Bifunctional t cell engagers targeting cd14 and cd3 and uses thereof

Abstract

This invention discloses a bifunctional T-cell connector targeting CD14 and CD3 and its application. Specifically, this invention provides a bifunctional T-cell connector targeting CD14 and CD3, comprising a backbone containing a constant region of an IgG antibody. The backbone includes two heavy chains and one light chain, wherein one heavy chain includes a heavy chain constant region CH1, CH2, and CH3, and the other heavy chain includes CH2 and CH3. The light chain includes CL. The bifunctional T-cell connector targeting CD14 and CD3 is a heterotrimer, specifically: a first subunit includes a variable region of the light chain of an anti-CD14 antibody and the light chain in the backbone; a second subunit includes a variable region of the heavy chain of an anti-CD14 antibody and one heavy chain in the backbone; and a third subunit includes a variable region of the heavy chain of an anti-CD3 antibody and a variable region of the light chain. The bifunctional T-cell connector of this invention exhibits excellent tumor-killing effects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a bifunctional T-cell connector that targets CD14 and CD3 and its application. Background Technology

[0002] Bifunctional T-cell engagers (BiTEs) can simultaneously recognize tumor cells and T cells, effectively mediating T-cell immune responses against tumors, representing a significant breakthrough in the treatment of hematological malignancies. Among these, blinatumomab targets CD19... + BiTE has shown significant efficacy in treating acute B-cell lymphoblastoma and has been approved for marketing. Nevertheless, its role in solid tumors is quite limited.

[0003] On the one hand, solid tumors lack suitable targets. On the other hand, the tumor microenvironment (TME) contains a large number of immunosuppressive cells (such as regulatory T cells, myeloid-derived suppressor cells, and tumor-associated fibroblasts), which can not only inhibit the activation and proliferation of tumor-infiltrating lymphocytes (TILs) and thus affect their anti-tumor activity, but also secrete substances that can form chemical or physical barriers to prevent the infiltration of T cells from the blood.

[0004] Therefore, developing novel bifunctional T-cell connectors that can effectively target tumor cells and increase T-cell infiltration is of great significance in this field. Summary of the Invention

[0005] This invention provides a bifunctional T-cell connector that targets CD14 and CD3 and its application.

[0006] In a first aspect of the invention, a bifunctional T-cell connector targeting CD14 and CD3 is provided, comprising a backbone including an IgG antibody constant region, the backbone comprising two heavy chains and one light chain, wherein one heavy chain includes heavy chain constant regions CH1, CH2, and CH3, the other heavy chain includes CH2 and CH3, and the light chain includes CL, wherein the bifunctional T-cell connector targeting CD14 and CD3 is a heterotrimer, specifically:

[0007] The first subunit includes a light chain variable region of the anti-CD14 antibody and a light chain in the backbone; the second subunit includes a heavy chain variable region of the anti-CD14 antibody and a heavy chain in the backbone; and the third subunit includes a heavy chain variable region and a light chain variable region of the anti-CD3 antibody.

[0008] Wherein, the light chain variable region of the anti-CD14 antibody of the first subunit and the heavy chain variable region of the anti-CD14 antibody of the second subunit constitute the antigen-binding domain of the anti-CD14 antibody, and the heavy chain variable region and the light chain variable region of the anti-CD3 antibody of the third subunit constitute the antigen-binding domain of the anti-CD3 antibody.

[0009] The IgG antibody is a human-derived IgG antibody.

[0010] The CH2 domains of the second and third subunits introduce LALA-PG mutations;

[0011] The two CH3 groups in the second and third subunits are designed with a knot structure and a hole structure respectively using the knot-into-hole (KIH) technique;

[0012] The amino acid sequence of the first subunit is shown in SEQ ID NO: 4, the amino acid sequence of the second subunit is shown in SEQ ID NO: 3, and the amino acid sequence of the third subunit is shown in SEQ ID NO: 1.

[0013] In a second aspect of the invention, a nucleic acid molecule is provided that encodes the bifunctional T-cell adaptor targeting CD14 and CD3.

[0014] In another preferred embodiment, the nucleic acid molecule is DNA or cDNA.

[0015] In a third aspect of the invention, an expression vector is provided, the expression vector comprising the nucleic acid molecule described in the second aspect of the invention.

[0016] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0017] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0018] In a fourth aspect of the invention, a host cell is provided, the host cell comprising the expression vector described in the third aspect of the invention, or having the nucleic acid molecule described in the second aspect of the invention integrated into its genome.

[0019] In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.

[0020] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0021] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0022] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0023] In a fifth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0024] (a) A bifunctional T-cell connector as described in the first aspect of the invention; and

[0025] (b) Coupled part: can detect markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0026] In another preferred embodiment, the conjugate is partially selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinoflavin (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0027] In a sixth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0028] (i) a bifunctional T-cell editor as described in the first aspect of the invention, a nucleic acid molecule as described in the second aspect of the invention, an expression vector as described in the third aspect of the invention, a host cell as described in the fourth aspect of the invention, or an immunoconjugate as described in the fifth aspect of the invention; and

[0029] (ii) Pharmaceutically acceptable carriers.

[0030] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0031] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating tumors, wherein the tumor is a CD14-positive tumor or a CD14-overexpressing tumor.

[0032] In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient, which is an antitumor drug.

[0033] In another preferred embodiment, the second active ingredient is selected from the group consisting of: chemotherapy drugs, targeted drugs, immunostimulants, antibody-drug conjugates, peptide drugs, and nucleic acid drugs.

[0034] In another preferred embodiment, the second active ingredient is atezolizumab.

[0035] In another preferred embodiment, the second active ingredient is an anti-PD-L1 antibody.

[0036] In a seventh aspect of the invention, the use of a bifunctional T-cell editor as described in the first aspect of the invention, a nucleic acid molecule as described in the second aspect of the invention, an expression vector as described in the third aspect of the invention, a host cell as described in the fourth aspect of the invention, an immunoconjugate as described in the fifth aspect of the invention, or a pharmaceutical composition as described in the sixth aspect of the invention, for preparing a medicament for treating tumors; and said tumors are tumors with high CD14 expression.

[0037] Among them, the tumor with high CD14 expression is bladder cancer.

[0038] In another preferred embodiment, the drug also contains other antitumor drugs.

[0039] In another preferred embodiment, the antitumor drug is selected from the group consisting of: chemotherapy drugs, targeted drugs, immunostimulants, antibody-drug conjugates, peptide drugs, nucleic acid drugs, or combinations thereof.

[0040] In an eighth aspect of the invention, a method for preparing the bifunctional T-cell connector described in the first aspect of the invention is provided, comprising the steps of:

[0041] (a) Under re-expression conditions, host cells as described in the fourth aspect of the present invention are cultured to express the bifunctional T cell adaptor;

[0042] (b) Isolate and purify the bifunctional T-cell connective described in (a).

[0043] In a ninth aspect of the invention, a method for treating a tumor is provided, comprising the steps of: administering to a subject in need a therapeutically effective amount of the bifunctional T-cell connector of the first aspect of the invention, a host cell as described in the fourth aspect of the invention, an immunoconjugate as described in the fifth aspect of the invention, or a pharmaceutical composition as described in the sixth aspect of the invention, or a combination thereof.

[0044] In another preferred embodiment, the tumor is a CD14-positive tumor.

[0045] In another preferred embodiment, the tumor is a tumor that highly expresses CD14.

[0046] In another preferred embodiment, the tumor with high CD14 expression is selected from the group consisting of bladder cancer, breast cancer, non-Hodgkin's lymphoma, ovarian cancer, non-small cell lung cancer, hepatocellular carcinoma, and laryngeal cancer, or combinations thereof.

[0047] In another preferred embodiment, the bladder cancer includes advanced high-grade bladder cancer.

[0048] In another preferred embodiment, the method further includes treating the subject with another disease treatment method.

[0049] In another preferred embodiment, the additional disease treatment methods are selected from the group consisting of: surgery, radiotherapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, adjuvant therapy, immunotherapy, or combinations thereof.

[0050] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0051] Figure 1 The molecular characterization analysis of CD14 / CD3 BiTE is shown. A: SDS-PAGE (non-reducing and reducing) and SEC-HPLC purity analysis. B: Thermal stability analysis and aggregation stability analysis. C: Average particle size analysis.

[0052] Figure 2 The affinity analysis of CD14 / CD3 BiTE is shown. A: Sensing map for affinity detection of human CD14 protein. B: Sensing map for affinity detection of human CD3 protein.

[0053] Figure 3 The cell-binding ability of CD14 / CD3 BiTE is shown. A: Binding with CD14high T24 cells. B: Binding with CD3 + Jurkat cell binding. C: Schematic diagram of T cell recruitment and cross-linking.

[0054] Figure 4 The in vitro T cell activation function of CD14 / CD3 BiTE is shown. A: Schematic diagram of Jurkat-NFAT reporter gene assay. B: Reporter gene activity assay. C: CD4 in PBMCs. + T cell activation. D: CD8 in PBMCs + T cell activation. E: IFN-γ secretion level. F: IL-2 secretion level. G: LDH release assay.

[0055] Figure 5 The in vivo antitumor efficacy of CD14 / CD3 BiTE is shown. A: Tumor growth curve of CDX model. B: Comparison of final tumor weight of CDX model. C: T cell infiltration in CDX model tumor detected by flow cytometry. D: Cytokine levels in CDX model tumor detected by CBA method. E: Tumor growth curve of PDX model.

[0056] Figure 6The effects of combining CD14 / CD3 BiTE with PD-L1 antibody are shown. A: Schematic diagram of PD-L1 expression upregulation in tumor cells after dual antibody therapy. B: Tumor growth curve of CDX model after combined therapy. C: Comparison of final tumor weight after combined therapy. D: Flow cytometry detection of T cell infiltration and activation in tumors after combined therapy. Detailed Implementation

[0057] Through extensive and in-depth research and screening, the inventors have, for the first time, proposed a bifunctional T-cell adjuvant targeting CD14 and CD3. Experiments demonstrate that this bifunctional T-cell adjuvant successfully mediates cross-linking between T cells and tumor cells, dose-dependently activates T cells and kills CD14-overexpressing tumor cells, and exhibits synergistic effects with PD-L1 antibodies. Based on these findings, this invention was completed.

[0058] the term

[0059] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0060] The term "about" can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101.

[0061] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0062] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0063] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0064] As used herein, the term “pharmaceuticalally acceptable carrier” refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0065] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0066] CD14

[0067] Human monocyte differentiation antigen CD14 is a 14 kDa glycoprotein that exists in a membrane-anchored form on the surface of monocytes and macrophages. It acts as an LPS receptor, mediating the activation of the TLR-MyD88-NF-κB pathway, thereby triggering an innate immune response. In recent years, numerous studies have shown that CD14 is highly expressed in advanced high-grade bladder cancer cells.

[0068] CD3

[0069] CD3 (Cluster of Differentiation 3) is a crucial protein complex that is consistently expressed on the surface of all T lymphocytes. Therefore, CD3 is the most specific and reliable "molecular identity card" for identifying T cells. In immunological research and clinical diagnosis, detecting CD3 to identify and count T cells is a fundamental and critical technique.

[0070] However, CD3 plays a role far beyond being a "label"; its core function is as a "transmitter" and "start switch" for T cell receptor signals.

[0071] The bifunctional T-cell connector of the present invention

[0072] As used herein, the terms "bifunctional T-cell connector of the present invention", "bifunctional T-cell connector of the present invention targeting CD14 and CD3", "T-cell connector of the present invention", and "CD14 / CD3 BiTE" are used interchangeably and all refer to the bifunctional T-cell connector described in the first aspect of the present invention.

[0073] The bifunctional T-cell adaptor of the present invention comprises three subunits:

[0074] The first subunit includes a light chain variable region of the anti-CD14 antibody and a light chain in the backbone; the second subunit includes a heavy chain variable region of the anti-CD14 antibody and a heavy chain in the backbone; and the third subunit includes a heavy chain variable region and a light chain variable region of the anti-CD3 antibody.

[0075] The anti-CD14 antibody or anti-CD3 antibody in the bifunctional T-cell connector of the present invention can be any antibody or antigen-binding fragment thereof with CD14 or CD3 specific binding ability.

[0076] Preferably, the amino acid sequence of the first subunit is shown in SEQ ID NO: 4, the amino acid sequence of the second subunit is shown in SEQ ID NO: 3, and the amino acid sequence of the third subunit is shown in SEQ ID NO: 1.

[0077] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of one light chain (L chain) and one heavy chain (H chain). In a general sense, the heavy chain can be understood as the larger polypeptide chain in the antibody, and the light chain as the smaller polypeptide chain. Each heavy chain consists of a variable region (VH) and a constant region (CH). The constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly denaturing regions (called complementarity-determining regions (CDRs)). The variable regions (VH and VL) of each heavy / light chain pair form the antibody-binding site. The term "antibody" is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different types of antibodies, such as IgG or its mutants, IgA1, IgA2, IgD, IgE or IgM antibodies.

[0078] As used herein, "antigen-binding fragment" refers to a full-length antibody, Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment that possesses antigen-binding activity. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant regions, and are the smallest antibody fragments possessing all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.

[0079] In this invention, the full-length antibody of this invention also includes its conserved variants, which refer to peptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the full-length antibody of this invention.

[0080] As used herein, unless otherwise stated, Fc refers to the Fc fragment of human immunoglobulin. The term "immunoglobulin Fc region" refers to the constant region of the immunoglobulin chain, particularly the carboxyl terminus or a portion thereof of the constant region of the immunoglobulin heavy chain. For example, the immunoglobulin Fc region may include a combination of two or more domains of the heavy chain CH1, CH2, and CH3 with an immunoglobulin hinge region. In a preferred embodiment, the Fc region of the immunoglobulin used includes at least one immunoglobulin hinge region, a CH2 domain, and a CH3 domain, preferably lacking the CH1 domain.

[0081] In one embodiment, the structure of the bifunctional T-cell connector of the present invention is as follows: Figure 1 The first molecule in A is shown in the schematic diagram, which includes three subunits. The first subunit includes the light chain variable region of the anti-CD14 antibody and the light chain in the backbone. The second subunit includes the heavy chain variable region of the anti-CD14 antibody and one heavy chain in the backbone. The third subunit includes the heavy chain variable region and the light chain variable region of the anti-CD3 antibody.

[0082] Wherein, the light chain variable region of the anti-CD14 antibody of the first subunit and the heavy chain variable region of the anti-CD14 antibody of the second subunit constitute the antigen-binding domain of the anti-CD14 antibody, and the heavy chain variable region and the light chain variable region of the anti-CD3 antibody of the third subunit constitute the antigen-binding domain of the anti-CD3 antibody.

[0083] The bifunctional T-cell linker of the present invention also includes variations of the above-described bifunctional T-cell linker. These variations include (but are not limited to): deletions, insertions, and / or substitutions of 1-5 amino acids (typically 1-3, more preferably 1); additions or deletions of one or more amino acids (typically up to 5, preferably up to 3, more preferably up to 1) at the C-terminus and / or N-terminus; or additions of amino acid fragments with smaller side chains (such as glycine, serine, etc.) as linkers at the N-terminus or C-terminus of the protein. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, adding or deleting one or more amino acids at the C-terminus and / or N-terminus generally does not alter the structure and function of the protein. Furthermore, the term also includes the polypeptides of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0084] The present invention also includes the active fragments, derivatives, and analogs of the above-described bifunctional T-cell connective. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the function or activity of the bifunctional T-cell connective of the present invention.

[0085] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusing a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, for example, polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (bifunctional T-cell connectives formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6His). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0086] A preferred class of active derivatives refers to polypeptides formed by replacing up to five, more preferably up to three, and even more preferably up to one amino acid with an amino acid of similar or analogous properties compared to the amino acid sequence of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0087] Table 1

[0088]

[0089] This invention also provides analogues of the bifunctional T-cell connective of the present invention. These analogues may differ from the peptides of the present invention in terms of amino acid sequence differences, differences in modifications that do not affect the sequence, or both. Analogues also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β-, γ-amino acids). It should be understood that the peptides of the present invention are not limited to the representative peptides exemplified above.

[0090] Furthermore, the bifunctional T-cell connector of the present invention can be modified. Modifications (typically without altering the primary structure) include chemically derived forms of the peptide, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the peptide to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include peptides modified to improve their resistance to proteolytic hydrolysis or optimize their solubility.

[0091] The term "polynucleotide of the present invention" can refer to a polynucleotide that includes encoding the bifunctional T-cell adaptor of the present invention, or it can also refer to a polynucleotide that includes additional coding and / or non-coding sequences.

[0092] This invention also relates to variants of the aforementioned polynucleotides that encode fragments, analogues, and derivatives of polypeptides or bifunctional T-cell connectives having the same amino acid sequence as those of this invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the bifunctional T-cell connective it encodes.

[0093] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0094] The bifunctional T-cell connector and polynucleotide of the present invention are preferably provided in an isolated form, and more preferably, purified to homogenization.

[0095] The full-length polynucleotide sequences of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0096] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0097] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0098] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0099] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE (RACE-cDNA end amplification) method is preferred. Primers used for PCR can be appropriately selected based on the sequence information disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0100] expression carrier

[0101] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or the coding sequences of the bifunctional T-cell connectors of the present invention, and methods for generating the polypeptides of the present invention via recombinant technology.

[0102] In this invention, a polynucleotide sequence encoding a bifunctional T-cell adaptor can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0103] In the method for preparing the bifunctional T-cell adaptor of the present invention, any suitable vector can be used, selected from pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, and the expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0104] Both eukaryotic and prokaryotic host cells can be used for the expression of the bifunctional T cell connector of the present invention. The eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably COS, CHO, NSO, sf9 and sf21 cells; the prokaryotic host cells are preferably one of DH5a, BL21(DE3) and TG1.

[0105] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the bifunctional T-cell adaptor of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0106] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0107] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0108] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces, Salmonella typhimurium bacterial cells, fungal cells such as yeast, and plant cells (such as ginseng cells).

[0109] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0110] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0111] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0112] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0113] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0114] The bifunctional T-cell connector disclosed in this invention can be separated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the bifunctional T-cell connector bound to the affinity column.

[0115] Using the above method, the bifunctional T cell connector can be purified into a basically homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis.

[0116] Pharmaceutical Composition

[0117] In this invention, a pharmaceutical composition comprising the bifunctional T-cell connective or its immunoconjugate is also provided.

[0118] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the bifunctional T-cell connector (or conjugate thereof) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents. The aforementioned bifunctional T-cell connector or its immunoconjugate can be formulated with pharmaceutically acceptable excipients to achieve more stable therapeutic effects. These formulations ensure the structural integrity of the amino acid core sequence of the bifunctional T-cell connector of the present invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). The formulation can be in various forms; typically, liquid formulations can be stably stored for at least one year at 2°C-8°C, and lyophilized formulations remain stable for at least six months at 30°C. The formulation can be a commonly used pharmaceutical preparation such as a suspension, injection, or lyophilized form, preferably an injection or lyophilized form.

[0119] For the pharmaceutical compositions of the present invention (such as aqueous injections or lyophilized formulations), pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic modifiers, and buffers. Surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleic, or octadecyl sarcosine; Pluronics; MONAQUAT™, etc., added in an amount that minimizes the tendency of protein granulation. Solution stabilizers can be sugars, including reducing and non-reducing sugars; amino acids, including monosodium glutamate or histidine; alcohols, including one or a combination of triols, higher sugar alcohols, propylene glycol, and polyethylene glycol. The amount of solution stabilizer added should be such that the final formulation is considered by a person skilled in the art to remain stable for a stable period of time. Isotonic modifiers can be one of sodium chloride and mannitol. Buffers can be one of TRIS, histidine buffer, and phosphate buffer.

[0120] When using the pharmaceutical composition, a safe and effective amount of the bifunctional T-cell connective or its immunoconjugate of the present invention is administered to a mammal, wherein the safe and effective amount is generally at least about 50 micrograms per kilogram of body weight, and in most cases does not exceed about 100 milligrams per kilogram of body weight, preferably between about 100 micrograms per kilogram of body weight and about 50 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise. Typically, the total dosage should not exceed a certain range, for example, an intravenous dose of 10 to 3000 mg / day / 50 kg, preferably 100 to 1000 mg / day / 50 kg.

[0121] The bifunctional T-cell connector and pharmaceutical formulations containing the present invention can be used as anti-tumor drugs for tumor treatment. The anti-tumor drugs referred to in the present invention are drugs that inhibit and / or treat tumors, and may include delaying the development of symptoms associated with tumor growth and / or reducing the severity of these symptoms. It further includes alleviating existing symptoms associated with tumor growth and preventing the occurrence of other symptoms, as well as reducing or preventing metastasis.

[0122] The above-mentioned bifunctional T-cell connectives and their drug formulations can also be administered in combination with other antitumor drugs for the treatment of tumors. These antitumor drugs used for combination administration include, but are not limited to: 1. Cytotoxic drugs (1) Drugs that act on the chemical structure of DNA: alkylating agents such as nitrogen mustard, nitrosamines, and methanesulfonates; platinum compounds such as cisplatin, carboplatin, and oxalate platinum; mitomycin C (MMC); (2) Drugs that affect nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta; thymidine synthase inhibitors such as fluorouracils (5FU, FT-207, capecitabine); purine nucleoside synthase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG; nucleotide reductase inhibitors such as hydroxyurea (HU); DNA polymerase inhibitors such as cytarabine (Ara-C) and Gemz; (3) Drugs that act on nucleic acid transcription Drugs: Drugs that selectively act on DNA templates and inhibit DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis, such as: Actinomycin D, daunorubicin, doxorubicin, epirubicin, aclarubicin, scintillans, etc.; (4) Drugs that mainly act on microtubule synthesis: Paclitaxel, Taxotere, Vinpocetine, Vinorelbine, Podophyllin, Homoharringtonine; (5) Other cytotoxic drugs: Asparaginase mainly inhibits protein synthesis; 2. Hormonal anti-estrogens: Tamoxifen, Droloxifene, Exemestane, etc.; Aromatase inhibitors: Aminoglutamate, Lantralon, Letrozole, Renin, etc.; Anti-androgens: Flutamiflu RH-LH agonists / antagonists: Noradrenaline, Enaton, etc.; 3. Biological response modifiers: Interferon that mainly inhibits tumors through the body's immune function; Interleukins other than IL-2; Thymopeptides; 4. Monoclonal antibodies: Rituximab (MabThera); Cetuximab (C225); Herceptin (Trastuzumab); Bevacizumab (Avastin); Yervoy (Ipilimumab); Pembrolizumab (Keytruda); Atezolizumab (Tecentriq); 5. Others include some drugs with unclear mechanisms and requiring further research; cell differentiation inducers such as retinoids; apoptosis inducers.

[0123] The main advantages of this invention include:

[0124] (a) The bifunctional T-cell connector of the present invention can effectively activate T cells and kill tumor cells.

[0125] (b) The bifunctional T-cell connector of the present invention has a synergistic effect when used in combination with PD-L1 antibody, thereby enhancing the anti-tumor effect.

[0126] (c) The bifunctional T-cell connector of the present invention has excellent stability and affinity characteristics, which is beneficial to targeting and safety.

[0127] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0128] Materials and methods

[0129] (I) Cell lines and animal models

[0130] (1) Cell lines: This study used T24 human bladder cancer cell line with high CD14 expression, Jurkat-NFAT-luc reporter gene cell line, human peripheral blood mononuclear cells (PBMC) and Jurkat T lymphocyte leukemia cell line.

[0131] (2) Animal model: NCG immunodeficient mice were selected, and tumor-bearing models were constructed by subcutaneous inoculation of T24 cells (CDX model) or tumor tissue derived from bladder cancer patients (PDX model). The mouse immune system was reconstructed by human PBMC at an appropriate time.

[0132] CDX model: Passaged CD14-positive T24 human bladder cancer cells were subcutaneously injected into the groin or back of immunodeficient NCG mice.

[0133] PDX model: Small pieces of tumor tissue from bladder cancer patients are directly transplanted into NCG mice, and the model is passaged 2-3 times to stabilize it.

[0134] (II) Experimental Design and Grouping

[0135] (1) In vitro functional experiments: including binding affinity test (SPR), flow cytometry binding verification, T cell activation reporter gene experiment, PBMC activation and cytokine detection.

[0136] (2) In vivo efficacy experiment:

[0137] CDX model efficacy evaluation: Tumor-forming mice were randomly divided into 6 groups, with 6 mice in each group:

[0138] Group 1: Isotype control antibody group (Control)

[0139] Group 2: Anti-CD14 control group

[0140] Group 3: Anti-CD3 control group

[0141] Group 4: Anti-CD14 control group combined with anti-CD3 control group (anti-CD14 + anti-CD3)

[0142] Group 5: CD14 / CD3 BiTE treatment group (low dose: 0.2 mg / kg)

[0143] Group 6: CD14 / CD3 BiTE treatment group (high dose: 1 mg / kg)

[0144] PDX model efficacy evaluation: Tumor-forming mice were randomly divided into 2 groups of 5 mice each.

[0145] Group 1: Isotype control antibody group (Control)

[0146] Group 2: CD14 / CD3 BiTE treatment group (high dose: 1 mg / kg)

[0147] Evaluation of combined drug use: Tumor-forming mice were randomly divided into 4 groups, with 6 mice in each group:

[0148] Group 1: Isotype control antibody group (Control)

[0149] Group 2: CD14 / CD3 BiTE monotherapy group

[0150] Group 3: PD-L1 antibody monotherapy group

[0151] Group 4: CD14 / CD3 BiTE + PD-L1 antibody combination group

[0152] (III) Observation Indicators and Sample Collection

[0153] (1) Tumor volume monitoring: Measure the tumor volume every 2-3 days and plot the growth curve.

[0154] (2) In vitro observation indicators: SPR affinity constant, flow cytometry binding rate, luciferase activity, expression of T cell activation markers (CD69 / CD25), and concentration of cytokines (IFN-γ, IL-2).

[0155] (3) In vivo sample analysis: The tumor weight was measured at the experimental endpoint, the tumor-infiltrating T cells were analyzed by flow cytometry, and the cytokine levels were detected by CBA chip.

[0156] (iv) Instruments and reagents

[0157] Main instruments: Surface plasmon resonance (SPR) instrument, flow cytometer, enzyme-linked immunosorbent assay (ELISA) reader, protein purification system, static light scattering instrument.

[0158] Reagents used: CD14 / CD3 BiTE (constructed and purified in our laboratory), human PBMCs, recombinant human CD14 and CD3 proteins, and cytokine detection kits.

[0159] (V) Statistical Analysis

[0160] All data were analyzed using GraphPad Prism software. Quantitative data are expressed as mean ± standard deviation. Comparisons between groups were performed using t-tests or one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0161] Example 1: Construction and purification of CD14 / CD3 BiTE

[0162] For the anti-CD3 terminus, the variable heavy chain (VH) and variable light chain (VL) of a humanized mouse anti-human CD3 antibody clone were linked together using a (GGGGS)3 hinge to design a single-chain antibody fragment (scFv), the specific sequence of which is shown in SEQ ID NO: 1. For the anti-CD14 terminus, the Fab fragment of a humanized mouse anti-human CD14 antibody was selected. The Fc region of the bispecific antibody was selected from the Fc fragment of human IgG1, modified using Knob-into-Hole (KIH) technology and salt bridge engineering. Furthermore, an LALA-PG mutation was introduced into the CH2 region.

[0163] The protein sequence of anti-CD3-scfv-Fc is as follows:

[0164] EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) (The bolded parts are VH, (GGGGS)3 and VL in order, and the underlined parts are CH2 and CH3)

[0165] Its signal peptide sequence is as follows: MGWSCIILFLVATATGVHS (SEQ ID NO: 2).

[0166] The protein sequence of anti-CD14-H-Fc is as follows:

[0167] QVQLQESGPGLVKPSETLSLTCTVSGYSITSDSAWNWIRQPPGKGLEWIGYISYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCVRGLRFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYP SDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3) (The bold part is VH, and the underlined part is CH1-CH3).

[0168] Its signal peptide sequence is as follows: MGWSCIILFLVATATGVHS (SEQ ID NO: 2).

[0169] The protein sequence of anti-CD14-L is as follows:

[0170] DIQLTQSPSSSLSASVGDRVTITCRASESVDSYVNSFLHWYQQKPGKAPKLLIYRASNLQSGVPSRFSGSGSRTDFTLTISSLQPEDVATYYCQQSNEDPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4) (The bold part is VL, and the underlined part is CL).

[0171] Its signal peptide sequence is as follows: MGWSCIILFLVATATGVHS (SEQ ID NO: 2).

[0172] The molecule was expressed in the HEK293 expression system and purified by methods such as Protein A affinity chromatography.

[0173] The results are as follows Figure 1 As shown, Figure 1 The first image in section A is a schematic diagram of the molecular structure of CD14 / CD3 BiTE finally constructed in this invention. SDS-PAGE and size exclusion chromatography (SEC-HPLC) results show that the constructed CD14 / CD3 BiTE has the expected molecular weight and high purity. Figure 1 (A) in the middle.

[0174] Full fluorescence scanning and static fluorescence scanning results indicate that this bispecific antibody exhibits good thermal stability and aggregation stability. Figure 1 (B in the middle).

[0175] Dynamic light scattering (DLS) analysis showed that its average particle size met expectations. Figure 1 The C in the figure indicates that the molecules are uniformly distributed in the solution and there is no obvious aggregation.

[0176] Comparative Example 1: In this embodiment, the inventors also attempted to combine other anti-CD3 antibodies or their antigen-binding fragments, or anti-CD14 antibodies or their antigen-binding fragments, but the results were not as good as the bifunctional T-cell connector formed by the anti-CD14 Fab antibody and the anti-CD3 single-chain antibody finally adopted in this invention. For example:

[0177] This invention also constructed other bifunctional T-cell connectives containing anti-CD3 single-chain antibodies, with the same sequence structure except for the different anti-CD3 single-chain antibody sequences.

[0178] Its anti-CD3-scfv-Fc protein sequence is as follows:

[0179] QVQLVQSGGGVVQPGRSLRLSKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYCARYYDDHYCLDYWGQGTPVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5) (The bolded parts are VH, (GGGGS)3 and VL in order, and the underlined parts are CH2 and CH3).

[0180] Example 2: CD14 / CD3 BiTE exhibits affinity characteristics that are beneficial for both targeting and safety.

[0181] 2.1 Experimental Methods

[0182] The affinity of CD14 / CD3 BiTE was analyzed using surface plasmon resonance (SPR).

[0183] 2.2 Experimental Results

[0184] like Figure 2 As shown, the SPR analysis results indicate that the CD14 end of CD14 / CD3 BiTE ( Figure 2 The A-terminal component exhibits a "fast binding, slow dissociation" characteristic, which is beneficial for its accumulation and retention at tumor sites. Meanwhile, its CD3 terminus (…) Figure 2 B) exhibits a "rapid dissociation" characteristic, which may help reduce the risk of cytokine storms caused by continuous T cell activation.

[0185] Furthermore, the inventors also analyzed the affinity of Comparative Example 1 in Example 1. The results showed that the CD3 terminus of Comparative Example 1 exhibited a "slow dissociation" characteristic, which may lead to excessive and sustained activation of T cells, thereby significantly increasing the potential risk of inducing a cytokine storm.

[0186] Example 3: CD14 / CD3 BiTE can effectively bind and mediate immune synapse formation at the cellular level.

[0187] 3.1 Experimental Methods

[0188] Experiment 1:

[0189] T24 cells (highly CD14-expressing) and Jurkat cells (CD3-positive) were collected separately.

[0190] Cells were incubated on ice with different concentrations of CD14 / CD3 BiTE (or control antibody).

[0191] After washing, add a fluorescently labeled secondary antibody against the Fc fragment (because CD14 / CD3 BiTE contains the Fc fragment and can be recognized).

[0192] The fluorescence intensity of cells was detected using flow cytometry. The stronger the fluorescence intensity, the greater the amount of CD14 / CD3 BiTE binding to the cells.

[0193] Experiment 2: T cell recruitment and immune synapse formation experiment

[0194] T cells and tumor cells were labeled with different colored fluorescent dyes (CFSE and CellTracker FarRed). After co-incubation with CD14 / CD3 BiTE, both were analyzed by flow cytometry. The presence of double-positive cell populations indicated that T cells and tumor cells were "bridged" together by CD14 / CD3 BiTE, forming a cell cross-link.

[0195] 3.2 Experimental Results

[0196] The results are as follows Figure 3 As shown, flow cytometry confirmed that CD14 / CD3 BiTE can interact with T24 cells that highly express CD14 (…Figure 3 A) and Jurkat cells expressing CD3 (in the middle) Figure 3 (B) Effective combination.

[0197] Further T cell recruitment experiments showed that this bispecific antibody successfully mediated the cross-linking of T cells with tumor cells. Figure 3 (C in the middle).

[0198] Example 4: CD14 / CD3 BiTE effectively activates T cells and promotes cytokine release in vitro.

[0199] 4.1 Experimental Methods

[0200] Experiment 1: Jurkat-NFAT reporter gene experiment

[0201] A specific Jurkat T cell line was used, transfected with a luciferase gene driven by the NFAT (a key transcription factor for T cell activation) response element. These reporter cells, T24 tumor cells, and varying concentrations of CD14 / CD3BiTE were co-cultured. Once activated, T cells initiated intracellular calcium flow and the NFAT signaling pathway, leading to luciferase expression.

[0202] Cells were lysed and luciferase substrate was added; the chemiluminescence intensity was then measured using a microplate reader. The luminescence intensity was directly proportional to the degree of T cell activation.

[0203] Experiment 2: PBMC Activation and Biomarker Detection

[0204] PBMCs, containing T cells, were isolated from peripheral blood of healthy individuals. PBMCs, CD14-positive T24 tumor cells, and CD14 / CD3 BiTE cells were co-cultured. After incubation for a specified time (e.g., 24-48 hours), cells were collected and labeled with fluorescent antibodies for CD4, CD8, CD69 (early activation markers), and CD25 (IL-2 receptor, a marker of activation and proliferation). CD4 was analyzed by flow cytometry. + and CD8 + Positive rates and average fluorescence intensity of CD69 and CD25 in the T cell population.

[0205] Experiment 3: Detection of Cytokine Release

[0206] In the same PBMC-tumor cell co-culture system as described above, the supernatant was collected. Using an ELISA (enzyme-linked immunosorbent assay) kit, the concentrations of IFN-γ and IL-2 in the supernatant were quantitatively determined based on the standard curve.

[0207] Experiment 4: In vitro tumor cell killing assay (LDH method)

[0208] PBMCs (effective cells) and CD14-positive T24 cells (target cells) were mixed in a specific ratio and co-cultured with CD14 / CD3 BiTE. After tumor cells were killed by T cells, their cell membranes ruptured, releasing intracellular lactate dehydrogenase (LDH) into the culture supernatant. LDH was added as a substrate, and a colored product was generated after the reaction. The absorbance was measured using a microplate reader.

[0209] The percentage of cytotoxicity is calculated using the formula: (Experimental group - spontaneous release from effector cells - spontaneous release from target cells) / (maximum release from target cells - spontaneous release from target cells) * 100%.

[0210] 4.2 Experimental Results

[0211] The results are as follows Figure 4 As shown, in the Jurkat-NFAT reporter gene experiment (see schematic diagram), Figure 4 In the A), CD14 / CD3 BiTE can dose-dependently activate T cells and induce luciferase expression ( Figure 4 (B in the middle).

[0212] In PBMC experiments, which more closely resemble physiological environments, this bispecific antibody significantly upregulated CD4. + ( Figure 4 C) and CD8 + T cells ( Figure 4 Early activation markers CD69 and CD25 on the surface of D) in the middle.

[0213] Simultaneously, ELISA testing confirmed that the key cytokine IFN-γ secreted by activated T cells (…) Figure 4 E) and IL-2 ( Figure 4 The level of F in the sample was significantly increased.

[0214] LDH release assay showed that BiTE could dose-dependently kill CD14-overexpressing T24 cells. Figure 4 (G in the middle).

[0215] Example 5: CD14 / CD3 BiTE exhibits potent antitumor activity in an in vivo model.

[0216] 5.1 Experimental Methods

[0217] In CDX and PDX model mice, respectively, the tumor volume reached approximately 100 mm. 3 Mice were randomly assigned to groups as designed in the experiment (e.g., control group, single-drug group, combination-drug group). The corresponding antibodies or PBS controls were administered periodically via intraperitoneal injection or tail vein injection.

[0218] Observation indicators:

[0219] Tumor volume: Measure the long and short diameters of the tumor every 2-3 days using calipers. Volume is calculated using the formula: Volume = (Long diameter × Short diameter) 2 Calculate ) / 2 and plot the growth curve.

[0220] Endpoint tumor weight: At the end of the experiment, the tumor was surgically removed and weighed.

[0221] Tumor tissue flow cytometry analysis: A portion of tumor tissue was digested into a single-cell suspension, and human CD45 was detected by flow cytometry. + Immune cells, CD3 + T cells and their subsets (CD4) + / CD8 + The infiltration ratio and activation state of ).

[0222] Detection of cytokines in the tumor microenvironment: A portion of tumor tissue homogenate or mouse serum was taken and CBA (cytokine microsphere detection) chip technology was used to simultaneously and quantitatively detect the concentration of multiple cytokines (such as IFN-γ, TNF-α, IL-2, etc.).

[0223] 5.2 Experimental Results

[0224] In the NCG mouse immune reconstitution CDX model, the CD14 / CD3 BiTE treatment group significantly inhibited tumor growth compared with the control group. Figure 5 (as shown in A and Table 2), the tumor inhibition rate exceeded 80%, and the endpoint of significantly reduced tumor weight ( Figure 5 (as shown in B and Table 2).

[0225] Table 2

[0226]

[0227] Note: All values ​​in the table are mean values.

[0228] Mechanistically, T cell infiltration was significantly increased in the tumor tissue of the treatment group. Figure 5 C in the middle), and the level of local inflammatory cytokines is higher ( Figure 5 (D in the middle).

[0229] In more clinically relevant PDX models, CD14 / CD3 BiTE also showed a clear tumor-suppressing effect. Figure 5 (E in the text).

[0230] Example 6: Synergistic effect of CD14 / CD3 BiTE combined with PD-L1 antibody

[0231] The experimental method is the same as in Example 5.

[0232] The PD-L1 antibody used in this embodiment is commercially available atezolizumab, which is a humanized IgG1 anti-PD-L1 monoclonal antibody.

[0233] The results are as follows Figure 6 As shown, after CD14 / CD3 BiTE treatment, the expression of PD-L1 in tumor cells was upregulated in a feedback manner. Figure 6 (A in the middle).

[0234] Based on this, the present invention explores combination drug therapy strategies.

[0235] The results showed that in the CDX model, the combination of CD14 / CD3 BiTE and PD-L1 antibody exhibited a stronger tumor-suppressive effect than either single-drug group. Figure 6 B in Figure 6 (C in Table 3).

[0236] Table 3

[0237]

[0238] Note: All values ​​in the table are averages. All data were measured at the end of the experiment.

[0239] Flow cytometry analysis further confirmed that the levels of T cell infiltration and activation in tumors were significantly increased in the combination therapy group. Figure 6 (D in Table 4).

[0240] Table 4

[0241]

[0242] discuss

[0243] This invention successfully constructed a novel bifunctional T-cell connective targeting CD14 and CD3, and systematically demonstrated that this molecule possesses excellent physicochemical properties, targeted binding ability, and can effectively activate T cells and kill bladder cancer cells. In immune reconstitution mouse models, this bispecific antibody exhibited significant tumor-suppressing effects in both CDX and PDX models.

[0244] Furthermore, this invention is the first to discover that this dual antibody therapy can induce PD-L1 feedback upregulation, and experiments have demonstrated that its combination with PD-L1 antibodies has a synergistic anti-tumor effect. This provides a novel and highly promising combination therapy strategy and solid preclinical evidence for subsequent treatment of CD14-positive bladder cancer, especially for combating drug resistance caused by PD-L1 upregulation.

[0245] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A bifunctional T-cell connector targeting CD14 and CD3, comprising a backbone including an IgG antibody constant region, the backbone comprising two heavy chains and one light chain, wherein one heavy chain includes heavy chain constant regions CH1, CH2, and CH3, the other heavy chain includes CH2 and CH3, and the light chain includes CL, characterized in that, The bifunctional T-cell connector targeting CD14 and CD3 is a heterotrimer, specifically: The first subunit includes a light chain variable region of the anti-CD14 antibody and a light chain in the backbone; the second subunit includes a heavy chain variable region of the anti-CD14 antibody and a heavy chain in the backbone; and the third subunit includes a heavy chain variable region and a light chain variable region of the anti-CD3 antibody. Wherein, the light chain variable region of the anti-CD14 antibody of the first subunit and the heavy chain variable region of the anti-CD14 antibody of the second subunit constitute the antigen-binding domain of the anti-CD14 antibody, and the heavy chain variable region and the light chain variable region of the anti-CD3 antibody of the third subunit constitute the antigen-binding domain of the anti-CD3 antibody. The IgG antibody is a human-derived IgG antibody. The CH2 domains of the second and third subunits introduce LALA-PG mutations; The two CH3 groups in the second and third subunits are designed with a knot structure and a hole structure respectively using the knot-into-hole technique. The amino acid sequence of the first subunit is shown in SEQ ID NO: 4, the amino acid sequence of the second subunit is shown in SEQ ID NO: 3, and the amino acid sequence of the third subunit is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bifunctional T-cell connector targeting CD14 and CD3 as described in claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 2.

4. A host cell, characterized in that, The host cell contains the expression vector of claim 3, or has the nucleic acid molecule of claim 2 integrated into its genome.

5. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) The bifunctional T-cell connector as claimed in claim 1; and (b) Coupling part: Detectable marker.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) First active ingredient: the bifunctional T cell editor as claimed in claim 1, the nucleic acid molecule as claimed in claim 2, the expression vector as claimed in claim 3, the host cell as claimed in claim 4, or the immunoconjugate as claimed in claim 5; and (ii) Pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition further contains a second active ingredient, which is an antitumor drug.

8. The pharmaceutical composition according to claim 7, characterized in that, The second active ingredient is an anti-PD-L1 antibody.

9. The use of the bifunctional T-cell editor of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3, the host cell of claim 4, the immunoconjugate of claim 5, or the pharmaceutical composition of claim 6, characterized in that, The drug is used to prepare a treatment for tumors; and the tumor is a tumor that highly expresses CD14. Among them, the tumor with high CD14 expression is bladder cancer.

10. A method for preparing the bifunctional T-cell connector of claim 1, characterized in that, Including the following steps: (a) Under expression conditions, the host cells of claim 4 are cultured to express the bifunctional T cell connective; (b) Isolate and purify the bifunctional T-cell connective described in (a).

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