucleic acid molecule according to claim 19, or the vector according to claim 20; Preferably, the host cell comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 11-15 or a multispecific antibody according to claim 16, or a vector comprising the nucleotide sequence; preferably, the host cell comprises Escherichia coli, yeast, insect cells, or mammalian cells; Preferably, the host cell comprises a nucleotide sequence encoding a chimeric antigen receptor according to claim 18 or a vector comprising the nucleotide sequence; preferably, the host cell comprises immune cells (such as human immune cells); preferably, the immune cells are selected from T cells and / or NK cells.

22. A pharmaceutical composition comprising the affinity molecule according to claim 10, an antibody or an antigen-binding fragment thereof according to any one of claims 11-15, a multispecific antibody according to claim 16, a chimeric antigen receptor according to claim 18, the isolated nucleic acid molecule according to claim 19, the vector according to claim 20, the host cell according to claim 21, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; preferably, the additional pharmaceutically active agent is a drug having antitumor activity, such as an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a specific antibody targeting tumor cells, or an immune checkpoint inhibitor; Preferably, the additional pharmaceutically active agent is an immune checkpoint inhibitor, such as a PD-1 / PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, or any combination thereof; Preferably, the additional pharmaceutically active agent is a PD-1 / PD-L1 inhibitor, such as a PD-1 or PD-L1 antibody.

23. Use of the affinity molecule according to claim 10, an antibody or an antigen-binding fragment thereof according to any one of claims 11-15, a multispecific antibody according to claim 16, a chimeric antigen receptor according to claim 18, the isolated nucleic acid molecule according to claim 19, the vector according to claim 20, the host cell according to claim 21, or the pharmaceutical composition according to claim 22, in the preparation of a drug for treating tumors; Preferably, the tumors include solid tumors or hematological tumors; Preferably, the tumor is a PILRα-positive tumor; Preferably, the tumor is selected from breast cancer, cholangiocarcinoma, esophageal cancer, glioblastoma, head and neck cancer, kidney cancer, sarcoma, thyroid cancer, and gastric cancer.

24. Use of the affinity molecule according to claim 10, the antibody or antigen-binding fragment thereof according to any one of claims 11-15, the multispecific antibody according to claim 16, the chimeric antigen receptor according to claim 18, the isolated nucleic acid molecule according to claim 19, the vector according to claim 20, the host cell according to claim 21, or the pharmaceutical composition according to claim 22, in the preparation of a medicament for regulating the immune response; Preferably, the immune response includes a T cell immune response; Preferably, the regulation of the immune response includes enhancing or stimulating the T cell immune response and / or enhancing T cell activity in a subject (such as a human).

25. Use of the affinity molecule according to claim 10, the antibody or antigen-binding fragment thereof according to any one of claims 11-15 as a PILRα / CD99 pathway inhibitor, or for the preparation of a PILRα / CD99 pathway inhibitor.

26. A method for screening a drug for treating a tumor or regulating the immune response, the method comprising: screening for a PILRα / CD99 pathway inhibitor; or, screening for a reagent capable of specifically binding to the stalk domain of PILRα; or, screening for a reagent that competes with the antibody or antigen-binding fragment thereof according to any one of claims 11-14 for binding to PILRα and / or binds to the same epitope on PILRα.

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