Use of small peptides or proteins for selective targeting of TREML1 / MD2 interactions and as vaccine adjuvants.

Abstract

A pharmaceutical composition for enhancing an immune response comprises a TREM-like transcript-1 (TREML1) extracellular domain (ECD) or stalk polypeptide. The TREML1 ECD or stalk polypeptide is derived from human or mouse TREML1. The pharmaceutical composition may further comprise an antigen as a vaccine, with the TREML1 ECD or stalk polypeptide functioning as an adjuvant or immune booster.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to polypeptide fragments derived from TREM-like transcript-1 (TREML1;TLT-1) protein and peptide fragments, and their use as vaccine adjuvants, immune boosters, or TLR (Tall-like receptor) agonists. [Background technology]

[0002] Background of the Invention TREML1 is found exclusively in platelets in human peripheral blood. Upon platelet activation, TREML1 is rapidly exposed on the platelet membrane, subsequently degraded, and released as a soluble fragment (sTREML1). Studies have shown that patients with sepsis, in contrast to healthy individuals, have elevated levels of soluble TREML1 in their plasma. Patients who died from sepsis had persistently high levels of soluble TREML1 in their plasma, while patients who survived sepsis showed decreased levels of soluble TREML1 in their plasma. In other diseases, such as acute respiratory distress syndrome (ARDS), acute coronary insufficiency syndrome, and coronary artery disease, high levels of soluble TREML1 plasma concentration have also been shown to be associated with negative outcomes. Therefore, monitoring plasma levels of soluble TREML1 can be an important prognostic indicator. We previously found that soluble TREML1 can directly bind to monocytes and modulate the immune response (WO2016197975A1). These results suggest that soluble TREML1 plays an important role in inflammation-related diseases.

[0003] MD2 (LY-96, lymphocyte antigen 96) associates with Toll-like receptor 4 (TLR4) on the cell surface, conferring broad responsiveness to endotoxin lipopolysaccharide (LPS), and thus linking the receptor to LPS signaling. However, it remains unclear whether MD2 and TLR4 can bind to other endogenous proteins to enable TLR signaling. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO2016197975A1 [Non-patent literature]

[0005] [Non-Patent Document 1] DW Yeh et al., “CpG-oligodeoxynucleotides developed for grouper toll-like receptor (TLR)21s effectively activate mouse and human TLR9s mediated immune responses,” Sci Rep, 2017, 7(1):17297 [Non-Patent Document 2] MRHutchinson et al., “Opioid Activation of Toll-Like Receptor 4 Contributes to Drug Reinforcement,” J. Neuorosci., 2012, 32(33):11187-11200 [Non-Patent Document 3] Hayden MS, West AP, Ghosh S (October 2006), "NF-κB and the immune response", Oncogene.25(51):6758-80 [Non-Patent Document 4] J. Takeda et al., "Anti-tumor immunity against CT26 colon tumor in mice immunized with plasmid DNA encoding beta-galactosidase fused to an envelope protein of endogenous retrovirus," Cell Immunol., (2000), 204(1):11-18 [Overview of the project]

[0006] Summary of the present invention Embodiments of the present invention are based on the unexpected discovery that the TREML1 extracellular domain (ECD) or its stalk can bind to the TLR4 / MD2 complex, resulting in modulation of TLR4 signaling. Furthermore, the TREML1 ECD can also enhance the medicated cellular response by TLR7 / 8 / 9 agonists. As a result of the TREML1 ECD or its stalk binding to the TLR4 / MD2 complex, the TREML1 ECD or its stalk can induce dendritic cell activation and maturation. Therefore, the TREML1 ECD or its stalk can act as a vaccine adjuvant or immune booster, or as a TLR agonist.

[0007] One aspect of the present invention relates to a pharmaceutical composition for boosting the immune response. A pharmaceutical composition according to one embodiment of the present invention comprises a TREM-like transcript-1 (TREML1) extracellular domain (ECD) or stalk polypeptide. The TREML1 ECD or stalk polypeptide may be derived from human or mouse TREML1. The TREML1 ECD or TREML1 stalk polypeptide has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0008] According to some embodiments of the present invention, the pharmaceutical composition further comprises a TLR agonist. Examples of TLR agonists include lipopolysaccharides, heat shock proteins, fibrinogen, heparan sulfate fragments, hyaluronic acid fragments, CpG (CpG-ODNs (CpG oligodeoxynucleotides), DW Yeh et al., "CpG-oligodeoxynucleotides developed for grouper toll-like receptor (TLR)21s effectively activate mouse and human TLR9s mediated immune responses," Sci Rep, 2017, 7(1):17297), R848 (4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol, ACS #144875-48-9), and various opioid drugs (e.g., morphine, remifentanil, MRHutchinson et al., "Opioid Activation of Toll-Like Receptor 4 Contributes to Drug Examples include "Reinforcement," J.Neuorosci., 2012, 32(33):11187-11200). According to some embodiments of the present invention, the pharmaceutical composition may further include an antigen as a vaccine, and TREML1 ECD or Stork polypeptide functions as an adjuvant or immune booster. The antigen may also be a marker for cancer.Cancers include colorectal cancer, breast cancer, lung cancer, melanoma, hepatocellular carcinoma, head and neck cancer, squamous cell carcinoma of the lung, ovarian cancer, uterine cancer, prostate cancer, stomach cancer, cervical cancer, esophageal cancer, bladder cancer, kidney cancer, brain cancer, bone cancer, pancreatic cancer, skin cancer, malignant melanoma of the skin or eye, rectal cancer, anal cancer, stomach cancer, testicular cancer, cancer of the Fallopian tube, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, para This may include kidney cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, medulloblastoma calcifying epithelioma, endometrial cancer, multiple myeloma, or T-cell lymphoma.

[0009] One aspect of the present invention relates to a method for enhancing an immune response. A method according to one embodiment of the present invention comprises administering one of the above-described pharmaceutical compositions to a subject requiring it.

[0010] Other aspects of the present invention are evident from the following description and the accompanying drawings. Brief explanation of the drawing [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows a schematic diagram of TREML1 illustrating the extracellular domain (ECD), transmembrane domain, and cytoplasmic domain. The ECD extends from residues 16 to 162 of TREML1, and the transmembrane domain extends from residues 163 to 183 of TREML1. The ECD contains a single IgV domain (residues 16-121) and a stalk region (residues 122-162). The cytoplasmic domain of TREML1, extending from residues 184 to 313, contains an immunotyrosine inhibitory motif (ITIM; residues 279-284). [Figure 2]Figure 2 shows the results of binding TREML1 ECD to various candidate proteins. The results demonstrate that recombinant TREML1 ECD can bind to immobilized MD2 and TLR4 in solid-phase binding assays. [Figure 3] Figures 3A and 3B show the results of MD2 binding to various proteins. The results demonstrate that human and mouse MD2 proteins can bind to immobilized TREML1 stalks and mTREML1 stalks, respectively, in solid-phase binding assays. [Figure 4] Figure 4 shows the results of various antibodies competing with TREML1 ECDs that bind to THP1 / XBlue / MD2 / CD14 monocytes expressing MD2 and CD14. THP1 is derived from human monocytes that naturally express many pattern recognition receptors, including Toll-like receptors. Both MD2 and CD14 are co-receptors of TLR4 and mediate LPS-induced responses. The results show that anti-MD2 antibodies (18H10) and anti-TLR4 antibodies (HTA125) can compete with TREML1 ECDs that bind to THP1 / XBlue / MD2 / CD14 monocytes. [Figure 5] Figures 5A and 5B demonstrate that TREML1 ECD can induce THP1 / XBlue / MD2 / CD14 monocyte activation. Figure 5A shows NF-κB secretion induced by TREML1 ECD upon activation of THP1 / XBlue / MD2 / CD14. Figure 5B shows TNF-α secretion induced by TREML1 ECD upon activation of THP1 / XBlue / MD2 / CD14. [Figure 6] Figure 6 shows that THREL1 ECD-induced THP1 / XBlue / MD2 / CD14 monocyte activation is reduced by treatment with anti-MD2 antibody (18H10) and anti-TLR4 antibody (HTA125), as evidenced by decreased TNF-α secretion. [Figure 7] Figures 7A and 7B demonstrate that TREML1 ECD can enhance cellular activation induced in intracellular TLRs (i.e., TLR7, TLR8, and TLR9). [Figure 8]Figure 8 shows that mTREML1 ECD and mTREML1 stalk peptide can induce dendritic cell maturation. [Figure 9] Figure 9A shows the treatment schedule in an animal model for testing the effect of the TREML1 stalk as an adjuvant for cancer vaccines. Figure 9B shows that the mTREML1 stalk as an adjuvant enhances the efficacy of cancer vaccines.

Mode for Carrying Out the Invention

[0012] Detailed Description Embodiments of the present invention relate to TREML1 ECD or a stalk peptide derived from TREML1 for use as an immune booster or a TLR agonist. The inventors of the present invention unexpectedly found that TREML1 ECD or its stalk can bind to MD2, and MD2 can associate with toll-like receptor 4 (TLR4) on the cell surface. MD2 interacts with TLR4 to confer responsiveness to a wide range of stimuli, such as endotoxin lipopolysaccharide (LPS). Activation of TLR4 causes intracellular signaling that increases the production of NF-κB and inflammatory cytokines (such as TNF-α), which activates the innate immune response. Furthermore, TREML1 ECD or its stalk has also been found to interact with TLR2, 7, 8, or 9. As a result of the interaction between TREML1 ECD or its stalk and TLR2 / 7 / 8 / 9, dendritic cell (DC) activation and maturation, and further enhancement of the immune response are brought about.

[0013] TREM-like transcript-1 (TREML1) is a member of the TREM family. As shown in Figure 1, TREML1 is a single transmembrane protein consisting of an extracellular domain (ECD, residues 16-162), a transmembrane domain (TMD, residues 163-183), and a cytoplasmic domain (residues 184-311) containing an immunotyrosine inhibitory motif (ITIM, residues 279-284). The ECD of TREML1 contains a single V-set immunoglobulin (Ig) domain (residues 16-121) and a stalk region (residues 122-162).

[0014] To identify potential binding partners for TREML1 ECD, the inventors evaluated the interaction between TREML1 ECD and candidate proteins using a solid-phase binding assay. In short, various candidate proteins and BSA (as a negative control) were coated onto 96-well plates, and TREML1 ECD at various concentrations was added to the wells. After binding and washing, anti-TREML1 antibody conjugated to horseradish peroxidase (HRP) was added to each well. After washing, the HRP substrate, TMB (3,3',5,5'-tetramethylbenzidine), was added. After the reaction, TREML1 binding was evaluated by measuring the absorbance at 450 nm. An exemplary set of results is shown in Figure 2, where recombinant TREML1 ECD binds to MD2 and TLR4 in a concentration-dependent manner. In contrast, TREML1 ECD does not bind to CD14 or BSA.

[0015] Furthermore, MD2(LY96) binds to TREML1 stalk in a concentration-dependent manner. In contrast, TLR4 and CD14 do not bind to TREML1 stalk (Figures 3A and 3B). Similar to MD2, CD14 also acts as a co-receptor for the detection of bacterial lipopolysaccharide (LPS) (together with the Toll-like receptor TLR4). These results indicate that TREML1 stalk directly binds to MD2 in the CD14 / MD2 / TLR4 receptor complex.

[0016] In addition to solid-phase binding assays, TREML1 ECD binding was further evaluated using THP1 / XBlue / MD2 / CD14 cells (InvivoGen, San Diego, USA) expressing MD2 and CD14. These cells (InvivoGen, San Diego, USA) are derived from the human monocyte THP1 cell line and express NF-κB and AP1-induced secreted embryonic alkaline phosphatase (SEAP) reporter genes.

[0017] As shown in Figure 4, TREML1 ECD can bind to THP1 / XBlue / MD2 / CD14 cells. Binding tests were also confirmed using anti-MD2 antibodies (18H10) and anti-TLR4 antibodies (HTA125) to compete with TREML1 for binding to THP1 / XBlue / MD2 / CD14 cells. The results of the binding competition are shown in Figure 4. Both anti-MD2 and anti-TLR4 antibodies can compete with TREML1 for binding to THP1 / XBlue / MD2 / CD14 cells, and an additive effect occurs when anti-MD2 and anti-TLR4 antibodies are used in combination.

[0018] The binding of TREML1 ECD to TLR4 / MD2 expressed by THP1 / XBlue / MD2 / CD14 cells suggests that TREML1 ECD should be able to activate monocytes. Indeed, as shown in Figures 5A and 5B, TREML1 ECD induces THP1 / XBlue / MD2 / CD14 monocyte activation, as evidenced by the induced activation of NF-κB (Figure 5A) and TNF-α (Figure 5B) secretion.

[0019] THRML1-mediated THP1 / XBlue / MD2 / CD14 monocyte activation is mediated by MD2 and TLR4, as evidenced by the ability of anti-MD2 antibody (18H10) and anti-TLR4 antibody (HTA125) to inhibit THRML1-induced TNF-α secretion. Figure 6 shows that THRML1-induced TNF-α secretion is specifically inhibited by anti-MD2 antibody (18H10), anti-TLR4 antibody (HTA125), or a combination thereof, while control IgG (MOPC137 and / or MPC-11) does not have this effect.

[0020] The TLR family plays a crucial role in pathogen recognition and the activation of innate immunity. They recognize pathogen-associated molecular patterns (PAMPs) expressed on infectious pathogens, which mediate the production of cytokines necessary for effective immunity. Various TLRs exhibit diverse expression patterns and can mediate diverse functions.

[0021] In addition to functioning through TLR4, we have also found that TREML1 ECD can enhance cellular activation induced by intracellular TLRs (i.e., TLR7, TLR8, and TLR9). For example, Figure 7A shows that agonists of R848 (TLR7 and TLR8 agonists) can induce TNF-α secretion. TNF-α secretion induced by R848, mediated by TLR7 / 8, is enhanced in the presence of TREML1 ECD. Similar results were observed for TLR9. As shown in Figure 7B, TNF-α secretion induced by ODN 2395 (TLR9 ​​agonist) is dose-dependently enhanced in the presence of TREML1 ECD. These results indicate that TREML1 ECD or Stork peptide can be used as agonists for TLR receptors. As agonists for these receptors, TREML1 ECD or Stork peptide can enhance the innate immune response.

[0022] The ability of dendritic cells (DCs) to modulate adaptive immunity is controlled by their maturation state and lifespan. TNF is a well-known maturation and survival factor for DCs. We have found that TREML1 is an even more effective factor for DC maturation. As shown in Figure 8, mouse TREML1 (mTREML1) ECD and mTREML1 stalk peptide can induce dendritic cell maturation, as evidenced by increased levels of DC maturation markers CD40, CD86, CD80, and MHC II. The effect of TREML1 ECD or its stalk peptide is significantly potent compared to that of TNF-α. These results suggest that TREML1 ECD or stalk peptide can also enhance adaptive immune responses in addition to enhancing innate immune responses.

[0023] The results above, taken together, indicate that TREML1 ECD or TREML1 stalks interact with MD2 / TLR4, enhancing TLR7, TLR8, and TLR9 activation and inducing DC maturation. TLRs are important regulators of both innate and adaptive immune responses. (Hayden MS, West AP, Ghosh S (October 2006), "NF-κB and the immune response," Oncogene. 25(51):6758-80). Interaction between TREML1 ECD (or its stalks) and MD2 / TLR4 or TLR7 / 8 / 9 leads to activation of NF-κB and TNF-α secretion.

[0024] The primary role of TNF-α is in regulating immune cells. NF-κB is a major transcription factor that regulates genes responsible for both innate and adaptive immune responses. Activation of either the T or B cell receptor leads to activation of NF-κB by distinct signaling components. Through a cascade of phosphorylation events, the kinase complex is activated, and NF-κB enters the nucleus to upregulate genes involved in T cell development, maturation, and proliferation. Therefore, the interaction between TREML1 ECD or Stork and MD2 / TLR4 can enhance both innate and adaptive immune responses. In this regard, certain TLR4 agonists have been used as immunomodulators or vaccine adjuvants. For example, MPL (monophosphoryl lipid A, a detoxified form of lipopolysaccharide) is used in commercially available vaccine formulations.

[0025] Furthermore, the interaction between TREML1 ECD or stalk and TLR4 / MD2 results in enhanced cell activation induced by TLR7 / 8 / 9. These effects, collectively, enhance dendritic cell (DC) activation and maturation. The ability to act via both the TLR4 / MD2 and TLR7 / 8 / 9 systems suggests that TREML1 ECD or its stalk is a potent immune booster. Based on these novel findings, embodiments of the present invention relate to reagents and methods for enhancing immune responses using TREML1 ECD or its stalk. Accordingly, TREML1 ECD or its stalk may be used in vaccines to enhance the immune response in a manner similar to that of an adjuvant. As an adjuvant or immune booster, TREML1 ECD or its stalk peptide can be used with an antigen, with or without another adjuvant or another TLR receptor agonist.

[0026] To test TREML1 ECD or its stalk as a vaccine adjuvant or immune booster, the inventors use a CT26 colon cancer animal model. Figure 9A shows the experimental protocol for this study. In short, CT26 colon cancer cells (3 × 10⁻¹⁶) 5The cells were subcutaneously injected into BALB / C mice on day 0. On day 6, the tumors were 30-100 mm in size. 3 When the tumors had grown, they were subcutaneously injected with a tumor vaccine (5E-GP70-15 / aluminum mixture; 100 μL / mouse, with or without the mouse TREML1 stalk tumor antigen). Vaccination was repeated on day 13. Tumor volume was measured every 3-4 days. GP70 is an endogenous ecotropic mouse leukemia virus equivalent to a human tumor-associated antigen. Protective immunity against CT26 cells was induced using a DNA vaccine containing the gp70 gene. (J. Takeda et al., "Anti-tumor immunity against CT26 colon tumor in mice immunized with plasmid DNA encoding beta-galactosidase fused to an envelope protein of endogenous retrovirus", Cell Immunol., (2000), 204(1):11-18).

[0027] As shown in Figure 9B, treatment with the tumor antigen 5E-GP70-15 / aluminum mixture alone resulted in a slight reduction in tumor volume compared to the control group (treated with aluminum alone), while the combination of the tumor antigen 5E-GP70-15 / aluminum mixture and mTREML1 Stork resulted in a dramatic reduction in tumor volume. These results indicate that TREML1 Stork can substantially enhance vaccine efficacy by enhancing the immune response. The unexpectedly strong enhancement of the immune response by TREML1 ECD or Stork appears to be due to the combined effects on both the innate and adaptive immune systems, namely the induction of antigen-presenting cell (APC) maturation by TREML1 ECD or Stork, and the interaction of TREML1 ECD or Stork with TLR4 / MD2 and / or TLR7 / 8 / 9, as described above.

[0028] According to embodiments of the present invention, TREML1 ECD or Stork peptide can be used alone as a TLR agonist to enhance the innate immune response. Furthermore, TREML1 ECD or Stork peptide can be used together with or without other adjuvants (e.g., alum) or other TLR agonists (e.g., monophosphoryl lipid A) as an adjuvant or immune booster in combination with antigens in vaccines or immunotherapies. Due to their adjuvant-like mechanism or action, these TREML1 ECD or Stork peptides can be used in immunotherapies or vaccines to treat cancers, not limited to any specific tumor. However, because TREML1 ECD or Stork peptide can function as a TLR agonist and enhance both innate and adaptive immune responses, it is expected to be more effective than conventional adjuvants. For these reasons, TREML1 ECD or Stork peptide can be used as a general immune booster in all types of vaccines or immunotherapies, including tumor immunotherapy. Examples of tumors or vaccines that can utilize ECD or Stork peptide include, but are not limited to, colorectal tumors (gp70-15 vaccine, carcinoembryonic antigen (CEA) vaccine, MUC-1 vaccine, beta-human chorionic gonadotropin (β-hCG) vaccine, guanylyl cyclase C (GUCY2C) vaccine, epidermal growth factor receptor (EGFR) vaccine, epithelial cell adhesion molecule (EpCAM) vaccine), breast cancer (MUC-1 vaccine, survival vaccine, telomerase vaccine, and epithelial tumor antigen vaccine), hepatocellular carcinoma (alpha-fetoprotein vaccine), ovarian cancer (CA-125), malignant melanoma (tyrosinase vaccine, and melanoma-associated antigen vaccine), virus-induced tumors (HPV types 16 and 18 E6 / E7, HBV, HCV, EBV vaccines), and various other tumors (RAS vaccine, p53 vaccine, β-catenin vaccine, CDK4 gene BCR-ABL protein vaccine, personalized genome vaccine, and neoantigen personalized vaccine).

[0029] The TREML1 ECD or Stork peptide used in embodiments of the present invention may be derived from human TREML1, mouse TREML1, or other animal TREML1. Some examples of TREML1 proteins or peptides used in embodiments of the present invention are shown in Table I.

[0030] [Table 1]

[0031] Embodiments of the present invention are further illustrated by the following specific examples. Those skilled in the art will understand that these examples are for illustrative purposes only, and that other modifications and variations are possible without departing from the scope of the invention.

[0032] Materials and methods Generation of recombinant TREML1 ECD and TREML1 stalk polypeptide Proteins and peptides for use in embodiments of the present invention can be produced by recombinant methods or chemical synthesis known in the art. For example, to produce recombinant TREML1 ECD, pET30-TREML1 encoding human or mouse TREML1 ECD (SEQ ID NO: 1 and SEQ ID NO: 2) having a polyhistidine tag at the N-terminus was expressed in Escherichia coli and purified using a Ni-NTA column (Novagen). The purity of the recombinant protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized using Coomassie blue staining, and was found to be over 95%. Endotoxin contamination of the purified protein was tested using a LAL assay (QCL-1000; Charles River Laboratories, Wilmington, Massachusetts, USA). All proteins were sterile, and the endotoxin concentration was below the detection limit (<0.1 EU / μg protein). The TREML1 stalk polypeptide was chemically synthesized by Kelowna International Scientific Inc. (Taipei, Taiwan).

[0033] Solid-phase bonding assay This study investigated protein-protein interactions. Recombinant TLR4 (5 ug / ml in PBS), MD2 (5 ug / ml in PBS), CD14 (5 ug / ml in PBS), and BSA (negative control) proteins were seeded separately on 96-well plates and incubated overnight at 4 degrees Celsius. A blocking buffer (2% BSA in PBS) was added to prevent nonspecific binding at room temperature for 2 hours. Serially diluted TREML1 ECD solutions (2.5 ug / ml to 156 ng / ml, 2-fold dilution) were added and incubated at room temperature for 2 hours. Anti-TREML1 antibody (1 ug / ml in PBS) was then added over 1 hour. Next, a secondary antibody with HRP labeling, anti-rat IgG-HRP, was added to the plate and incubated at room temperature for 30 minutes. The plate was washed three times with 0.05% tween20 PBS buffer (PBST) between each step. Finally, TMB substrate was added and incubated for 10–15 minutes to detect the HRP-labeled antibody. Subsequently, a 1N HCl stop solution was added to halt the reaction. The absorbance at OD450 / 540 nm was measured using a microplate reader.

[0034] For TREML1 or mTREML1 stalks that bind to human or mouse MD2 protein, TREML1 stalks or mTREML1 stalks (5 ug / ml in PBS) were separately coated on 96-well plates. Recombinant hTLR4, hMD2, hCD14, or mMD2 were sequentially diluted and added to the plates. Protein binding was detected using the anti-His tag antibody-HRP as the detection antibody.

[0035] Preparation of human leukocytes Human peripheral blood samples were collected from healthy volunteer donors by inducing bleeding into an ACD vacutainer tube via venous puncture. Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque Plus (GE17-1440-03) according to the manufacturer's instructions.

[0036] Human peripheral blood mononuclear cells (PBMCs) were pretreated with or without hTREML1 (10 ug / ml, 5 ug / ml, and 1 ug / ml) and incubated with 5% CO2 at 37°C for 30 minutes. After treatment, the cells were stimulated with TLR agonists containing R848 (InvivoGen #tlrl-r848) and ODN2395 (InvivoGen #tlrl-2395) and incubated with 5% CO2 at 37°C for 18 hours. The supernatant of the cell culture medium was collected for TNF-α measurement.

[0037] THP1 / XBlue / MD2 / CD14 cell line The THP1 / XBlue / MD2 / CD14 cell line was derived from the human monocyte THP1 cell line and purchased from InvivoGen. The cells were cultured at 37°C with 5% CO2 in RPMI1640 containing 10% thermally inactivated FBS and antibiotics.

[0038] in vitro assay To examine the efficacy of anti-TLR4 antibody (HTA125, BioLegend #312814) and anti-MD2 antibody (18H10, ThermoFisher #MA5-33351) on TREML1 ECDs that bind to THP1 / XBlue / MD2 / CD14 cells, cells were pretreated with anti-MD2 antibody, anti-TLR4 antibody, IgG control, or a combination thereof, at 37°C for 15 minutes with 5% CO2. After antibody treatment, cells were treated with TREML1 ECD (10 ug / ml) and incubated for 1 hour. Cells were washed with PBS containing 1% FBS, stained with AF647-anti-human TREML1 antibody, and subsequently subjected to flow cytometry analysis.

[0039] NF-κB activation in THP1 / XBlue / MD2 / CD14 cells To investigate the effects of anti-TLR4 and anti-MD2 antibodies on the TLR response induced by TREML1 ECD, THP1 / XBlue / MD2 / CD14 cells were pretreated with anti-TLR4 antibody, anti-MD2 antibody, or IgG control for the indicated groups and incubated at 37°C and 5% CO2 for 15 minutes. After pretreatment, THP1 / XBlue / MD2 / CD14 cells were stimulated with TREML1 ECD (10 ug / ml) and incubated in wells of a 96-well plate at 37°C and 5% CO2 for 18 hours at 1 × 10⁶ times per well. 6 Cells were seeded at a concentration of cells / ml (200 μl per well). To determine NF-κB activation, the supernatant of the culture medium was collected and evaluated with QUANTI-Blue® reagent (InvivoGen) according to the manufacturer's instructions.

[0040] Cytokine analysis Cell supernatant and plasma samples were harvested and stored at -80°C. Human TNFα concentration was detected using the TNF-α DuoSet ELISA kit (R&D, Inc. #DY210) according to the manufacturer's instructions. The signal was expressed using 3,3',5,5'-tetramethylbenzidine, and the absorbance at 450 nm was detected using a microplate reader.

[0041] Flow cytometry analysis Place the cells in PBS containing 1% FBS, 2 × 10 6 The cells were resuspended at a density of cells / ml, and nonspecific binding of Ab was blocked for 10 minutes at room temperature using Human Fc Block (BD Biosciences #564220). AF647 anti-human TREML1 (clone 268420) (R&D #FAB2394R) antibody was used as the detection antibody for TREML1 bound to the cell surface. Dead cells were removed according to Fixable Viability Stain 780 (FVS780) (BD Horizon #565388) staining. Binding analysis was performed on a CytoFLEX Flow Cytometer (Beckman Coulter), and the collected data were analyzed using Kazula software.

[0042] Generation of dendritic cells derived from mouse bone marrow Femur and tibia were harvested from female C57BL / 6 mice, and excess muscle tissue was removed. The bones were disinfected with 70% ethanol, then washed with RPMI-1640 medium, and both ends were cut off. The bone marrow was flushed out with RPMI-1640 medium using a 26G needle syringe. To disperse cell aggregates, the bone marrow suspension was pipetted several times in and out and washed twice with RPMI-1640 medium by centrifugation. The R10 culture medium consisted of RPMI-1640 containing antibiotics, 10% inactive fetal bovine serum, and 2-mercaptoethanol (50 μM, Sigma USA). On day 0, after cell counting, leukocytes were collected in sterile 10 mm diameter Petri dishes at a rate of 2 × 10⁶ cells per 10 ml of R10 medium containing 200 U / ml rmGM-CSF. 6 Cells were seeded at the specified concentration and incubated at 37°C with 5% CO2. On day 3, 10 ml of R10 medium containing another 200 U / ml rmGM-CSF was added. On day 6, half of the supernatant was removed, centrifuged to collect the cell pellet, and resuspended in the first plate containing 10 ml of R10 medium with 200 U / ml GM-CSF. On day 8, non-adherent cells were collected by gently pipetting and centrifuged at 300 g for 5 minutes. To obtain purer dendritic cells, macrophages (F4 / 80) were added. high The population of ) was removed with anti-F4 / 80 MicroBeads UltraPure (130-110-443, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. In short, the cell pellet was resuspended and thoroughly mixed with anti-F4 / 80 MicroBeads UltraPure in MACS buffer. This mixture was incubated in the dark at 4°C for 15 minutes. After washing the cells with MACS buffer, the cells were resuspended in MACS buffer, filtered through a 30 μm nylon mesh, and added to a column washed with MACS buffer, and then F4 / 80 +Cells were separated. Unlabeled cells were collected and washed with R10 medium. To freeze the cells, the cell pellet was counted and resuspended at a concentration of 1×10 7 cells / ml in 2 ml of CELLBANKER2 (#11891, ZENOAQ) per cryopreservation vial and stored at -80 °C.

[0043] BMDCs were thawed for the DC maturation experiment. These cells were seeded at 1×10 6 cells / 2 ml in R10 medium containing 200 U / ml of rmGM-CSF in the wells of a 6-well tissue culture plate and cultured to recover cell viability at 37 °C with 5% CO2. After 6 hours, the BMDCs were treated with various fragments of the mTREML1 protein and cultured at 37 °C with 5% CO2. The next day, the resuspended cells were collected and analyzed by flow cytometry.

[0044] The cells were centrifuged at 1800 rpm for 3 minutes in PBS containing 1% FBS and 0.1% sodium azide (PBSBA), and then split at a concentration of 1×10 5 cells / 100 μl of PBSBA in the flow tube. Before staining the surface markers of the DCs, the cells were treated with mouse Fc blocker to avoid antibody non-specific binding at room temperature for 30 minutes. The cells were incubated with the following antibodies: PE anti-mouse CD11c antibody (BioLegend #117308), Brilliant Violet 421™ anti-mouse I-A / I-E antibody (BioLegend #107631), BV786 rat anti-mouse CD40 (BD #140891), Alexa Fluor® 488 anti-mouse CD86 antibody (BioLegend #105018), or PE / Dazzle™ 594 anti-mouse CD80 antibody (BioLegend #104737) at 4 °C for 30 minutes. After washing with PBSBA, the samples were analyzed with a CytoFlex flow cytometer using CytExpert software.

[0045] Mouse All genetically wild-type experimental BALB / c and C57BL / 6 mice were purchased from National Laboratory Animal Center (Taiwan). All experimental procedures for animal experiments complied with the regulations of the local Institutional Animal Care and Use Committee (IACUC).

[0046] Tumor research To establish a mouse colon cancer model with the same gene, BALB / c mice were subjected to a procedure on the dorsal side, measuring 3 × 10⁶ mice per mouse. 5 CT26 cells were subcutaneously injected. The volume of the inoculated tumor was 30-100 mm². 3 When the mice reached a certain stage, they were randomly divided into cohorts for various treatments. Tumor antigen vaccines were administered subcutaneously according to the indicated time schedule. During each animal study, tumor dimensions were monitored by caliper measurement, and tumor volume was calculated using the equation v = π / 6 (length) × (width), where length is the longest diameter of the tumor and width is the shortest diameter. The tumor load size was 3000 mm. 3 If the threshold was exceeded, the mouse was considered to have died in the survival study and was euthanized using CO2 or cervical dislocation.

[0047] Embodiments of the present invention have been described using a limited number of examples. Those skilled in the art will understand that these examples are for illustrative purposes only, and that other modifications and variations are possible without departing from the scope of the invention. Accordingly, the scope of protection is limited only to the appended claims.

Claims

1. A pharmaceutical composition comprising a compound for use as a vaccine adjuvant or in enhancing an immune response, wherein the compound comprises a TREM-like transcript-1 (TREML1) extracellular domain (ECD) or a TREML1 stalk polypeptide.

2. The pharmaceutical composition according to claim 1, wherein the TREML1 ECD or TREML1 stalk polypeptide is obtained from human or mouse TREML1.

3. The pharmaceutical composition according to claim 1 or 2, wherein the TREML1 ECD or TREML1 stalk polypeptide has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

4. A pharmaceutical composition according to any one of claims 1 to 3, further comprising a carrier.

5. The pharmaceutical composition according to claim 4, further comprising a TLR agonist, wherein the TLR agonist comprises lipopolysaccharide, heat shock protein, fibrinogen, heparan sulfate, hyaluronic acid, CpG oligodeoxynucleotide, 4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol (R848), morphine, or remifentanil.

6. The pharmaceutical composition according to claim 4 or 5, further comprising an antigen, for use as a vaccine.

7. The pharmaceutical composition according to claim 6, wherein the antigen includes a cancer antigen.

8. The aforementioned cancer antigens are found in colorectal cancer, breast cancer, lung cancer, melanoma, hepatocellular carcinoma, head and neck cancer, squamous cell carcinoma of the lung, ovarian cancer, uterine cancer, prostate cancer, stomach cancer, cervical cancer, esophageal cancer, bladder cancer, kidney cancer, brain cancer, bone cancer, pancreatic cancer, skin cancer, malignant melanoma of the skin or eye, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, cancer of the Fallopian tube, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, The pharmaceutical composition according to claim 7, wherein the antigen is a cancer selected from the group consisting of urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic or acute leukemia including chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, medulloblastoma calcifying epithelioma, endometrial cancer, multiple myeloma, and T-cell lymphoma.

9. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the manufacture of a pharmaceutical for enhancing the immune response.

10. Use of the pharmaceutical composition according to any one of claims 6 to 8 in the manufacture of a vaccine.

11. A pharmaceutical composition comprising a compound for inducing cell activation, wherein the compound comprises a TREM-like transcript-1 (TREML1) extracellular domain (ECD) or a TREML1 stalk polypeptide, and the cell activation comprises monocyte activation and / or dendritic cell activation and / or maturation.

12. The pharmaceutical composition according to claim 11, wherein the cell activation includes cell activation induced by intracellular TLRs.

13. The pharmaceutical composition according to claim 11 or 12, wherein inducing cell activation includes the secretion of TNF-α and / or NF-κB.

14. A pharmaceutical composition according to any one of claims 11 to 13, further comprising a carrier.

15. The pharmaceutical composition according to claim 14, further comprising a TLR agonist, wherein the TLR agonist comprises lipopolysaccharide, heat shock protein, fibrinogen, heparan sulfate, hyaluronic acid, CpG oligodeoxynucleotide, 4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol (R848), morphine, or remifentanil.

16. The pharmaceutical composition according to claim 14 or 15, further comprising an antigen, for use as a vaccine.

17. The pharmaceutical composition according to claim 16, wherein the antigen includes a cancer antigen.

18. The aforementioned cancer antigens are found in colorectal cancer, breast cancer, lung cancer, melanoma, hepatocellular carcinoma, head and neck cancer, squamous cell carcinoma of the lung, ovarian cancer, uterine cancer, prostate cancer, stomach cancer, cervical cancer, esophageal cancer, bladder cancer, kidney cancer, brain cancer, bone cancer, pancreatic cancer, skin cancer, malignant melanoma of the skin or eye, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, cancer of the Fallopian tube, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, The pharmaceutical composition according to claim 17, wherein the antigen is a cancer selected from the group consisting of urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic or acute leukemia including chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, medulloblastoma calcifying epithelioma, endometrial cancer, multiple myeloma, and T-cell lymphoma.

19. A pharmaceutical composition for cell activation according to any one of claims 11 to 15, wherein the cell activation includes activation of monocytes and / or activation and / or maturation of dendritic cells.

20. A pharmaceutical composition according to any one of claims 16 to 18 for use as a vaccine.

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