Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: (A) Schematic of the phage display workflow. The extracellular domain of TREM2 was used as the target for selection from a cysteine-constrained cyclic peptide M13 phage library. Four rounds of biopanning, including binding, washing, elution, and amplification, were performed to enrich TREM2-binding clones. (B) Top enriched peptide sequences identified from rounds 2-4. Filled circles indicate detection of a given sequence in the corresponding round, while open circles indicate absence. All sequences conform to a cysteine-constrained cyclic peptide scaffold. (C) Phage ELISA validation of selected peptides. Binding signals are presented as the ratio of signal obtained in TREM2-coated wells relative to control wells lacking protein (E/C). Values above 1 indicate preferential binding to TREM2. Data are shown as mean ± SEM (n=3).

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques: Selection, Binding Assay, Amplification, Clone Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Control

Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: (A) IL-1β secretion in human iPSC-derived microglia following Aβ 1-42 oligomer challenge in the presence of TREM2-6 or TREM2-12 (5, 10, and 25 µM). VG-3927 (5 μM) was used as a positive control. Data are normalized to the Aβ-treated vehicle control and expressed as percent change. (B) Loss of peptide-mediated suppression of IL-1β secretion in TREM2 knockout (KO) microglia confirms TREM2-dependent activity. (C) Modulation of secreted ApoE levels in human microglia following Aβ exposure and peptide treatment (25 µM). ApoE levels are normalized to vehicle-treated controls. Data are presented as mean ± SD (n = 5). Statistical significance was determined by one-way or two-way ANOVA with Dunnett’s post hoc test. ns , not significant; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) relative to vehicle treatment.

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques: Derivative Assay, Positive Control, Control, Knock-Out, Activity Assay

Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: Quantification of PSD95 levels in human iPSC-derived neuron-microglia co-cultures following Aβ 1-42 oligomer exposure and treatment with TREM2-6 or TREM2-12 (5, 10, and 25 µM). VG-3927 (5 μM) was used as a positive control. PSD95 levels are expressed as percent rescue relative to Aβ-treated vehicle controls. Data are presented as mean ± SD (n = 5). Statistical significance was assessed by one-way ANOVA with Dunnett’s post hoc test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) relative to vehicle treatment.

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques: Derivative Assay, Positive Control

Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: (A) Time-dependent root-mean-square deviation (RMSD) of the TREM2 receptor in the free form (cyan line) and in complex with T6 (purple line) and T12 (yellow line) over a 100 ns MD simulation. (B) RMSD of the T6 and T12 over a 100-ns MD simulation.

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques:

Journal: bioRxiv

Article Title: Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

doi: 10.64898/2026.04.22.720287

Figure Lengend Snippet: B) Structural snapshot clustered from the late stage of the MD simulation of T12 (A) and T6 (B) complexed with TREM2. The receptor is shown in grey cartoon, T12 is shown in blue sticks, and T6 is shown in orange sticks. (C and D) Views of the binding interface between T12 (C) / T6 (D) and TREM2 with the key interacting residues on both sides and yellow dashed lines that demonstrate stable hydrogen bonds and salt bridges.

Article Snippet: A 10 μM solution of the His-tagged TREM2 ectodomain (SinoBiological, 11084-H08H) was labeled using the RED-maleimide 2 nd generation dye (NanoTemper, MO-L014) according to the manufacturer’s protocol (labeling buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20), resulting in a 0.90 μM stock of labeled protein (“TREM2-maleimide-dye”) in storage buffer (HBSP: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) with a final degree of labeling (DOL) of 0.596.

Techniques: Binding Assay

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs impact the response of microglial TREM2 and autophagy to Aβ. A Western blot analysis of TREM2-related protein levels in cortex of APP/PS1 mice and the corresponding quantification results (n = 4). B Relative mRNA levels of DAP12 and CD68 in cortex (n = 5). C Western blot analysis of autophagy-related protein levels in cortex and the corresponding quantification results (n = 4). D Relative mRNA levels of BECLIN1 and LAMP1 in cortex (n = 5). Western blot analysis of TREM2-related E and autophagy-related F Protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 4 h in the presence of Aβ and the corresponding quantification results (n = 3). Western blot analysis of TREM2-related G and autophagy-related H protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 12 h in the presence of Aβ and the corresponding quantification results (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs suppress TREM2 activation through binding to TREM2. A Molecular docking results of L-Leucine and KICA binding to TREM2 extracellular domain. B Biolayer interferometry (BLI) analysis of interaction of BCAAs or BCKAs with TREM2. BCAAs and BCKAs was titrated from 15.63 to 500 μM and 31.25 to 500 μM, respectively. C LC–MS/MS spectra of TREM2 immunoprecipitated L-Leucine- 13 C of BV2 cells after BCAAs isotope treatment for 1 h. D Thermal stabilization of TREM2 protein evaluated by cellular thermal shift assay (CETSA) after treating BV2 cells with BCAAs/ BCKAs for 4 h (n = 3). E Representative images of binding of fluorescein labeled-Aβ to cell surface TREM2 in BV2 cells after treatment of BCAAs/ BCKAs for 2 h (n = 3, Scale bars, 40 μm). F Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of S1P for 4 h (n = 3, Scale bars, 40 μm). G Western blot analysis of phosphorylation levels of SYK in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of sphingosine-1-phosphate (S1P) for 4 h (n = 3). Data in D , E , F and G are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Thermal Shift Assay, Labeling, Western Blot, Phospho-proteomics

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs reduce the expression and recycling of microglial TREM2 via inhibiting autophagy. Western blot analysis of TREM2-related A and autophagy-related B protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3). C Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). D Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). Western blot analysis of TREM2-related E and autophagy-related F protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3). G Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3, Scale bars, 40 μm). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test or Student’s t test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Labeling

Journal: Frontiers in Pharmacology

Article Title: Artesunate Therapy Alleviates Fracture-Associated Chronic Pain After Orthopedic Surgery by Suppressing CCL21-Dependent TREM2/DAP12 Inflammatory Signaling in Mice

doi: 10.3389/fphar.2022.894963

Figure Lengend Snippet: Fracture-associated behavioral allodynia and spinal over-expressions of CCL21, TREM2, and DAP12 after orthopedic surgery in mice. The development of mechanical allodynia was assessed by paw withdrawal mechanical threshold (A) and paw withdrawal mechanical frequency to 0.16 g filament (B) in the von Frey test after sham surgery and tibial fracture ( n = 8). The development of cold allodynia was assessed by cold response scoring (C) in acetone test after sham surgery and tibial fracture ( n = 8). The spinal dorsal horn L 4 - 5 segments were collected for biochemical experiments. (D–F) ELISA identified the increased levels of spinal CCL21, TREM2, and DAP12 proteins after tibial fracture ( n = 5). All the data are expressed as mean ± SEM and analyzed by two-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. group sham surgery.

Article Snippet: Recombinant CCL21 (Abcam, ab201361, United Kingdom), a neutralizing antibody against CCL21 (anti-CCL21, R&D Systems, AF457, United States) and a neutralizing antibody against TREM2 (anti-TREM2, R&D Systems, 1729-T2, United States), was dissolved in normal saline or 10% DMSO for i.t.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Artesunate Therapy Alleviates Fracture-Associated Chronic Pain After Orthopedic Surgery by Suppressing CCL21-Dependent TREM2/DAP12 Inflammatory Signaling in Mice

doi: 10.3389/fphar.2022.894963

Figure Lengend Snippet: Intrathecal pre-administration of artesunate reduces fracture-associated spinal over-expressions of CCL21, TREM2, and DAP12 and microglia activation. Intrathecal artesunate (100 μg) was administered daily for three consecutive days on days 4, 5, and 6 after tibial fracture with orthopedic surgery. All biochemical data were collected on day 7 after sham and fracture surgeries. (A–C) ELISA identified that pretreatment with artesunate downregulated the increased levels of spinal CCL21, TREM2, and DAP12 proteins after tibial fracture. (D,E) Immunohistochemistry staining showed representative photomicrographs of the marker of microglia activation (Iba1) in the spinal dorsal horn after fracture intervention and artesunate treatment (scale bar, 50 μm). All biochemical results are expressed as mean ± SEM ( n = 3–5) and analyzed by one-way ANOVA with Bonferroni post hoc comparisons. # p < 0.05 vs. group sham + DMSO, * p < 0.05 vs. group fracture + DMSO.

Article Snippet: Recombinant CCL21 (Abcam, ab201361, United Kingdom), a neutralizing antibody against CCL21 (anti-CCL21, R&D Systems, AF457, United States) and a neutralizing antibody against TREM2 (anti-TREM2, R&D Systems, 1729-T2, United States), was dissolved in normal saline or 10% DMSO for i.t.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Marker

Journal: Frontiers in Pharmacology

Article Title: Artesunate Therapy Alleviates Fracture-Associated Chronic Pain After Orthopedic Surgery by Suppressing CCL21-Dependent TREM2/DAP12 Inflammatory Signaling in Mice

doi: 10.3389/fphar.2022.894963

Figure Lengend Snippet: Induction of acute pain by exogenous CCL21 and attenuation of allodynia by artesunate. Artesunate (i.t., 100 μg, indicated by a red arrow) was injected 60 min prior to recombinant CCL21 (i.t., 0.1 μg) application. (A,B) Exogenous CCL21-induced acute inflammatory pain was alleviated by the pre-application of artesunate. All behavioral results are mean ± SEM ( n = 8) and analyzed by two-way ANOVA with Bonferroni post hoc comparisons. All biochemical data were collected at hour 12 after intrathecal injection. (C) ELISA identified that pretreatment with artesunate downregulated the increased levels of spinal TREM2 and DAP12 proteins after exogenous CCL21 exposure. All biochemical results are expressed as mean ± SEM ( n = 5) and analyzed by one-way ANOVA with Bonferroni post hoc comparisons. # p < 0.05 vs. group DMSO, * p < 0.05 vs. group recombinant CCL21 (0.1 μg) + DMSO.

Article Snippet: Recombinant CCL21 (Abcam, ab201361, United Kingdom), a neutralizing antibody against CCL21 (anti-CCL21, R&D Systems, AF457, United States) and a neutralizing antibody against TREM2 (anti-TREM2, R&D Systems, 1729-T2, United States), was dissolved in normal saline or 10% DMSO for i.t.

Techniques: Injection, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Artesunate Therapy Alleviates Fracture-Associated Chronic Pain After Orthopedic Surgery by Suppressing CCL21-Dependent TREM2/DAP12 Inflammatory Signaling in Mice

doi: 10.3389/fphar.2022.894963

Figure Lengend Snippet: Spinal neutralization of TREM2 reduces fracture-associated chronic pain and CCL21-induced acute pain. (A,B) A neutralizing antibody against TREM2 (anti-TREM2, i.t., 2 μg, indicated by a red arrow) was injected on day 14 after tibial fracture. Behavioral test showed the attenuation of the established fracture-associated mechanical allodynia and cold allodynia by anti-TREM2. (C,D) Anti-TREM2 (i.t., 2 μg, indicated by a red arrow) was injected 60 min prior to recombinant CCL21 (i.t., 0.1 μg). Exogenous CCL21-induced acute inflammatory pain was alleviated by the pre-application of anti-TREM2. All results are mean ± SEM ( n = 8) and analyzed by two-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant CCL21 (Abcam, ab201361, United Kingdom), a neutralizing antibody against CCL21 (anti-CCL21, R&D Systems, AF457, United States) and a neutralizing antibody against TREM2 (anti-TREM2, R&D Systems, 1729-T2, United States), was dissolved in normal saline or 10% DMSO for i.t.

Techniques: Neutralization, Injection, Recombinant

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 1 TREM2 is a potential marker of colorectal cancer (CRC). (A) Kaplan-Meier analysis representing the association of TREM2 expression with overall survival in CRC patients from The Cancer Genome Atlas cohort; ≥ 50% quantile of TREM2 expression is defined as the high-expressing group, and others are considered in the low-expressing group. (B) Immunohistochemistry staining representing TREM2 (brown) expression in CRC tissues. (C) Bar plots summarizing the immunohistochemistry semi-quantitative analysis; score 1, ≤ 25%; 2, 26–50%; 3, 51–75%; 4, > 75%. Scale bar: 100 μm; magnification: 200× (n = 4). (D) Relative TREM2 mRNA levels in CRC patients (n = 4). (E) CRC tissues or normal tissues were double stained with anti-CD206 (marked macrophages) (green) and anti-TREM2 (red) antibodies, and then observed by fluorescent microscopy. DAPI, blue (n = 10). Scale bars: 20 μm. Data are expressed as means ± SD; * P < 0.05, **P < 0.01, by unpaired Student’s t test (C-D). TREM2, triggering-receptor-expressed on myeloid cells 2; CD, cluster of differentiation; DAPI, 4′,6-diamidino-2-phenylindole

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Marker, Expressing, Immunohistochemistry, Staining, Microscopy

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Scheme 1 Schematic illustration of targeted delivery of a PD1-TREM2 scFv by CAR-T cells to enhances anti-tumor efficacy in colorectal cancer. First, the lentivirus encoding CART-CEA.sBsAb was generated using a three-plasmid lentiviral packing system. Mouse T cells were transduced with the encoding CART-CEA.sBsAb, and the successful preparation of autocrine PD-1-TREM2 scFv CAR-T cells (CART-CEA.sBsAb) was achieved. The anti-tumor mechanism of CART-CEA.sBsAb was evaluated in a subcutaneous CRC mouse model. The PD-1-TREM2 scFv secreted by CAR-T cells remained localized within tumors and blocked the PD-1/PD-L1 pathway and the binding of ligands to TREM2 receptors present on MDSCs and TAMs; the percentages of MDSCs and TAMs were decreased, and the levels of TNF-α, IFN-γ, IL-2, granzyme B, and perforin were enhanced. Finally, the intratumoral proportion and cytotoxic ability of CART-CEA.sBsAb increased

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Generated, Plasmid Preparation, Transduction, Binding Assay

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 2 Construction and characterization of the bi-specific scFv antibody (BsAb). (A) Schematic representation of PD-1-scFv, TREM2-scFv, and BsAb. (B) SDS-PAGE analysis of PD-1 scFv, TREM2 scFv, and BsAb derived from the supernatants of CHO cells using nickel column purification and concentration. (C) Flow cytometric histograms demonstrating His-tag detection of PD-1 scFv, TREM2 scFv, and BsAb binding to 293T stably expressing PD-1 (293T-PD-1). The 293T-PD-1 cells were treated with PD-1 scFv, TREM2 scFv, and BsAb at 200 µg/mL and MFI of His was detected by flow cytometry. Bar plots summarizing the data are shown on the right. (D) Flow cytometric histograms demonstrating His-tag detection of PD-1 scFv, TREM2 scFv, and BsAb binding to TREM2 on the RAW 264.7 cells. PD-1 scFv, TREM2 scFv, and BsAb at 200 µg/mL and MFI of His was detected by flow cytometry. Bar plots summarizing the data are shown on the right. (E-F) Relative viability of MC38 cells, mouse primary hepatocytes, and mouse primary intestinal cells treated with PD-1 scFv, TREM2 scFv, and BsAb at 200 µg/mL for 24 h analyzed using the CCK-8 kit. Data are expressed as means ± SD; * P < 0.05, **P < 0.01 (n = 3), by unpaired Student’s t test (C-D). PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; RAW 264.7, Mouse Mononuclear Macrophages Cells; 293T, human embryonic kidneys; CCK, cell counting kit; SD, standard deviation

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: SDS Page, Derivative Assay, Nickel Column, Purification, Concentration Assay, Binding Assay, Stable Transfection, Expressing, Flow Cytometry, CCK-8 Assay, Polyacrylamide Gel Electrophoresis, Cell Counting, Standard Deviation

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 3 The bi-specific scFv antibody (BsAb) delays tumor growth and modulates intratumoral cytokines and the proportions of infiltrating immune cells, MDSCs, and M2 TAMs. (A) Schematic representation of the animal experiment. Six-week-old C57BL/6J mice were subcutaneously injected with 2 × 106 MC38 cells expressing CEA. On day 7, mice received intravenous injection of PD-1 scFv, TREM2 scFv, or the BsAb at 200 µg/mouse daily; the PBS-treated group served as the control. (B) Bioluminescence images of mice treated with PD-1 scFv, TREM2 scFv, and BsAb at days 7, 14, and 21 (n = 4). (C) Biolumi nescence signals of individual groups at day 7, 14, and 21 are shown on the Y-axis. The PBS group is indicated as black, the PD-1 scFv group is in orange, the TREM2 scFv group is in blue, and the BsAb group is in red. (D-E) Tumor volume and body weight of mice subjected to the indicated treatments (n = 4). (F-H) MC38-CEA tumors excised from individual groups of sacrificed mice on day 23. Tumor volume and weight of mice subjected to the indicated treat ments (n = 4). (I) Effect of different treatments on the survival of tumor-bearing mice. (J-L) Effect of different treatment modalities on the infiltration of CD8-positive T cells (J), MDSCs (K), and M2 TAMs (L). Proportions of tumor-infiltrating CD8-positive T cells, MDSCs, and M2 TAMs were detected by flow cytometry on day 23. All cells gated on CD45, CD45 + CD11b Gr1 + were marked as MDSCs, whereas cells gated at CD45 + CD11b F4/80 CD206 were marked as M2 TAMs. Representative plots are shown on the right (n = 4). Data are expressed as means ± SD; * P < 0.05; **P < 0.01, by unpaired Student’s t test and one-way ANOVA. scFv, single-chain fragment variable; CEA, carcinoembryonic antigen; PD-1, programmed death-1; TREM2, triggering-receptor- expressed on myeloid cells 2; PBS, phosphate buffered saline; MDSCs, myeloid-derived suppressor cells; TAMs, tumor associated macrophages; CD, cluster of differentiation

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Injection, Expressing, Control, Flow Cytometry, Saline, Derivative Assay

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 4 Construction and characterization of bi-specific scFv antibody (BsAb)- secreting CAR-T cells. (A) Schematic representation of Mock, PD-1 scFv, TREM2 scFv, and BsAb-secreting CAR constructs targeting CEA. CART-CEA was constructed as a control. (B) Efficient transduction of all constructs in mouse primary T cells as observed by fluorescence microscopy. Scale bar: 50 μm. (C, D) Flow cytometric assay analysis (C) and bar plot summary (D) indicating the transduction efficiency of all constructs in mouse primary T cells. (E) Western blot detecting the His-tag for CAR protein in mouse primary T cells. (F) Cytotoxicity of all constructed CAR-T cells against MC38 cells. All groups of CAR-T cells were co-cultured with MC38 cells at 1:1, 4:1, 8:1, and 16:1, ratios. MC38 cell lysis was detected by an LDH cytotoxicity kit. Data are expressed as means ± SD; * P < 0.05; **P < 0.01, by one-way ANOVA. CAR-T, chimeric antigen receptor-modified-T; PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; CEA, carcinoembryonic antigen; LDH, lactate dehydrogenase

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Construct, Control, Transduction, Fluorescence, Microscopy, Flow Cytometry, Western Blot, Cell Culture, Lysis, Modification

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 5 Anti-tumor effects of armed CAR-T cells in tumor-bearing mice. (A) Time-course diagram of the animal experiment. Six-week-old C57BL/6J mice were subcutaneously injected with 2 × 106 MC38 cells expressing CEA. On day 7, mice from all groups were individually injected with 1 × 107 constructed CAR-T cells through the tail vein. Tumor imaging was performed once a week. (B) Bioluminescence images of mice treated with CART-CEA, CART-CEA. sPD-1scFv, CART-CEA.sTREM2 scFv, and CART-CEA.sBsAb at days 7, 14, and 21; PBS treatment served as the control (n = 4). (C) Bioluminescence signals of mice in each group at days 7, 14, and 21 are shown on the Y-axis. The PBS group is shown in black, the CART-CEA group in purple, the CART-CEA.sPD-1 scFv group in orange, the CART-CEA.sTREM2 scFv group in blue, and the CART-CEA.sBsAb group in red. (D) Comparison of tumor volume over time in each group of tumor-bearing mice. (E) Weight development of mice during treatment in each group. (F) Effect of different treatments on the survival of tumor-bearing mice. Data are expressed as means ± SD; * P < 0.05; **P < 0.01, by unpaired Student’s t test. CAR-T, chimeric antigen receptor-modified-T; CEA, carcinoembryonic antigen; PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; PBS, phosphate-buffered saline

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Injection, Expressing, Construct, Imaging, Control, Comparison, Modification, Saline