A method of treating cancer using lymphotoxin beta receptor agonists.

Abstract

This disclosure relates, for example, to a method of treating cancer by administering a lymphotoxin beta receptor (LTBR) agonist in combination with adoptive cell therapy (ACT). The combination of LTBR agonists and ACT exhibits unexpected synergistic antitumor efficacy in inducing potent and sustained tumor control in subjects with cancer.

Description

[Technical Field]

[0001] Sequence List The sequence listing for this application is submitted electronically as an XML file in ST.26 format, with the filename "SeqList-11483", created on February 27, 2024, and measuring 2,682 bytes. This submitted sequence listing is part of this specification and is incorporated herein by reference in its entirety.

[0002] This disclosure relates, in general terms, to a method for treating cancer using, for example, a lymphotoxin beta receptor agonist in combination with adoptive cell therapy. [Background technology]

[0003] Manipulating the immune system has become a crucial tool in the fight against cancer. Immune cells migrate through secondary lymphoid tissues such as lymph nodes, where they are exposed to chemokines and cytokines. Lymphocytes such as B cells and T cells enter the lymph nodes through specialized blood vessels called high endothelial venules (HEVs), which are located in the region between the B cell zone and the T cell zone. The T cell zone is CD4 + and CD8 + This includes T cells and a subset of dendritic cells (DCs). See Non-Patent Literature 1. Tertiary lymphoid structures (TLSs) are ectopic lymphoid formations that occur within inflammatory, infectious, or tumor tissue. TLSs contain HEVs and B-cell follicles surrounded by T-cell zones and are characterized by high chemokine expression. The presence of TLSs and HEVs in solid tumors is positively correlated with patient survival in many cancer types and may predict a good response to immune checkpoint blockade. See Non-Patent Literature 2.

[0004] Lymphotoxin beta receptors (LTBRs) play a central role in the development and homeostasis of lymph nodes and secondary lymphoid organs by regulating the expression of several homeostatic lymphocyte cytokines (e.g., CCL19, CCL21, CXCL13) and adhesion molecules (ICAM-1, VCAM-1, MADCAM1) via the NF-Kappa B pathway. See Non-Patent Literature 3. LTBRs are activated by two different trimer ligands, LIGHT and lymphotoxin alpha-1 beta-2 (LTa1b2 or LTα1β2). Activation of LTBRs by their ligands leads to ectopic formation of tertiary lymphoid structures (TLSs). See Non-Patent Literature 4. The presence of TLSs in the tumor microenvironment typically correlates with immune infiltration and is associated with a favorable prognosis; therefore, treatment with LTBR agonists can induce an antitumor immune response and improve current cancer immunotherapy. See Non-Patent Literature 5.

[0005] Adoptive cell therapy (ACT) uses the subject's own immune cells (or donor immune cells) to treat diseases such as cancer. Generally, ACT involves the transfer of genetically modified T lymphocytes into the subject. Some examples of ACT include the use of engineered chimeric antigen receptors (CARs) or T cell receptors (TCRs). Generally, CARs contain a variable region of an antibody fragment or a tumor-associated antigen (TAA)-specific binding domain, bound to the cytoplasmic domain of a T cell signaling molecule via a hinge and transmembrane domain. The most common lymphocyte activation portion includes a T cell costimulatory domain in series with a T cell effector function-inducing portion. CAR-mediated ACT allows CAR-grafted T cells to directly recognize and attack TAAs on target tumor cells.

[0006] ACT using TCRs involves manipulating T cells to express a specific TCR, which is a heterodimer having two subunits. Each subunit contains a constant region that anchors a receptor to the cell membrane and a hypervariable region that performs antigen recognition. TCRs can recognize tumor-specific proteins both inside and outside the cell. In TCR therapy, T cells are collected from the blood of a subject or donor, then genetically modified to express a newly engineered TCR, and then administered to the subject to target the subject's cancer. TCRs have been reported to mediate cell death, increase B cell proliferation, and limit cancer development and severity.

[0007] Due to the inherent complexity of live cell cultures and some patient-to-patient variability, ACT agents tended to have limited success due to their fluctuating clinical activity. Therefore, there is a need to improve the antitumor activity of ACT. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Mueller et al., Nat. Rev. Immunol., 9:618~629 (2009) [Non-Patent Document 2] Sautes-Fridman et al., Nat. Rev. Cancer., 19:307~325 (2019) [Non-Patent Document 3] Schneider et al., Immunol. Rev., 202:49~66 (2004) [Non-Patent Document 4] Schrama et al., Immunity 14:111-121 (2001) [Non-Patent Document 5] Tang et al., Cell. Mol. Immunol. 14:809~18 (2017) [Overview of the project] [Problems that the invention aims to solve]

[0009] The disclosed technology addresses one or more of the aforementioned needs. The applicant has shown herein that LTBR agonism yields the remarkable effect of increasing the antitumor efficacy of administered adoptive cell therapy. Accordingly, in one embodiment, the disclosed technology is a method for treating cancer, comprising administering a therapeutically effective dose of a lymphotoxin beta receptor (LTBR) agonist in combination with a therapeutically effective dose of adoptive cell therapy (ACT) to a subject in need, wherein the combination administration results in an increase in the efficacy and duration of the antitumor response compared to a subject treated with ACT as monotherapy. In another embodiment, the disclosed technology is a method for increasing the efficacy of adoptive cell therapy (ACT), comprising (a) selecting a subject having cancer; and (b) administering a therapeutically effective dose of ACT in combination with a therapeutically effective dose of a lymphotoxin beta receptor (LTBR) agonist to the subject, wherein the combination administration results in an increase in the efficacy and duration of the antitumor response compared to a subject treated with ACT as monotherapy. [Means for solving the problem]

[0010] In some embodiments of the disclosed method, the LTBR agonist is an antibody. In some embodiments of the disclosed method, the ACT comprises immune cells selected from T cells, tumor-infiltrating lymphocytes, and natural killer (NK) cells. In some embodiments, the immune cells comprise a modified T cell receptor (TCR) for tumor-associated antigens (TAAs), or a chimeric antigen receptor (CAR) for TAAs. In some embodiments, TAA is AFP, ALK, BAGE protein, BCMA, BIRC5 (Survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE Proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, Mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PL The following are selected: AC1, PRLR, PRAME, PSMA(FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0011] In some embodiments, the disclosed method further includes administering an additional therapeutic agent or treatment. In some embodiments, the additional therapeutic agent or treatment is selected from radiotherapy, surgery, checkpoint inhibitors, chemotherapeutic agents, cancer vaccines, vascular endothelial growth factor (VEGF) antagonists, angiopoietin-2 (Ang2) inhibitors, transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens, Bacillus Calmette-Guerin vaccine, granulocyte-macrophage colony-stimulating factor (GM-CSF), cytotoxins, interleukin-6 receptor (IL-6R) inhibitors, interleukin-4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-12, IL-21, IL-15, antibody-drug conjugates, anti-inflammatory drugs, and combinations thereof. In some embodiments, the checkpoint inhibitor is selected from inhibitors of PD-1, PDL-1, PDL-2, LAG-3, CTLA-4, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD80, CD86, CD160, CD276, GAL9, HAVCR2, IDO1, IDO2, KIR, LAIR1, macrophage receptors with collagen structures (MARCO), phosphatidylserine (PS), TIGHT, VISTA, and VTCN1. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PDL-1, PDL-2, LAG-3, or CTLA-4.

[0012] In some embodiments of the disclosed method, the cancer is selected from adrenal cancer, anal cancer, autonomic ganglion cancer, biliary tract cancer, bladder cancer, hematological cancer, bone cancer, brain cancer, breast cancer, meningeal cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, eye cancer, esophageal cancer, fallopian tube cancer, stomach cancer, genital cancer, head and neck cancer, kidney cancer, colorectal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, peritoneal cancer, pituitary cancer, placental cancer, pleural cancer, prostate cancer, rectal cancer, kidney cancer, salivary gland cancer, skin cancer, small intestine cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, upper respiratory tract and gastrointestinal cancer, urinary tract cancer, uterine cancer, vaginal cancer, and vulvar cancer. In some embodiments, the cancer expresses CXCL13, CCL19, CCL21, or LT-alpha (LTα, LTa). In some embodiments, cancer expresses LTα.

[0013] In some embodiments of the disclosed method, the administration of the combination produces one or more therapeutic effects selected from increased tumor-specific HEV formation, increased dendritic cell and T cell infiltration, enhanced T cell activation in the tumor microenvironment, increased expression of TLS-related chemokines, delayed tumor growth, reduced tumor cell count, tumor regression, extended survival, partial response, and complete response. In some embodiments of the disclosed method, the therapeutic effective dose of the LTBR agonist includes 0.005 mg / kg to 10 mg / kg of the subject's body weight. In some embodiments of the disclosed method, the therapeutic effective dose of ACT is 1 × 10⁻⁶ 6 Contains one or more immune cells.

[0014] In some embodiments of the disclosed method, the LTBR agonist and / or ACT are administered to the subject in one or more doses. In some embodiments of the disclosed method, the LTBR agonist and / or ACT are administered intravascularly, subcutaneously, intraperitoneally, or intratumorally. In some embodiments of the disclosed method, the LTBR agonist is administered before or after administration of ACT. In some embodiments of the disclosed method, the LTBR agonist is administered concurrently with administration of ACT. In some embodiments of the disclosed method, the LTBR agonist and ACT are provided in separate compositions. In some embodiments of the disclosed method, the LTBR agonist and ACT are provided in a single composition.

[0015] In one embodiment, the present disclosure provides a method for treating cancer, comprising administering a therapeutically effective dose of a lymphotoxin beta receptor (LTBR) agonist in combination with a therapeutically effective dose of a CTLA4 inhibitor to a subject in need, wherein the combination administration results in an increased efficacy and duration of antitumor response compared to a subject treated with the CTLA4 inhibitor as monotherapy. In some embodiments, the CTLA4 inhibitor is an antibody or its antigen-binding fragment that specifically binds to CTLA4. Examples of anti-CTLA4 antibodies used include, but are not limited to, ipilimumab and REGN4659. As disclosed herein, the combination of an anti-CTLA-4 antibody and an LTBR agonist and / or LTα expression delayed tumor progression and promoted complete tumor regression.

[0016] In another aspect, the disclosed technique is a method for increasing tumor-specific HEV formation in subjects requiring it, comprising (a) selecting subjects having cancer; and (b) administering a therapeutically effective dose of ACT in combination with a therapeutically effective dose of a lymphotoxin beta receptor (LTBR) agonist to the subjects, wherein the combination administration results in increased tumor-specific HEV formation, as well as increased efficacy and duration of the antitumor response, compared to subjects treated with ACT as monotherapy.

[0017] In another aspect, the disclosed technique is a method for increasing the expression of TLS-related chemokines in subjects requiring it, comprising (a) selecting subjects having cancer; and (b) administering a therapeutically effective dose of ACT in combination with a therapeutically effective dose of a lymphotoxin beta receptor (LTBR) agonist to the subjects, wherein the combination administration results in increased expression of TLS-related chemokines, as well as increased efficacy and duration of the antitumor response, compared to subjects treated with ACT as monotherapy.

[0018] Other embodiments of this disclosure will become apparent from the following detailed description. [Brief explanation of the drawing]

[0019] [Figure 1-1] As described in Example 1, this is a series of graphs showing the expression analysis of multiple TLS-related chemokines in MC38 tumors. [Figure 1-2] Same as above. [Figure 2-1] Figures 2A-2C illustrate the effects of LTBR agonism on DC and T cell infiltration and activation, as described in Example 2. Figure 2A is a series of graphs showing the quantification of DC and T cell infiltration and activation in response to LTBR agonism in vivo. Figure 2B is a graph showing the results of the bone marrow-derived DC (BMDC) assay. Figure 2C is a series of graphs showing the results of the antigen presentation assay using pre-treated BMDC and OT-I T cells. The bar graphs show the mean ± SEM, and each point represents one biological copy. All experiments were performed in triplicates. Unpaired Student's t-tests (two-sided) were used to compare the two groups. A p-value < 0.05 was considered significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). All statistical analyses were performed using GraphPad Prism software. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 3-1] Figures 3A-3C illustrate the increased antitumor efficacy of T cell-based therapy when combined with an LTBR agonist, as described in Example 3. Figure 3A is a graph showing CD8 T cell depletion in the Colon26 tumor model. Figure 3B is a series of graphs showing the antitumor effect in the Colon26 tumor model. Figure 3C is a series of graphs showing immune cell infiltration in the Colon26 tumor model. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 4] These are schematic diagrams and graphs illustrating the antitumor efficacy of LTBR agonism combined with mouse anti-hCD20 CAR T cell therapy in the MC38-hCD20 tumor model described in Example 4. [Figure 5] This is a series of graphs showing the enhanced tumor response to anti-CTLA-4 treatment in TLS+ Colon26 tumors, as described in Example 6. [Figure 6-1] This is a series of graphs showing the enhanced tumor response to anti-CTLA-4 treatment in the MC38-OVA tumor model described in Example 6. [Figure 6-2] Same as above. [Figure 7] This figure shows an exemplary model of multifaceted immunomodulation of TME by LTBR agonism. [Figure 8] Figures 8A–8B are a series of graphs showing the characterization of TA-HEC and TA-EC isolated from Colon26 tumors 10 days after treatment with LTBR agonist or isotype control antibody (n=6) by flow cytometry. Figure 8A shows the frequency of TA-HEC and MFI in PNAd. Figure 8B shows the endothelial inflammation markers E-selectin, P-selectin, ICAM-1, and VCAM-1. [Figure 9]Figures 9A-9B are a series of graphs showing the expression of the HEV marker MAdCAM-1, as well as the endothelial inflammation markers ICAM-1, VCAM-1, and P-selectin (n=3), quantified by flow cytometry. Endothelial cells were activated in vitro for 18 hours by treatment with 1 μg / ml LTBR agonist or isotype control antibody and / or 10 ng / ml TNFα, 10 ng / ml IL-1β, or 10 μg / ml LPS. Mean ± SEM values ​​are shown. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. Figure 9A shows SVEC4-10 cells. Figure 9B shows b.End3 cells. [Figure 10-1] Figure 10A is a heatmap showing RT-qPCR analysis of TLS-related chemokine expression in tumor lysates of MC38 tumors treated with LTBR agonists (n=9) or isotype control antibodies (n=7) for 7 days. Logarithmically transformed Z-scores of chemokine expression are plotted. Each column represents an individual tumor. Figure 10B is a series of graphs showing the quantification of tumor-infiltrating CD45+ immune cells, T cells, and DCs in MC38 tumors (n=5) treated for 7 days by flow cytometry. Figure 10C is a series of graphs showing RT-qPCR analysis of TLS-related chemokine expression in tumor lysates of MC38 tumors treated with LTBR agonists (n=12) or isotype control antibodies (n=11) for 10 days. Figure 10D is a series of graphs showing the quantification of tumor-infiltrating CD45+ immune cells, B, T, DC, and NK cells in Colon 26 tumors (n=15) treated for 10 days, as measured by flow cytometry. Figures 10B-10D show mean ± SEM values. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 10-2] Same as above. [Figure 11]Figures 11A and 11B are a series of graphs (n=15) showing the characterization of tumor-infiltrating DCs and T cells in Colon 26 tumors by flow cytometry after 10 days of treatment with LTBR agonists or isotype-controlled antibodies. Mean ± SEM values ​​are shown. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. Figure 11A shows the frequencies of CD40+DCs, CD83+DCs, and CD40+CD83+DCs. Figure 11B shows the frequencies of Ki67+, TCF1+PD1+, and SLAMF6-Tim3+ cells in CD4+ or CD8+ T cells. Figure 11C is a graph showing the quantitative analysis of MHCI, MHCII, CD80, CD86, CD40, CD83, and CD8 expression by flow cytometry in MutuDC1940 cells treated with PBS or 10 μg / ml LPS for 2 hours, followed by treatment with 10 μg / ml LTBR agonist or isotype control antibody for 18 hours (n=5). Mean ± SEM values ​​are shown. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 12-1]Figures 12A-12C are schematic diagrams and graphs (n=5) showing the analysis of BMDC activation and licensing by LTBR agonist treatment in vitro. Figure 12A is a schematic diagram of BMDC differentiation, treatment with OT-I CD8+ T cells and co-culture with them, and subsequent quantification by flow cytometry. Figure 12B is a graph showing the percentage of MHCII+CD11c+BMDC cells in in vitro culture. Mean ± SEM values ​​are shown. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. Figure 12C shows the frequency of CD83+BMDC cells. Mean ± SEM values ​​are shown. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. Figures 12D-E are a series of graphs (n=5) showing the characterization of tumor-infiltrating DCs and T cells in MC38 tumors after 10 days of treatment with LTBR agonists or isotype-controlled antibodies, as determined by flow cytometry. Mean ± SEM values ​​are shown. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. Figure 12D shows the MFI of MHCII and CD40 in DCs. Figure 12E shows the frequencies of Ki67+CD4+ T cells, IFNg+CD4 T cells, Ki67+CD8+ T cells, and IFNg+CD8+ T cells. [Figure 12-2] Same as above. [Figure 13-1]Figure 13A is a pair of graphs showing the cell viability of Colon26 and MC38-OVA cells treated in vitro for 24 hours with LTBR agonist antibodies at 0, 10, 100, and 1000 ng / mL (n=3). Figure 13B is a graph showing the change in Colon26 tumor size of individual mice from day 0 to day 7 of LTBR agonist or isotype control antibody treatment in SRG mice (n=4-5); D0 is the start day of treatment. Figure 13C is a graph showing the change in Colon26 tumor size of individual mice from day 0 to day 13 of LTBR agonist treatment with and without CD8 T cell depletion by anti-CD8α antibody (n=8). Figure 13D is a graph showing the change in Colon26 tumor size of individual mice from day 0 to day 11 of LTBR agonist treatment and / or PNAd blockade by MECA-79 antibody (n=8). Figures 13E–13F are a series of graphs showing the quantification of B and T cells in tumors, peripheral blood, and lymph nodes of Colon26 tumor-bearing mice treated with LTBR agonists and / or MECA-79 antibody, as measured by flow cytometry (n=7). Figure 13E shows the percentage of CD45+ immune cells and CD19+ B cells in the tumor, as well as the frequency of CD19+ B cells among CD45+ cells in the tumor, blood, and lymph nodes. Figure 13F shows the percentage of CD4+ and CD8+ T cells in the tumor, as well as the frequency of central memory (CM) cells (CD62L+CD44+) among the CD4+ and CD8+ T cell subsets in the tumor, blood, and lymph nodes. Figures 13A, 13E–13F: Mean ± SEM. Figures 13A-13F: *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above. [Figure 14]Figure 14A is a graph showing the change in Colon26 tumor size in individual mice from day 0 to day 10 after treatment with an LTBR agonist and / or anti-PD-1 antibody (n=14). Data are pooled from two independent experiments. Response rate (RR) is defined as a reduction of more than 30% in tumor size. Figure 14B is a series of graphs showing the quantification of tumor-infiltrating CD45+ immune cells, CD4+ T cells, NK cells, and CD103+ CD11b-1 type DCs in Colon26 tumors treated with an LTBR agonist and / or anti-PD-1 antibody by flow cytometry (n=5). Mean ± SEM values ​​are shown. Figures 14A~14B: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 15] Figure 15A is a graph showing the change in MC3S-hCD20 tumor size in individual mice from day 0 to day 16 of LTBR agonist and / or hCD20 CAR T treatment (n=9). D0 is the start day of treatment. Figure 15B is a pair of graphs showing the immunohistochemical analysis of the number of CD4+ and CD8+ T cells in tumors collected at the end of the study (n=4-5). Figures 15A-15B: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 16] These are a pair of graphs showing the number and area of ​​B cell aggregates in chemokine-expressing Colon 26 tumors after 10 days of treatment with LTBR agonists or isotype control antibodies, quantified by immunohistochemistry (n=10-12). [Figure 17] A pair of graphs showing the identification of B cell aggregates and TA-TLSs in LTα-expressing Colon26 tumors after 10 days of immunofluorescence staining of B cells (B220+), T cells (CD3+), and HEV (PNAd+) treated with isotype control or LTBR agonist antibody. The number and area of ​​intratumoral B cell aggregates or TLSs (from two independent experiments, n=4 in the isotype control group and n=12 in the LTBR agonist-treated group) are plotted. Mean ± SEM is shown. [Figure 18]This is a series of graphs showing individual tumor growth curves for Colon26 tumors expressing EV or LTα after four doses (indicated by arrows) of isotype-controlled or LTBR agonist antibody treatment. [Figure 19A] This is a UMAP plot of all cells isolated from EV control or LTα-expressing Colon26 tumors after 7 days of LTBR agonist or isotype-controlled antibody treatment, color-coded by unsupervised clustering. [Figure 19B] This is a UMAP plot of all cells isolated from EV control or LTα-expressing Colon26 tumors after 7 days of LTBR agonist or isotype-controlled antibody treatment, color-coded by LTBR expression. [Figure 19C] This is a UMAP plot of ECs from control or LTα-expressing Colon26 tumors after 7 days of treatment with an LTBR agonist or isotype control antibody, color-coded by unsupervised clustering. [Figure 19D] This chart shows the proportion of EC clusters in different treatment groups. [Figure 19E] This is a UMAP plot of stromal cells, color-coded using unsupervised clustering. [Figure 19F] This chart shows the proportion of stromal cell clusters in different treatment groups. [Figure 20A] Figure 20A is a chart showing the change in tumor size in Colon26 tumors expressing either an empty vector (EV) control or LTα in individual mice from day 0 to day 17 after treatment with an LTBR agonist and / or anti-CTLA-4 antibody (n=7-8). The response rate (RR) is defined as a reduction of more than 30% in tumor size. [Figure 20B] Figure 20B is a chart showing the change in tumor size of control MC38-OVA tumors in individual mice treated with an LTBR agonist and / or anti-CTLA-4 antibody (n=6-11). [Figure 20C]Figure 20C is a chart showing the change in tumor size of LTα-expressing MC38-OVA tumors in individual mice treated with an LTBR agonist and / or anti-CTLA-4 antibody (n=13). RR, the response rate, is defined as a reduction of more than 30% in tumor size. TF: percentage of tumor-free mice. Figures 20A-20C: Day 0 is the start date of anti-CTLA-4 treatment. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 21] Figures 21A-21C are schematic diagrams and graphs showing the analysis of OT-I CD8+ T cell activation in MC38-OVA tumor-bearing mice treated with FTY720. Figure 22A is a schematic diagram of the administration of the LTBR agonist, FTY720, and adoptive transfer of OT-I CD8+ T cells; tumors were collected 12 days after LTBR agonist treatment for flow cytometry analysis. Figure 22B is a graph showing the frequency of proliferating cells among OT-I CD8+ T cells in MC38-OVA tumors. Figure 22C is a graph showing the frequency of CD69+ proliferating cells among OT-I CD8+ T cells in MC38-OVA tumors. Mean ± SEM values ​​are shown (n=5-7). Statistical significance was evaluated by one-way ANOVA using Tukey's multiple comparison test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 22-1]Figures 22A-22H are a series of graphs showing the characterization of T cells in MC38-OVA tumor-bearing mice treated with FTY720 and adopted by OT-I CD8+ T cell transfer. Figure 22A is a graph showing the quantification of CD8+ T cells in the blood by flow cytometry. Figure 22B is a graph showing the quantification of OT-I CD8+ T cells in the blood by flow cytometry. Figure 22C is a graph showing the quantification of proliferating OT-I CD8+ T cells in the blood by flow cytometry. Figure 22D is a graph showing the quantification of CD8+ T cells in tumor-perfusing lymph nodes by flow cytometry. Figure 22E is a graph showing the quantification of OT-I CD8+ T cells in tumor-perfusing lymph nodes by flow cytometry. Figure 22F is a graph showing the quantification of proliferating OT-I CD8+ T cells in tumor-perfusing lymph nodes by flow cytometry. Figure 22G is a graph showing the quantification of OT-I CD8+ T cells in MC38-OVA tumors by flow cytometry. Figure 22H is a graph showing the quantitative analysis of proliferating OT-I CD8+ T cells in MC38-OVA tumors by flow cytometry. Mean ± SEM is shown (n=5~7). Statistical significance was evaluated by one-way ANOVA using Tukey's multiple comparison test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 22-2] Same as above. [Figure 22-3] Same as above. [Figure 23-1]Figures 23A-23F are a series of graphs showing the characterization of T cells (DCs) in MC38-OVA tumor-bearing mice treated with FTY720 and adoptively transferred OT-I CD8+ T cells. Figure 23A is a graph showing the quantification of DCs in MC38-OVA tumors by flow cytometry. Figure 23B is a graph showing the quantification of CD40+ DCs in MC38-OVA tumors by flow cytometry. Figure 23C is a graph showing the quantification of the percentage of CD40+ cells in DCs in MC38-OVA tumors by flow cytometry. Figure 23D is a graph showing the quantification of DCs in tumor-perfusing lymph nodes by flow cytometry. Figure 23E is a graph showing the quantification of CD40+ DCs in tumor-perfusing lymph nodes by flow cytometry. Figure 23F is a graph showing the quantification of the percentage of CD40+ cells in DCs in tumor-perfusing lymph nodes by flow cytometry. Mean ± SEM is shown (n=5-7). Statistical significance was assessed by one-way ANOVA using Tukey's multiple comparison test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 23-2] Same as above. [Figure 24] Figure 24A is a graph showing the frequency of proliferating OT-I CD8+ T cells in tumor-perfusing lymph nodes. Figure 24B is a graph showing the frequency of CD69+ proliferating OT-I CD8+ T cells in tumor-perfusing lymph nodes. Mean ± SEM is shown (n=5~7). Statistical significance was evaluated by one-way ANOVA with Tukey's multiple comparison test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 25]This graph shows the quantitative counts of CD45+ immune cells, CD19+ B cells, and CD3+, CD4+, CD8+ T cells in the peripheral blood of Colon26 tumor-bearing mice treated with anti-CD8α or isotype control antibodies immediately prior to LTBR agonist treatment, as measured by flow cytometry. Mean ± SEM is shown (n=16). Statistical significance was assessed by two-way ANOVA using Sidak's multiple comparison test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 26] Figures 26A and 26B are a series of graphs showing the combination of LTBR agonism and intratumoral chemokine expression. Figure 26A is a graph showing the day 3 growth rate versus day 1 growth rate of engineered Colon26 cell lines transduced with an empty vector (EV), Cxcl13, or Ccl19 in vitro (n=6). Statistical significance was assessed by one-way ANOVA with Dunnett's multiple comparison test. Figure 26B is a graph showing the day 3 growth rate versus day 1 growth rate of engineered Colon26 cell lines transduced with an empty vector (EV) or LTα in vitro (n=8). Mean ± SEM is shown. Statistical significance was assessed by unpaired two-sided Student's t-test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Figure 27-1]Figures 27A–E are a series of graphs showing ICB treatment in a colorectal tumor model with TA-TLS induced by a combination of LTBR agonism and intratumoral LTα expression. Figure 27A shows the tumor volume at the start of anti-PD-1 treatment for LTα-expressing Colon26 tumors treated with an LTBR agonist and anti-PD-1. Figure 27B shows the change in tumor size in individual mice from day 0 to day 19 of anti-PD-1 treatment for LTα-expressing Colon26 tumors treated with an LTBR agonist and anti-PD-1. TF, tumor-free mouse. Figure 27C shows the tumor volume at the start of anti-CTLA-4 treatment for empty vector (EV) or LTα-expressing Colon26 tumors treated with an LTBR agonist and anti-CTLA-4. Figure 27D shows the tumor volume at the start of anti-CTLA-4 treatment for MC38-OVA tumors treated with an LTBR agonist and anti-CTLA-4. Figure 27E shows the tumor volume at the start of anti-CTLA-4 treatment for LTα-expressing MC38-OVA tumors treated with LTBR agonists and anti-CTLA-4. ISO, isotype control. Mean ± SEM shown; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 27-2] Same as above. [Figure 27-3] Same as above. [Modes for carrying out the invention]

[0020] This disclosure relates, for example, to a method of treating cancer by administering a lymphotoxin beta receptor (LTBR) agonist in combination with adoptive cell therapy (ACT). As disclosed herein, the combination of an LTBR agonist and ACT demonstrates unexpected synergistic antitumor efficacy in inducing potent and sustained tumor control in subjects with cancer.

[0021] Methods for treating cancer This disclosure includes a method for treating cancer, comprising administering a therapeutically effective dose of an LTBR agonist in combination with a therapeutically effective dose of ACT to a subject in need thereof. This disclosure also includes a method for selecting a subject having cancer and a method for increasing the efficacy of ACT when treating cancer by administering a therapeutically effective dose of an LTBR agonist in combination with a therapeutically effective dose of ACT to the subject.

[0022] The applicant hereby demonstrates that LTBR agonism promotes HEV formation and upregulates the expression of CXCL13, CCL19, and CCL21 chemokines, thereby increasing T cell, B cell, and DC tumor infiltration. LTBR agonism was also found to enhance DC-mediated T cell activation through a direct effect on DC activation and maturation. Activated T and B cells expressing LTBR ligands can further amplify this process to achieve sustained antitumor immunity and potential TLS formation. Figure 7 shows a model of the multifaceted immunomodulation of TME by LTBR agonism based on the results herein. LTBR agonism resulted in increased T cell and DC infiltration and activation in solid tumors, thereby enhancing the antitumor effect of ACT.

[0023] As used herein, terms such as “to treat” and “to treat” mean to alleviate symptoms, to temporarily or permanently eliminate the cause of symptoms, to delay or inhibit tumor growth, to reduce tumor cell load or tumor volume, to promote tumor regression, to cause tumor shrinkage, necrosis, and / or disappearance, to prevent tumor recurrence, to prevent or inhibit metastasis, to inhibit the growth of metastatic tumors, and / or to extend the survival of the subject.

[0024] As used herein, the expression “subjects in need of it” refers to human or non-human mammals exhibiting one or more symptoms or signs of cancer and / or diagnosed with cancer and in need of treatment. The term “subject” includes subjects having primary or metastatic tumors (advanced malignancies). In certain embodiments, the expression “subjects in need of it” includes subjects having tumors that are resistant to or refractory to conventional treatments (e.g., chemotherapy) or are poorly controlled by conventional treatments (e.g., chemotherapy). This expression also includes subjects having tumors for which conventional chemotherapy is not recommended, for example, due to toxic side effects. For example, this expression also includes subjects who have received one or more cycles of chemotherapy and experienced toxic side effects.

[0025] As used herein, the terms “tumor” or “cancer” refer to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system.

[0026] In some embodiments, the disclosed methods are used to treat or inhibit the growth of tumors, including but not limited to adrenal cancer, anal cancer, autonomic ganglion cancer, biliary tract cancer, bladder cancer, hematological cancer, bone cancer, brain cancer, breast cancer, meningeal cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, eye cancer, esophageal cancer, fallopian tube cancer, stomach cancer, genital cancer, head and neck cancer, kidney cancer, colorectal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, peritoneal cancer, pituitary cancer, placental cancer, pleural cancer, prostate cancer, rectal cancer, kidney cancer, salivary gland cancer, skin cancer, small intestine cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, upper respiratory tract and gastrointestinal cancer, urinary tract cancer, uterine cancer, vaginal cancer, and vulvar cancer. The terms “tumor,” “cancer,” and “malignant tumor” are used interchangeably herein.

[0027] In some embodiments, cancer expresses CXCL13, CCL19, CCL21, or LTα.

[0028] In some embodiments, the disclosed method results in an increase in the efficacy and duration of the antitumor response. A method according to this aspect of the disclosure comprises selecting a subject having cancer and administering to the subject a therapeutically effective dose of an LTBR agonist in combination with a therapeutically effective dose of ACT. In certain embodiments, the method provides an increase in tumor inhibition of about 20%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80% compared to a subject treated with ACT as monotherapy.

[0029] In certain embodiments, the method provides an increase in the duration of the antitumor response by approximately 20%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80% compared to subjects treated with ACT as monotherapy. In certain embodiments, administration of an LTBR agonist in combination with ACT increases the response and duration of the response in subjects by more than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, or greater than 50% compared to untreated subjects or subjects treated with ACT as monotherapy.

[0030] In certain embodiments, the disclosed method results in a delay in tumor growth and development, for example, tumor growth can be delayed by about 3 days, longer than 3 days, about 7 days, longer than 7 days, longer than 15 days, longer than 1 month, longer than 3 months, longer than 6 months, longer than 1 year, longer than 2 years, or longer than 3 years compared to an untreated subject or a subject treated with ACT monotherapy.

[0031] In certain embodiments, administration of any of the combinations disclosed herein prevents tumor recurrence and / or extends the survival of a subject by, for example, 1–5 days, 5 days, 10 days, 15 days, 15 days, 15 days, 1 month, 3 months, 6 months, 12 months, 18 months, 24 months, 36 months, or 48 months compared to the survival of an untreated subject, or a subject treated with ACT as monotherapy, or a subject treated with ACT in combination with a non-LTBR agonist.

[0032] In certain embodiments, administration of an LTBR agonist in combination with ACT to a subject with cancer results in the complete disappearance of all traces of tumor cells ("complete response"). In certain embodiments, administration of an LTBR agonist in combination with ACT to a subject with cancer results in a reduction of at least 30% or more of tumor cells or tumor size ("partial response"). In certain embodiments, administration of an LTBR agonist in combination with ACT to a subject with cancer results in the complete or partial disappearance of tumor cells / lesions, including new measurable lesions. Tumor reduction is measured by any method known in the art, e.g., by radiography, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis.

[0033] In certain embodiments, administration of an LTBR agonist in combination with ACT to subjects with cancer results in an improved overall response rate compared to untreated subjects or subjects treated with ACT monotherapy.

[0034] In certain embodiments, administering a therapeutically effective dose of disclosed ACT and LTBR agonists to subjects with cancer results in an increase in overall survival (OS) or progression-free survival (PFS) compared to subjects treated with ACT as monotherapy.

[0035] In certain embodiments, PFS increases by at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, or at least 3 years compared to subjects treated with ACT as monotherapy.

[0036] In a particular embodiment, OS is increased by at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, or at least 3 years compared to subjects treated with ACT as monotherapy.

[0037] In certain embodiments, the additional therapeutic effects of the disclosed combination therapy include increased tumor-specific HEV formation, increased dendritic cell and T cell infiltration, enhanced T cell activation in the tumor microenvironment, and / or increased expression of TLS-related chemokines.

[0038] LTBR agonist As used herein, “lymphotoxin beta receptor,” “LTBR,” or “LTβR” refers to tumor necrosis factor receptor superfamily member 3 (TNFRSF3), a cell surface receptor for lymphotoxins involved in apoptosis and cytokine release. It is a member of the tumor necrosis factor receptor superfamily. LTBR is expressed in many different cell types, including epithelial and myeloid cells.

[0039] As used herein, “LTBR agonist” refers to any substance that binds to LTBR and results in an increase in signaling similar to that induced by the binding of its native ligand. LTBR agonists may take various appropriate forms, including native ligands, proteins, peptides, peptide mimes, nucleic acids, small molecules, or antibodies.

[0040] In some embodiments, LTBR agonists may include intrinsic ligands such as LTα1β2 and LIGHT. LTα1β2 is primarily expressed on activated lymphocytes, natural killer cells, and lymphoid tissue-inducing cells. LIGHT has a similar expression pattern to LTα1β2 but is also expressed on immature dendritic cells.

[0041] In some embodiments, the LTBR agonist may include an antibody or its antigen-binding fragment that specifically binds to LTBR, ​​such as human LTBR. Non-limiting examples of anti-LTBR agonizing antibodies include BHA10, CBE11, and BS-1. See, for example, Mackay et al., J.Immunol., 159:3299~3310 (1997); Hu et al., Carcinogenesis, 34:1105~1114 (2013), WO2018119118;US7429644.

[0042] As used herein, "agonist" refers to an agent that binds to a receptor and induces a response within the cell. Agonists mimic the action of an endogenous ligand, such as a hormone, and produce a physiological response similar to that produced by the endogenous ligand. "Partial agonist" refers to an agent that binds to a receptor and induces a partial response within the cell. Partial agonists produce only a partial physiological response to the endogenous ligand.

[0043] As used herein, “antibody” refers to an immunoglobulin molecule (i.e., a “complete antibody molecule”) consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains linked together by disulfide bonds, as well as its polymer (e.g., IgM) or its antigen-binding fragment. Each heavy chain consists of a heavy chain variable region (HCVR) and a heavy chain constant region. Each light chain consists of a light chain variable region (LCVR) and a light chain constant region. The HCVR and LCVR regions are further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each of the HCVR and LCVR consists of three CDRs. The term “antibody” also includes the antigen-binding fragment of a complete antibody molecule.

[0044] As used herein, “antigen” refers to any substance against which the immune system produces antibodies or a specific cell-mediated immune response. A disease-associated antigen is any substance associated with any disease against which the immune system produces antibodies or a specific cell-mediated response.

[0045] As used herein, “antigen-binding fragments” of antibodies, “antigen-binding moieties” of antibodies, etc., include any naturally occurring, enzymatically obtained, synthesized, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from a complete antibody molecule using any suitable standard technique, such as protein digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated, chemically or by using molecular biological techniques, for example, to position one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids.

[0046] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated CDRs such as the CDR3 peptide), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR graft antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other manipulated molecules such as shark variable IgNAR domains are also included within the expression “antigen-binding fragment” as used herein.

[0047] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. V L domains associated with V H domains in an antigen-binding fragment having V H and V L domains can be positioned relative to each other in any suitable arrangement. For example, the variable regions can be dimers and can contain V H -V H 、V H [[ID=1分]]-V L 、またはV L -V L dimers. Alternatively, the antigen-binding fragment of an antibody can contain monomeric V H or V L domains.

[0048] Adoptive cell therapy (ACT) As used herein, the terms "adoptive cell therapy", "ACT", or "adoptive immunotherapy" are used interchangeably and refer to the administration of modified immune cells to a subject having cancer. "Immune cells" (also referred to interchangeably herein as "immune effector cells") are part of the subject's immune system and refer to cells that assist in the fight against cancer in the subject's body. Non-limiting examples of immune cells for use in the disclosed methods include T cells, tumor infiltrating lymphocytes, and natural killer (NK) T cells. The immune cells can be autologous or allogeneic to the subject being treated.

[0049] As used herein, the terms "T cell" and "T lymphocyte" are used interchangeably. T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 + T cells), CD4 +T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + This can be a T cell, or any other subset of T cells. Other exemplary populations of T cells suitable for use in a particular embodiment include naive T cells and memory T cells. Also mentioned are “natural killer T (NKT) cells” or “NKT cells,” which refer to a special population of T cells that not only express a semi-invariant abT cell receptor but also express various molecular markers typically associated with NK cells, such as NK1.1. + and NK1.G, and CD4 + CD4, CD8 + Examples include CD8 cells.

[0050] The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also mentioned are "gamma-delta T cells (γδT cells)," which refer to a special population of a small subset of T cells that have different TCRs on their surface. Unlike most T cells, where the TCR consists of two glycoprotein chains denoted as a and b-TCR chains, the TCR in γδT cells consists of g and d chains. γδT cells can play a role in immune surveillance and immunomodulation, are an important source of IL-17, and have a robust CD8 + It has been found that it induces a cytotoxic T cell response. Also mentioned are "regulatory T cells" or "Tregs," which refer to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4 + T cells, IL-10 producing CD4 + This may also include T cells, specifically Foxp3-negative regulatory T cells.

[0051] T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, and tumors. In some embodiments, T cells can be obtained from blood units collected from a subject using several techniques known to those skilled in the art, such as FICOLL isolation. In one embodiment, T cells derived from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets.

[0052] The disclosed immune effector cells, such as T cells, are isolated using known methods and then genetically modified (to form modified immune cells), or the immune cells are activated and expanded, or, in the case of precursors, differentiated in vitro before genetic modification. In some embodiments, immune effector cells, such as T cells, are genetically modified using the TCR or CAR described herein (e.g., transduced with a viral vector containing nucleic acids encoding a TCR or CAR), and then activated and expanded in vitro. Techniques for activating and expanding T cells are known in the art and are suitable for use with the disclosed techniques. See, for example, US6,905,874;US6,867,041;US6,797,514;WO2012079000;US2016 / 0175358. TCR-expressing or CAR-expressing immune effector cells suitable for use in the disclosed methods are prepared according to known techniques shown in the art.

[0053] For use in the disclosed manner, immune cells are modified with a TCR or CAR for tumor-associated antigens (TAAs). In other words, non-limiting examples of ACTs for use in the disclosed manner include modified TCRs for tumor-associated antigens (TAAs), or chimeric antigen receptors (CARs) for TAAs.

[0054] TAA may originate from any of the following cancers, including but not limited to adrenal cancer, anal cancer, autonomic ganglion cancer, biliary tract cancer, bladder cancer, hematological cancer, bone cancer, brain cancer, breast cancer, meningeal cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, eye cancer, esophageal cancer, fallopian tube cancer, stomach cancer, reproductive tract cancer, head and neck cancer, kidney cancer, colorectal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, peritoneal cancer, pituitary cancer, placental cancer, pleural cancer, prostate cancer, rectal cancer, kidney cancer, salivary gland cancer, skin cancer, small intestine cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, upper respiratory tract and gastrointestinal cancer, urinary tract cancer, uterine cancer, vaginal cancer, and vulvar cancer.

[0055] In certain embodiments, TAA is AFP, ALK, BAGE protein, BCMA, BIRC5 (Survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A 3, hTERT, LMP2, MAGE protein (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, P The following proteins are selected: AX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA(FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0056] As used herein, “T cell receptor” refers to an isolated TCR polypeptide that specifically binds to TAA, or a TCR expressed on an isolated immune cell (e.g., a T cell). The TCR binds to an epitope on a small antigenic determinant on the surface of an antigen-presenting cell associated with a major histocompatibility complex (MHC; in mice) or human leukocyte antigen (HLA; in humans) complex (e.g., found in tumor-associated antigens). The TCR also refers to an immunoglobulin superfamily member having a variable binding domain, constant domain, transmembrane region, and short cytoplasmic tail that can specifically bind to an antigen peptide bound to an MHC receptor (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, 1997).

[0057] As used herein, the term “polypeptide” refers to any polymer that is essentially composed of any of the 20 naturally occurring amino acids, regardless of its size. The term “protein” is often used in reference to relatively large proteins, and “peptide” is often used in reference to small polypeptides, although the use of these terms in this art is often redundant. Unless otherwise specified, the term “polypeptide” generally refers to proteins, polypeptides, and peptides. Peptides useful according to this disclosure are generally between about 0.1 and 100 KD or above, or up to about 1000 KD, preferably between about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, and 50 KD, as determined by standard molecular sizing techniques such as centrifugation or SDS-polyacrylamide gel electrophoresis.

[0058] The TCR is found on the cell surface and is generally composed of heterodimers having α and β chains (also known as TCRα and TCRβ, respectively) or γ and δ chains (also known as TCRγ and TCRδ, respectively). Similar to immunoglobulins, the extracellular portion of the TCR chain (e.g., α and β chains) contains two immunoglobulin regions: a variable region (e.g., TCR variable α region or Vα, and TCR variable β region or Vβ; typically amino acids 1-116 at the N-terminus based on Kabat numbering) and a constant region adjacent to the cell membrane (e.g., TCR constant domain α or Cα, typically amino acids 117-259 based on Kabat, TCR constant domain β or Cβ, typically amino acids 117-295 based on Kabat). Also, similar to immunoglobulins, the variable domain contains a CDR delimited by a framework region (FR). In some embodiments, the TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex. The sources of the TCRs of this disclosure may be derived from various animal species, such as humans, mice, rats, rabbits, or other mammals. In some embodiments, the source of the TCRs of this disclosure is a mouse genetically engineered to produce a TCR containing human alpha and beta chains (see, for example, WO2016 / 164492).

[0059] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid sequence within the antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). Representative conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, the ABM definition, and the IMGT definition. See, for example, Kabat, 1991, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al., 1997, J.Mol.Biol.273:927~948 (Chothia numbering scheme); Martin et al., 1989, Proc.Natl.Acad.Sci.USA 86:9268~9272 (ABM numbering scheme); and Lefranc et al., 2003, Dev.Comp.Immunol.27:55~77 (IMGT numbering scheme). Publicly available databases are also available for identifying CDR sequences within antibodies.

[0060] The TCRα and TCRβ polypeptides (and similarly the TCRγ and TCRδ polypeptides) are linked to each other via disulfide bonds. Each of the two polypeptides constituting the TCR contains an extracellular domain including a constant region and a variable region, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and cytoplasmic tail are also part of the constant region). The variable region of the TCR determines its antigen specificity and, like immunoglobulins, contains three CDRs. The TCR is expressed on most T cells in the body and is known to be involved in the recognition of MHC restriction antigens. The TCRα chain contains covalently linked Vα and Cα regions, while the β chain contains a Vβ region covalently linked to the Cβ region. The Vα and Vβ regions form pockets or clefts that can bind antigens in the context of major histocompatibility complex (MHC) (or HLA in humans).

[0061] The term "HLA" refers to the human leukocyte antigen (HLA) system or complex, a gene complex that codes for MHC proteins in humans. These cell surface proteins play a role in regulating the immune system in humans. HLAs corresponding to MHC class I (A, B, and C) present peptides from within the cell. The term "HLA-A" refers to the group of human leukocyte antigens (HLAs) encoded by the HLA-A locus. HLA-A is one of the three major types of human MHC class I cell surface receptors. This receptor is a heterodimer, consisting of a heavy α chain and a smaller β chain. The α chain is coded by the variant HLA-A gene, and the β chain (β2-microglobulin) is an invariant β2-microglobulin molecule. The term "HLA-A2" is used to refer to the group of human leukocyte antigens (HLA-A2) * HLA-A (also known as "HLA-A 01") is a specific group of MHC class I alleles at the HLA-A locus; the α chain is HLA-A * It is encoded by the O2 gene, and the β chain is encoded by the β2-microglobulin or B2M locus.

[0062] TCRs are detection molecules with excellent specificity and exhibit vast diversity, similar to antibodies. The general structure of TCR molecules, as well as techniques for constructing and using such molecules, including their binding to peptides:MHC, are described in PCT / US98 / 04274, PCT / US98 / 20263, and WO99 / 60120.

[0063] For example, non-human animals (e.g., rodents, e.g., mice or rats) are genetically engineered to express human or humanized TCRs containing a variable domain encoded by at least one human TCR variable region gene segment. See, for example, WO2016 / 164492. For example, Veloci-T® mouse technology (Regeneron) provides genetically modified mice that enable the production of fully human therapeutic TCRs against tumor and / or viral antigens and is used to produce TCRs suitable for use with the disclosed technology. Those skilled in the art can obtain altered TCR sequences using standard mutagenesis techniques in conjunction with the assays described herein and test them for specific binding affinity and / or specificity. Useful mutagenesis techniques known in the art include, but are not limited to, de novo gene synthesis, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linker-scanning mutagenesis, and PCR-based site-specific mutagenesis.

[0064] In some embodiments, a method for constructing a TCR for TAA includes: immunizing a non-human animal (e.g., a rodent, e.g., mouse or rat), such as a genetically engineered non-human animal containing an unreconstituted human TCR variable locus in its genome, with a specific peptide derived from TAA; enabling the animal to initiate an immune response to the peptide; isolating peptide-responsive T cells from the animal; determining the nucleic acid sequence of the human TCR variable region expressed by the T cells; cloning the human TCR variable region into a nucleotide construct containing the nucleic acid sequence of the human TCR constant region, thereby operably linking the human TCR variable region to the human TCR constant region; and expressing peptide-specific human T cell receptors from the constructs, respectively. The steps of isolating T cells, determining the nucleic acid sequence of the human TCR variable region expressed by the T cells, cloning the human TCR variable region into a nucleotide construct containing the nucleic acid sequence of the human TCR constant region, and expressing human T cell receptors are carried out using standard techniques known to those skilled in the art.

[0065] As used herein, an HLA-presenting peptide (such as an HLA-A2-presenting peptide) may refer to a peptide bound to an HLA protein, such as an HLA protein expressed on the cell surface. Therefore, a TCR that binds to an HLA-presenting peptide binds to the HLA-bound peptide and, in some cases, to the HLA itself. The interaction with HLA may confer specificity to binding to peptides presented by a particular HLA. In some embodiments, the TCR may bind to an isolated HLA-presenting peptide. In some embodiments, the TCR may bind to an HLA-presenting peptide on the cell surface.

[0066] As used herein, “chimeric antigen receptor” or “CAR” refers to an antigen-binding protein comprising an immunoglobulin antigen-binding domain (e.g., an immunoglobulin variable domain) and the TCR constant domain or a portion thereof, which is administered to a target as chimeric antigen receptor T cell (CAR-T) therapy. As used herein, the “constant domain” of the TCR polypeptide comprises the membrane-proximal TCR constant domain and may also include the TCR transmembrane domain and / or the TCR cytoplasmic tail. For example, in some embodiments, the CAR is a dimer comprising a first polypeptide comprising an immunoglobulin heavy chain variable domain linked to the TCRβ constant domain, and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a κ or λ variable domain) linked to the TCRα constant domain.

[0067] As used herein, “variable region” refers to the alpha or beta chain variable region that is directly involved in the binding of the TCR to the antigen. As used herein, the term “constant domain” refers to the alpha and beta chain constant regions that are not directly involved in the binding of the TCR to the antigen but exhibit various effector functions.

[0068] CARs are typically artificially constructed hybrid proteins or polypeptides that include an antigen-binding domain of another antibody agent linked to an scFv or T cell signaling domain. In the context of this disclosure, CARs target tumor-associated antigens. Features of CARs include the ability to redirect T cell specificity and responsiveness to selected targets in a non-MHC-restrictive manner using the antigen-binding properties of a monoclonal antibody. Non-MHC-restrictive antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen treatment, thereby circumventing the primary mechanism of tumor escape. When used in the ACTs disclosed herein, immune cells can be manipulated to express CARs by any known method, including, for example, transfection using RNA and DNA, both of which are known in the art.

[0069] In some embodiments, immunoeffector cells expressing TCRs or CARs are first harvested from the culture medium and then formulated by washing and concentrating the cells in a medium and container system suitable for administration in a therapeutically effective dose ("pharmaceutically acceptable" carrier). Suitable infusion media may be any isotonic media formulation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% dextrose or Ringer's lactate solution in water may also be used. The infusion media may be supplemented with human serum albumin.

[0070] The therapeutically effective number of immune cells administered in the disclosed method is typically 10 2 It is more than one cell, for example, 10 6Less than or equal to 10 8 Less than or equal to 10 9 A number of cells or fewer, or 10 10 This refers to more than one cell. The number and / or type of cells administered to the target depends on the ultimate intended use for treatment.

[0071] The TCRs and CARs of this disclosure may be recombinant, meaning that they can be prepared, expressed, isolated, or obtained as recombinant DNA technologies by techniques or methods known in the art, such techniques or methods include, for example, DNA splicing and transgenic expression. Recombinant TCRs or CARs can be expressed in non-human mammals (including transgenic non-human mammals, e.g., transgenic mice) or cells (e.g., CHO cells) expression systems, or isolated from recombinant combinatorial human antibody libraries.

[0072] Combination therapy In some embodiments, the disclosure involves administering a combination therapy comprising a therapeutically effective dose of an LTBR agonist and a therapeutically effective dose of ACT to a subject with cancer. In some embodiments, the disclosed combination therapy exhibits synergistic antitumor efficacy in various cancer types. In some embodiments, the disclosed combination therapy increases the efficacy of ACT administered to a subject with cancer compared to a subject treated with ACT as monotherapy, thereby treating the cancer more effectively.

[0073] With respect to pharmaceutical compositions, the disclosed LTBR agonists and / or ACTs are formulated with one or more pharmaceutically acceptable carriers, excipients, and / or diluents. Pharmaceutical compositions comprising the disclosed LTBR agonists and / or ACTs are formulated for specific uses, such as pharmaceutical and veterinary use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.), as well as the excipients, diluents, and / or carriers used, depend on the intended therapeutic use and preferred mode of administration of the LTBR agonist and / or ACT.

[0074] The pharmaceutical compositions of this disclosure may comprise either an LTBR agonist or an ACT, or both, and the LTBR agonist and ACT may be formulated as separate compositions or as a single composition. Such pharmaceutical compositions may be administered to a subject by various routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically, or topically. In some embodiments, the pharmaceutical composition may be administered intravenously or subcutaneously to a subject. The pharmaceutical compositions may be conveniently provided in unit dosage forms comprising a predetermined amount of the disclosed LTBR agonist and / or ACT per dose.

[0075] In some embodiments, the disclosed method further includes the administration of an additional therapeutic agent or treatment. In some embodiments, the additional therapeutic agent or treatment may include, but is not limited to, CTLA-4, PD-1, PD-L1, PD-1-PD-L1, PD-1-PD-L2, T cell immunoglobulin and mucin 3 (TIM3 or HAVCR2), galectin 9-TIM3, phosphatidylserine-TIM3, lymphocyte activator gene 3 protein (LAG3), MHC class II-LAG3, 4-1BB-4-1BB ligand, OX40-OX40 ligand, GITR, GITR ligand-GITR, CD27, CD70-CD27, TNFRSF25, TNFRSF25-TL1 A, CD40L, CD40-CD40 ligand, HVEM-LIGHT-LTA, HVEM, HVEM-BTLA, HVEM-D160, HVEM-LIGHT, HVEM-BTLA-CD160, CD80, CD80 -PDL-1, PDL2-CD80, CD244, CD48-CD244, CD244, ICOS, ICOS-ICOS ligand, B7-H3, B7-H4, VISTA, TMIGD2, HHLA2-TMIGD2, BT Butyrophylline including L2, Siglec family, TIGIT and PVR family members, KTR, ILT and LIR, KG2D and KG2A, MICA and MICB, CD244, CD28, CD86-CD28, CD86-CTLA, CD80-CD28, phosphatidylserine, TEVI3, phosphatidylserine-TEVI3, SIRPA-CD47, neuropilin, CD160, CD30, and CD155 (for example) This includes, for example, CTLA-4 or PD1 or PD-L1), as well as other immunomodulators, such as immune checkpoint inhibitors (e.g., antibodies) that target immune checkpoint receptors such as interleukin-2 (IL-2), indoleamine 2,3-dioxygenase (IDO), IL-10, transforming growth factor-β (TGFp), CD39, CD73, adenosine-CD39-CD73, and CXCR4-CXCL12.

[0076] Additional therapeutic agents or non-limiting examples of additional treatments include radiation, surgery, cancer vaccines, CD47 inhibitors, antagonists of other T cell co-inhibitors or ligands (e.g., antibodies against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), vascular endothelial growth factor (VEGF) antagonists [e.g., aflibercept as described in US7,087,411, or other VEGF inhibitory fusion proteins such as "VEGF-Trap," or anti-VEGF]. GF antibodies or their antigen-binding fragments (e.g., bevacizumab or ranibizumab), or small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib), Ang2 inhibitors (e.g., nesbakumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), agonists against costimulatory receptors (e.g., against glucocorticoid-induced TNFR-related proteins) Agonists, antibodies against tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), vaccines (e.g., Bacillus calmet-Guérin, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphate) Examples include lophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), radiotherapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, anti-DS6-DM4 ADC), anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), antioxidants and other nutritional supplements, as well as combinations thereof.

[0077] In some embodiments, additional therapeutic agents or treatments include anticancer drugs. As used herein, “anticancer drug” means any agent useful for treating cancer, including, but not limited to, cytotoxins and drugs such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazines, hydroxyureas, asparaginases, corticosteroids, mitotanes (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radiopharmaceuticals. As used herein, “cytotoxin or cytotoxic agent” also means chemotherapeutic agents and any substance that acts harmfully on cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbiastine, coichicin, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues.

[0078] As used herein, “therapeutic agent or treatment” means a molecule, compound, or means that, when administered to a subject, produces some beneficial effect. Beneficial effects may include enabling diagnostic decisions; improving a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally counteracting a disease, symptom, disorder, or pathological condition.

[0079] In some embodiments, the combined administration of LTBR agonists and ACTs with additional therapeutic agents or treatments results in improved antitumor efficacy, reduced side effects of one or both of the primary treatments, and / or a reduction in the dosage of one or both of the primary treatments.

[0080] This disclosure also provides kits comprising at least one LTBR agonist and at least one ACT (e.g., anti-TAA TCR or CAR-modified immune cells). The kits typically include labels and instructions for use indicating the intended use of the kit contents. As used herein, the term “label” includes any documents or recorded materials provided on, within, or with the kit, or otherwise accompanying the kit. In some embodiments, this disclosure provides a kit for treating a cancer-affected subject, the kit comprising: (b) at least one LTBR agonist in a therapeutically effective dose; at least one ACT in a therapeutically effective dose; and (b) instructions for use of a combination of doses in any of the methods disclosed herein.

[0081] Administration regimen This disclosure includes a method comprising administering a combination of an LTBR agonist and / or ACT to a subject with cancer at a dose frequency that achieves a therapeutic response. In some embodiments, the LTBR agonist and / or ACT are administered to the subject in one or more doses, as long as a therapeutic response is achieved.

[0082] In the disclosed method, ACT is administered to the subject in combination with an LTBR agonist. As used herein, the expression “in combination” means that ACT is administered before, after, or concurrently with the LTBR agonist. This expression includes sequential or concurrent administration of the LTBR agonist and ACT. As used herein, “sequential” administration means that each dose of the selected treatment is administered to the subject at different time points, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). For illustrative purposes, sequential administration may include administering an initial dose of ACT (or LTBR agonist), followed by one or more secondary doses of LTBR agonist (or ACT), and optionally thereafter, one or more tertiary doses of ACT (or LTBR agonist). For illustrative purposes, sequential administration may include administering to a subject an initial dose of ACT (or LTBR agonist), followed by one or more secondary doses of LTBR agonist (or ACT), and optionally, one or more tertiary doses of LTBR agonist (or ACT) thereafter.

[0083] Dosage Generally, the amount of LTBR agonist and / or ACT administered to a subject according to the method of this disclosure is a therapeutically effective dose. As used herein, “therapeutically effective dose” means the amount of LTBR agonist combined with ACT that results in one or more of the following: (a) reduction in the severity or duration of cancer symptoms; (b) enhanced inhibition of tumor growth, or increased tumor necrosis, tumor shrinkage, and / or tumor disappearance; (c) delay in tumor growth and development; (d) inhibition, delay, or cessation of tumor metastasis; (e) prevention of recurrence of tumor growth; (f) extension of survival in a subject with cancer; (g) reduced use or need for conventional anticancer treatment (e.g., reduced or elimination of the use of chemotherapeutic agents or cytotoxic agents) compared to an untreated subject or a subject treated with ACT as monotherapy; (h) increased tumor-specific HEV formation; (i) increased dendritic cell and T cell infiltration; (j) enhanced T cell activation in the tumor microenvironment; and / or (k) increased expression of TLS-related chemokines.

[0084] In some embodiments, the therapeutically effective dose of ACT is approximately 1 × 10⁻⁶ 6 The above 5 x 10 6 The above is 1 x 10 7 The above 5 x 10 7 The above is 1 x 10 8 The above 5 x 10 8 The above is 1 x 10 9 The above 5 x 10 9 This may include immunoeffector cells expressing modified TCRs or CARs against tumor-associated antigens, administered in quantities equal to or greater than the above.

[0085] In some embodiments, a therapeutically effective dose of LTBR agonist may be approximately 0.05 mg to approximately 600 mg of LTBR agonist, such as 100 mg, 250 mg, or 350 mg. In some embodiments, the amount of LTBR agonist administered to a subject may be 0.005 mg / kg to 10 mg / kg relative to the subject's body weight, for example, 1 mg / kg, 3 mg / kg, or 5 mg / kg.

[0086] Where used herein, the singular forms “a,” “an,” and “it” include the plural unless the context clearly indicates otherwise. Where used herein, the terms “including,” “comprising,” “containing,” and “having,” and their variations, are intended to encompass the items and their equivalents listed therein, as well as additional subject matter, unless otherwise specified. Where used herein, phrases such as “in one embodiment,” “in various embodiments,” and “in some embodiments” are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may refer to the same embodiment unless the context clearly indicates otherwise. Where used herein, the terms “and / or” or “ / ” mean any one of the items related to that term, any combination of those items, or all of those items.

[0087] Where used herein, when applied to one or more of the values ​​of interest, the terms “approximately” or “about” refer to a value similar to the stated reference value. In some embodiments, unless otherwise specified or evident from the context, the terms “approximately” or “about” refer to a range of values ​​that are 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction from the stated reference value (except where such a number exceeds 100% of the possible value).

[0088] This disclosure is merely illustrative of the principles of the disclosed technology. Any examples shown herein are not intended to limit, but merely illustrate a number of possible embodiments of the appended claims. Those skilled in the art will readily recognize a variety of modifications and changes that can be made without following the exemplary embodiments and the applications illustrated and described herein, and without departing from the true spirit and scope of the following claims. All references cited and / or discussed herein are incorporated herein by reference in whole and by reference individually, to the same extent as such references are incorporated herein by reference in whole. [Examples]

[0089] The disclosed technology is described below by the following examples. The use of these and other examples throughout this specification is illustrative only and does not in any way limit the scope and meaning of the invention or any illustrated form. Similarly, the invention is not limited to any particular preferred embodiment described herein. In fact, modifications and variations of the invention will be obvious to those skilled in the art by reading this specification and can be made without departing from its spirit and scope. Thus, the invention is limited only by the terms of the claims, along with the entire scope of the equivalents of the granted claims. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is Celsius, room temperature is approximately 25°C, and pressure is atmospheric pressure or near atmospheric pressure. [Examples]

[0090] Induction of tumor-specific high endothelial venules (HEVs) and tertiary lymphoid structures (TLS) by LTBR agonism. This example relates to LTBR agonism that induces TLS, including HEVs, T cells, B cells, and TLS-related chemokines. Treatment with a single LTBR agonist mAb induced tumor-specific MECA-79+ HEV formation in MC38-OVA tumors. The induced HEVs were associated with locally increased T cell infiltration. HEV formation was absent in normal organs, and the effects shown by gene expression analysis were minimal.

[0091] The MC38-OVA cell line was created by transduction of ovalbumin into MC38 mouse colorectal tumor cells. In the MC38-OVA tumor model, 1 × 10⁶ MC38-OVA mouse colorectal tumor cells were subcutaneously transplanted into the right flank of C57BL / 6 mice. The tumor size was 50–100 mm. 3Upon reaching a certain stage, mice were randomized to a treatment group and administered either an anti-mouse LTBR agonist (5G11, Abcam) mAb or an isotype control (rat IgG2a, 2A3) mAb at 2 mg / kg intraperitoneally twice a week for a total of two doses. After one week of treatment, mice were sacrificed, and excised tumor samples were fixed in 10% neutral buffered formalin at 4°C for 48 hours, then replaced with 70% ethanol, embedded in paraffin, and sectioned for immunofluorescence staining. High endothelial venules (HEVs) were stained with MECA-79 antibody, which recognizes HEV-specific PNAd epitopes. T cells and endothelial cells were stained with anti-CD3 and anti-CD31 antibodies, respectively.

[0092] MC38-OVA tumors in mice treated with LTBR agonist antibodies showed HEV formation, while tumors in mice treated with isotype control antibodies did not. HEV formation did not occur in normal organs, and the effect observed by gene expression analysis was minimal. MC38-OVA tumors in mice treated with LTBR agonist antibodies also showed increased T cell infiltration compared to mice treated with isotype controls.

[0093] In the second experiment, a Colon26 tumor model was used. 1 × 10 6 Colon26 mouse colorectal tumor cells were subcutaneously transplanted into the right flank of BALB / c mice. The tumors were 50-100 mm in size. 3 Upon reaching a certain stage, mice were randomized to a treatment group and administered either an anti-mouse LTBR agonist (5G11) mAb or an isotype control (rat IgG2a, 2A3) mAb at 2 mg / kg intraperitoneally twice a week for a total of three doses. After 10 days of treatment, mice were sacrificed, and excised tumor samples were fixed in 10% neutral buffered formalin at 4°C for 48 hours, then replaced with 70% ethanol, embedded in paraffin, and sectioned for immunofluorescence staining. High endothelial venules (HEVs) were stained with MECA-79 antibody, which recognizes HEV-specific PNAd epitopes. T cells and B cells were stained with anti-CD3 and anti-B220, respectively.

[0094] Tumor-associated TLSs indicate HEV formation and the presence of B and T cells. Tumor-associated B cell aggregates or tertiary lymphoid structures (TLSs) induced by LTBR agonist antibody treatment were classified according to their location relative to the tumor, i.e., adjacent, peripheral, or intratumor. LTBR agonist antibody treatment induced TLS-like structures, primarily in the peripheral region, in approximately 20% of the treated Colon26 tumors.

[0095] Expression analysis of multiple TLS-related chemokines in MC38 tumors (Figure 1). In the MC38 tumor model, 1 × 10⁻¹⁶ 6 Individual MC38 mouse colorectal tumor cells were subcutaneously transplanted into the right flank of C57BL / 6 mice. The tumors were 50-100 mm in size. 3 Upon reaching a certain level, mice were randomized to a treatment group and administered either an anti-mouse LTBR agonist (5G11) mAb or an isotype control (rat IgG2a, 2A3) mAb at a dose of 2 mg / kg via intraperitoneal injection twice a week for a total of two doses.

[0096] After one week of treatment, the mice were sacrificed, and RNA was extracted from excised tumor samples using TRIzol® reagent (Life Technologies) and MagMAX®-96 (Thermo Fisher Scientific) for microarray total RNA isolation kits, according to the manufacturer's instructions. Pre-amplified cDNA was prepared from tumor lysates using the SuperScript® III First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), according to the manufacturer's protocol. Subsequently, qPCR was performed using the pre-amplified cDNA against the target gene on a CFX96® real-time system instrument (Biorad). qPCR analysis was performed using SsoAdvanced Universal SYBR® Green Supermix (Biorad).

[0097] Relative gene expression was calculated as the multiplicative change in target gene expression relative to the housekeeping gene Actb. The bar graph shows the mean ± SEM, with each point representing one tumor. All reactions were performed in pairs. An unpaired Student's t-test (two-sided) was used to compare the two groups. A p-value < 0.05 was considered statistical significance. * :p<0.05; ** :p<0.01; *** :p<0.001; **** (p<0.0001). All statistical analyses were performed using GraphPad Prism software.

[0098] The target genes were mouse homologs of published human 12-chemokine gene signatures that correlated with the presence of TLS and improved patient survival in colorectal cancer, melanoma, and breast cancer patients. Treatment with LTBR agonist mAb significantly upregulated the expression of multiple TLS-related chemokines in MC38 tumors (Figure 1). [Examples]

[0099] LTBR agonism promoted the infiltration and activation of DCs and T cells. This example quantitatively demonstrated the effects of treatment with LTBR agonist antibodies in vivo and in vitro. LTBR agonists resulted in in vivo infiltration and activation of DCs and T cells into tumors compared to treatment with isotype control antibodies. In vitro, LTBR agonist treatment directly upregulated activation and maturation markers in BMDCs and enhanced in vitro DC-mediated OT-I CD8 T cell activation.

[0100] Quantification of DC and T cell infiltration and activation in vivo (Figure 2A). In the MC38 tumor model, 1 × 10⁻⁶ 6Individual MC38 mouse colorectal tumor cells were subcutaneously transplanted into the right flank of C57BL / 6 mice. When the tumor reached 50–100 mm3, the mice were randomized to a treatment group and administered either an anti-mouse LTBR agonist (5G11) mAb or an isotype control (rat IgG2a, 2A3) mAb at a dose of 2 mg / kg intraperitoneally twice a week for a total of three doses.

[0101] After 10 days of treatment, mice were sacrificed, tumors were excised and finely chopped, and incubated with enzymes A, D, and R (Tumor Dissociation Kit, Miltenyi) in combination with mechanical dissociation using a gentleMACS Dissociator (Miltenyi). Single-cell suspensions were prepared from the dissociated tumors and stained with an antibody cocktail at 4°C for 30 minutes. Tumor-infiltrating immune cell data were acquired using an LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo software.

[0102] Immune cells were stained with anti-CD45 antibody. Bone marrow cells were identified by CD45+CD11b+ staining. Dendritic cells were identified by CD45+CD11c+MHCII+F4 / 80- staining, in addition to the activation markers MHC class II, CD40, and CD86. CD4 and CD8 T cells were detected by CD45+CD3+CD4+CD8- and CD45+CD3+CD4-CD8+ staining, respectively, in addition to the activation markers Ki67 and IFNg.

[0103] Tumor-infiltrating immune cells were quantified as a percentage of total viable cells. The bar graph shows the mean ± SEM, with each point representing one tumor. ISO bars labeled "ISO" indicate the value of isotype treatment. Bars labeled "LTBR" indicate the value of LTBR agonist treatment. An unpaired Student's t-test (two-sided) was used to compare the two groups. A p-value < 0.05 was considered statistically significant. * :p<0.05; ** :p<0.01; *** :p<0.001; ****(p<0.0001). All statistical analyses were performed using GraphPad Prism software.

[0104] Treatment with LTBR agonist mAbs enhanced dendritic cell and T cell tumor infiltration and activation in MC38 tumors. Consistent results were observed in a Colon26 tumor model (data not shown).

[0105] Bone marrow-derived DC (BMDC) assay (Figure 2B). To test the direct effects of LTBR agonism on DC activation and DC-mediated T cell activation, an antigen presentation assay was used with bone marrow-derived DCs and primary CD8+ T cells isolated from the spleen of OT-I mice. Briefly, bone marrow cells were isolated from the femur and tibia of wild-type C57BL / 6 mice and differentiated in vitro in 6-well plates for 8 days by treatment with recombinant human FLT3L protein 200 ng / mL. The bone marrow-derived cells were then dissociated and plated for 18 hours in 96-well plates containing anti-mouse LTBR agonist (5G11) mAb or isotype control (rat IgG2a, 2A3) mAb 160 μg / mL.

[0106] To evaluate dendritic cell (DC) activation, these treated cells were dissociated, prepared as single-cell suspensions, and stained at 4°C for 30 minutes with an antibody cocktail containing DC markers (CD11c+MHCII+) and DC activation and maturation markers (CD83, MHCI, MHCII, CD80, CD86, CD40, ICOSL). Data were acquired using an LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo software.

[0107] The amount of bone marrow-derived dendritic cells (DCs) was quantified as the percentage of CD11c+MHCII+ cells in a mixed bone marrow-derived culture. The percentage of CD83+ cells and the median fluorescence intensity (MFI) of the DC activation marker were measured in CD11c+MHCII+ DCs and are shown as the fold change in DCs treated with an LTBR agonist compared to DCs treated with an isotype control.

[0108] Treatment with LTBR agonist mAbs increased the percentage of DCs and CD83+ mature DCs in bone marrow-derived mixed cultures, and upregulated the expression of DC activation markers in BMDCs.

[0109] Antigen presentation assay using pre-treated BMDCs and OT-I T cells (Figure 2C). In the antigen presentation assay, BMDCs treated with isotype control or LTBR agonist mAb were further incubated with 10 ng / mL of OVA peptide (SIINFEKL) for 2 hours and co-cultured with primary OT-I CD8+ T cells in a 1:5 ratio for 3 days. OT-I CD8+ T cells were isolated from the spleen of OT-I mice possessing a transgenic T cell receptor designed to recognize the OVA peptide SIINFEKL using the EasySep® mouse CD8+ T cell isolation kit (STEMCELL Technologies). In the antigen presentation assay, BMDCs can present the OVA peptide along with MHCI molecules, activating OT-I SIINFEKL-specific CD8+ T cells.

[0110] After 3 days of co-culture, CD8+ T cell activation was evaluated by the percentage of CD44+ proliferating cells among CD8+ T cells and the median fluorescence intensity (MFI) of activation markers (CD44, TNFα, and GZMB) in CD44+ proliferating CD8+ cells. T cell proliferation was measured using the CellTrace® Far Red Cell Proliferation Kit.

[0111] Pretreatment of BMDCs with an LTBR agonist mAb increased the percentage of CD44+ proliferating CD8+ cells and enhanced the expression of activation (CD44) and cytotoxic (TNFα and GZMB) markers. [Examples]

[0112] LTBR agonism enhanced the antitumor efficacy of PD-1 blockade. This example demonstrated the antitumor efficacy of PD-1 blockade. LTBR agonist treatment attenuated tumor growth in a CD8 T cell-dependent manner. The combination of LTBR agonist and anti-PD1 antibody treatment showed enhanced immune cell infiltration and antitumor effects in the Colon26 tumor model.

[0113] CD8 T cell depletion in the Colon26 tumor model (Figure 3A). In the Colon26 tumor model, 1 × 10⁶ Colon26 mouse colorectal tumor cells were subcutaneously transplanted into the right flank of BALB / c mice on day 0. To deplete CD8+ T cells, mice were intraperitoneally administered anti-CD8a (cl.2.43; BioXCell) or isotype control (rat IgG2a, 2A3) mAb 10 mg / kg on days -2 and 0, and then twice a week until the end of the study.

[0114] Mice were randomized to a treatment group and administered either an anti-mouse LTBR agonist (5G11) mAb or an isotype control (rat IgG2a, 2A3) mAb at a dose of 2 mg / kg intraperitoneally on day 8. Subsequently, a total of four doses were administered twice a week. CD8+ T cell depletion was confirmed on day 8, immediately prior to the administration of the LTBR agonist mAb, by quantitative analysis of CD8+ T cells in the blood using flow cytometry.

[0115] Tumor volume was calculated from caliper measurements using the formula L × W × W × 0.5, where L is the longest dimension and W is the vertical dimension. The antitumor effect of LTBR agonist mAb treatment was dependent on the presence of CD8+ T cells (Figure 3A).

[0116] Antitumor effect in the Colon26 tumor model (Figure 3B). In the Colon26 tumor model, 1 × 10⁻¹⁶ 6 Colon26 mouse colorectal tumor cells were subcutaneously transplanted into the right flank of BALB / c mice. The tumors were 50-100 mm in size. 3Upon reaching a certain level, mice were randomized to a treatment group and administered 2 mg / kg of anti-mouse LTBR agonist (5G11) mAb and / or anti-mouse PD-1 (RPM1-14, BioXCell) mAb or isotype control (rat IgG2a, 2A3) mAb intraperitoneally twice a week for a total of four doses.

[0117] Tumor volume was calculated from caliper measurements using the formula L × W × W × 0.5, where L is the longest dimension and W is the vertical dimension. As shown in Figure 3B, monotherapy with anti-PD-1 mAb or LTBR agonist mAb showed a moderate antitumor effect, but combination therapy with LTBR agonist mAb and anti-PD-1 mAb enhanced the antitumor efficacy of the monotherapy, with 3 out of 8 tumors disappearing on day 28.

[0118] Immune cell infiltration in a Colon26 tumor model (Figure 3C). In a repeat study of combined treatment with an LTBR agonist and anti-PD-1 in a Colon26 tumor model, mice were sacrificed after 10 days of treatment, tumors were excised and finely chopped, and incubated with enzymes A, D, and R (Tumor Dissociation Kit, Miltenyi) in combination with mechanical dissociation using a gentleMACS Dissociator (Miltenyi). Single-cell suspensions were prepared from the dissociated tumors and stained with an antibody cocktail at 4°C for 30 minutes. Tumor-infiltrating immune cell data were acquired using an LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo software.

[0119] Immune cells were identified by anti-CD45 staining, and CD4 and CD8 T cells were identified by CD45+CD3+CD4+CD8- and CD45+CD3+CD4-CD8+ staining, respectively. Dendritic cells were detected as CD45+CD11c+MHCII+F4 / 80-CD11b+2 type DCs and CD45+CD11c+MHCII+F4 / 80-CD11b-CD103+1 type DCs. Natural killer (NK) cells were labeled by CD45+CD3-NKp46+ staining.

[0120] Tumor-infiltrating immune cells were quantified relative to the number of tumor cells. The bar graph shows the mean ± SEM, with each point representing one tumor. An unpaired Student's t-test (two-sided) was used to compare the two groups. A p-value < 0.05 was considered statistically significant. * :p<0.05; ** :p<0.01; *** :p<0.001; **** (p<0.0001). All statistical analyses were performed using GraphPad Prism software. Combination therapy with anti-PD-1 mAb and LTBR agonist mAb significantly increased tumor infiltration by immune cells. [Examples]

[0121] LTBR agonism increased the antitumor efficacy of T-cell therapy. LTBR agonism enhanced the antitumor efficacy of mouse hCD20 CAR-T therapy in an MC38-hCD20 tumor model.

[0122] The MC38-hCD20 cell line was created by transducing human CD20 into MC38 mouse colorectal tumor cells. To generate mouse hCD20 CAR T cells, mouse CD3 T cells were isolated from the spleen of wild-type C57BL / 6 mice, activated in vitro using IL-2 and CD3 / CD28 beads, and transduced with a retrovirus containing a CAR sequence for hCD20, including the CD3z and 4-1BB intracellular signaling domain (SEQ ID NO: 1) (Table 1). Mouse CAR T cells targeting unrelated antigens were used as a control (CTRL).

[0123] [Table 1]

[0124] In the MC38-hCD20 tumor model, three days before tumor transplantation (-3 days), mice underwent lymphocyte depletion chemotherapy by intraperitoneal administration of cyclophosphamide 250 mg / kg, and on day 0, 1 × 10⁶ 6100 MC38-hCD20 cells were subcutaneously transplanted into the right flank of C57BL / 6 mice. Six days after tumor transplantation (day 6), the mice were randomized to four treatment groups: the control group received intraperitoneal injection of isotype control (rat IgG2a, 2A3) mAb 5 mg / kg and intravenous injection of 3 million CTRL CAR T cells; the LTBR agonist group received intraperitoneal injection of anti-mouse LTBR agonist (5G11) mAb 5 mg / kg and intravenous injection of 3 million CTRL CAR T cells; the anti-hCD20 CAR T group received intraperitoneal injection of isotype control mAb 5 mg / kg and intravenous injection of 3 million anti-hCD20 CAR T cells; and the combination group received intraperitoneal injection of anti-mouse LTBR agonist mAb 5 mg / kg and intravenous injection of 3 million anti-hCD20 CAR T cells.

[0125] Anti-mouse LTBR agonist mAbs and isotype control mAbs were administered twice a week for a total of four doses (days 6, 9, 13, and 16). Tumor volume was calculated from caliper measurements using the formula L × W × W × 0.5, where L is the longest dimension and W is the vertical dimension. Combination therapy with LTBR agonists and hCD20 CAR T showed a significantly higher antitumor effect compared to either treatment alone (Figure 4). [Examples]

[0126] Induction of robust tumor-associated TLS by a combination of LTBR agonism and LTα expression This example describes robust induction of tumor-associated TLSs by combined treatment with an LTBR agonist antibody and forced expression of cytokines / chemokines by Colon26 tumors. In vivo tumor screening revealed that combinations of LTBR agonism and local expression of CXCL13, CCL19, CCL21, or LTα induced the formation of TLS-like structures in Colon26 tumors. Combinations of CXCL13 and CCL19 / CCL21 expression with LTBR agonism induced B-cell clusters and immature TLSs, respectively. Combining LTBR agonism with LTα expression demonstrated robust TLS induction.

[0127] In vivo screening of cytokine / chemokine-expressing Colon26 tumor models (Table 2). To perform in vivo screening of TLS-inducing factors, a panel of Colon26 cell lines was generated by lentiviral transduction to express different TLS-related cytokines, as shown in the left column of Table 2. 1 × 10 6 Colon26 cells expressing a specific cytokine were subcutaneously transplanted into the right flank of BALB / c mice.

[0128] The tumor is 50-100 mm 3 Upon reaching a certain stage, mice were randomized to a treatment group and administered either an anti-mouse LTBR agonist (5G11) mAb or an isotype control (rat IgG2a, 2A3) mAb at 2 mg / kg intraperitoneally twice a week for a total of three doses. After 10 days of treatment, mice were sacrificed, and excised tumor samples were fixed in 10% neutral buffered formalin at 4°C for 48 hours, then replaced with 70% ethanol, embedded in paraffin, and sectioned for immunofluorescence staining. High endothelial venules (HEVs) were stained with MECA-79 antibody, which recognizes HEV-specific PNAd epitopes. T cells, B cells, and dendritic cells were stained with anti-CD3, anti-B220, and anti-CD11c antibodies, respectively.

[0129] As summarized in Table 2, the combination of LTBR agonism and local expression of CXCL13, CCL19, CCL21, or LTα induced the formation of B-cell aggregates / TLS-like structures in Colon 26 tumors.

[0130] [Table 2]

[0131] The combination of LTBR agonist treatment and CXCL13 expression induced dispersed TLS-like structures primarily within tumors and without HEVs. CCL21 and CCL19 expression showed similar effects, inducing immature TLS-like structures without T cell zones. In particular, the combination of LTBR agonist and LTα expression induced robust TLS formation in both Colon26 (BALB / c) and MC38 (C57BL / 6) tumor models. Activated T cells and B cells were identified by co-expression of Ki67 and CD3 or B220, respectively. PDPN+ fibroblast-like reticular (FRC) networks and dendritic cells were found in T cell zones; regulatory T cells were also observed within TLS detected by CD4 and FOXP3 expression. [Examples]

[0132] Tumor-associated TLS formation enhanced the tumor response to anti-CTLA-4 treatment. This example relates to the effects of a combination of treatment with an LTBR agonist and anti-CTLA-4 on the growth of Colon26 and MC38 tumors generated to express LTα. Anti-CTLA-4 treatment promoted complete regression of TLS+ Colon26 tumors, and the combination of an LTBR agonist and anti-CTLA-4 treatment showed an antitumor effect benefit, particularly in LTα-expressing tumors in the MC38-OVA model.

[0133] TLS+ Colon26 tumor (Figure 5). Anti-CTLA-4 treatment promoted complete regression of TLS+ Colon26 tumors. In the TLS+ Colon26 model, 1 × 10⁶ LTα-expressing Colon26 cells were subcutaneously transplanted into the right flank of BALB / c mice on day 0. All tumor-bearing mice were intraperitoneally injected with the anti-mouse LTBR agonist (5G11) mAb 2 mg / kg on days 10, 13, 17, and 21. Mice were deconditioned to the treatment group on day 17 and intraperitoneally injected with anti-PD-1 (RPM1-14) mAb 10 mg / kg and / or anti-CTLA-4 (9D9) mAb 5 mg / kg on days 17, 21, 25, and 28.

[0134] Tumor volume was calculated from caliper measurements using the formula L × W × W × 0.5, where L is the longest dimension and W is the vertical dimension. Anti-PD-1 mAb treatment resulted in tumor remission in 3 out of 7 mice, while anti-CTLA-4 mAb treatment resulted in the elimination of all TLS+ Colon26 tumors. Follow-up studies showed that the combination of anti-CTLA-4 mAb with an LTBR agonist and / or LTα expression promoted complete regression of large (approximately 200 mm3) Colon26 tumors (Figure 5).

[0135] MC38-OVA tumor model (Figure 6). LTBR agonists and anti-CTLA-4 treatment showed a combined antitumor benefit, particularly in LTα-expressing tumors in the MC38-OVA model. To further analyze the combined effects of anti-CTLA-4 mAbs with LTBR agonists and LTα expression, a triple combination study was replicated in MC38-OVA models with greater resistance to anti-CTLA-4 treatment.

[0136] MC38-OVA cells were transduced using a lentivirus carrying an empty vector (EV) or LTα to create MC38-OVA cell lines expressing EV and LTα. In the MC38-OVA tumor model, 1 × 10⁻¹⁶ cells were transduced. 6 MC38-OVA cells expressing EV or LTα were subcutaneously transplanted into the right flank of C57BL / 6 mice. The tumor was approximately 100 mm. 3 Upon reaching a certain level, mice were randomized to a treatment group and administered either an anti-mouse LTBR agonist (5G11) mAb or an isotype control (rat IgG2a, 2A3) mAb at 2 mg / kg, and / or an anti-CTLA-4 (9D9) mAb at 5 mg / kg, intraperitoneally twice a week for a total of four doses.

[0137] Tumor volume was calculated from caliper measurements using the formula L × W × W × 0.5, where L is the longest dimension and W is the vertical dimension. Treatment with anti-CTLA-4 mAb and LTBR agonist mAb showed a combined effect, particularly delaying tumor progression in LTα-expressing TLS+ tumors (Figure 6). [Examples]

[0138] LTBR-mediated immunomodulation in the tumor microenvironment promotes antitumor responses. The presence of high endothelial venules (HEVs) and tertiary lymphoid structures (TLSs) in solid tumors correlates with a favorable prognosis in many cancer types and is associated with a good treatment response to immune checkpoint blockade (ICB). However, the molecular mechanisms underlying intratumoral HEV and TLS formation, as well as their contribution to the antitumor response, remain unclear. Lymphotoxin beta receptor (LTBR) signaling is a key regulator of lymph node organogenesis, and combining anti-angiogenic treatment with ICB treatment can increase tumor-associated HEV formation. This example demonstrates that LTBR signaling modulates the tumor microenvironment through multiple mechanisms to promote the antitumor T cell response. Systemic activation of the LTBR pathway by agonist antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-associated chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in a syngeneic tumor model. In vitro studies confirmed the direct effects of LTBR agonism on DC activation and maturation, as well as associated DC-mediated T cell activation. Monotherapy with LTBR agonists inhibited syngeneic tumor growth in a CD8 T cell and HEV-dependent manner and enhanced the antitumor effects of anti-PD-1 and CAR T therapies. In vivo tumor screening of TLS-inducing cytokines revealed that the combination of LTBR agonism and lymphotoxin alpha (LTα) expression promoted robust intratumoral TLS induction and enhanced the tumor response to anti-CTLA-4 treatment. In summary, these studies highlight the crucial role of LTBR signaling in modulating the tumor microenvironment and will be useful for future therapeutic strategies to enhance immune cell infiltration and activation in solid tumors.

[0139] introduction Immune checkpoint blockade (ICB) has emerged as a promising strategy for activating antitumor cytotoxic T cells, highlighted by unprecedented patient responses across multiple cancer types. However, not all patients respond to ICB, and a lack of sufficient immune cell infiltration into the tumor constitutes one potential limiting factor. High endothelial venules (HEVs) are specialized vessels that mediate the transport of lymphocytes to lymph nodes (LNs) and Peyer's patches. High endothelial cells are characterized by high expression of adhesion molecules, ligands for lymphocyte homing receptors, including L-selectin (CD62L) (Gerard 2012). HEVs are specifically labeled by MECA-79 antibody staining of peripheral lymph node adresin (PNAd) in both mice and humans (Streeter 1988, Berg, 1991). PNA in human tumors + The presence of HEVs is frequently associated with T cell and B cell infiltration, good overall survival, and response to ICB treatment (Martinet 2011, Martinet 2012, Asrir 2022, Hua 2022). Mouse tumor studies have demonstrated that tumor-associated HEVs (TA-HEVs) may function as a major entry point for lymphocyte infiltration into tumors (Asrir 2022). Tertiary lymphoid structures (TLSs) are ectopic lymphoid formations that occur in inflammatory or tumor tissue. TLSs are associated with HEVs, as well as Cxcl13 + Follicular dendritic cells (FDCs) and Ccl19 +Because it contains characteristic B and T cell aggregates organized by fibroblast-like reticular cells (FRCs), it is structurally similar to secondary lymphoid tissue (Sautes-Fridman 2019). The presence of TLS in solid tumors is generally associated with a good prognosis and a good response to immunotherapy (Sautes-Fridman 2019, Cabrita 2020, Helmink 2020, Petitprez 2020). Tumor-associated TLS (TA-TLS) has been shown to function as a site for B cell maturation to antitumor antibody-producing plasma cells in renal cell carcinoma (Maylan 2022). Furthermore, TLS-associated mature dendritic cells (DCs) correlate with effector-memory T cell phenotype and long-term survival in non-small cell lung cancer (NSCLC) (Goc 2014), suggesting in situ T cell priming in TA-TLS.

[0140] Lymphotoxin beta receptor (LTBR) signaling plays a crucial role during LN organogenesis in LTBR knockout mice lacking LNs and Peyer's patches (Futterer 1998). Mechanistically, LTBR signaling induces the expression of adhesion molecules and lymphoid chemokines in stromal cells of LN primordia, promoting immune cell cluster formation, structural separation, and niche maturation (Onder 2018). Sustained LTBR signaling is required for the expression of PNAd scaffold proteins and core enzymes in high-endothelial cells, and is therefore critical to the homeostatic regulation of HEV differentiation and function (Browning 2005). TLS formation replicates LN development and is dependent on LTBR signaling in both autoimmune and tumor contexts (Browning 2008, Rodriguez 2021). LTBR activation, when combined with ICB and / or anti-angiogenic treatment, induced TA-HEV differentiation and maturation while enhancing the tumor response (Asrir 2022, Hua 2022, Allen 2017). Despite the crucial role of LTBR signaling in HEV and TLS formation, the underlying mechanisms of LTBR agonism-mediated antitumor effects remain poorly understood. We investigated the antitumor immune response induced by systemic LTBR activation via agonist antibody treatment in a syngeneic mouse tumor model. LTBR agonist treatment promoted tumor-specific HEV formation, upregulated TLS-related chemokine expression, and resulted in increased immune cell infiltration into tumors. LTBR agonism showed a direct effect on DC licensing in vitro and enhanced DC and T cell activation, as well as the antitumor effects of immunotherapy, in vivo. Finally, combining LTBR agonism with LTα expression significantly increased TA-TLS induction and enhanced the tumor response to anti-CTLA-4 treatment.

[0141] material and method Mice: 8-10 week old female BALB / c and C57BL / 6J wild-type mice, Prkdc scid (B6.Cg-Prkdc scid / SzJ) SCID mouse, TNFR1- / - Mouse (C57BL / 6-Tnfrsf1a) tm1Imx The C57BL / 6J (C57BL / 6-Tg(TcraTcrb)1100Mjb / J) and OT-I mice were purchased from Jackson Labs. Sirpa in the background on the C57BL / 6J. hu / hu Rag2 - / - Il2rg - / - SRG mice were generated using VelociGene technology. All animals were maintained under sterile conditions.

[0142] Cells and cell lines: Cancer cell lines Colon26 (HCT876), MC38 parent (HCT510), and variant cell lines expressing cytoplasmic ovalbumin (MC38-OVA, HCT1434), and human CD20 (MC38-hCD20, ACL20303), 4T1 (HCT456), and B16-F10 (HCT652) were obtained from the Regeneron cell bank. The pancreatic cancer cell line FC1242 was provided by David Tuveson, CSHL. Colon26 and MC38-OVA cell lines expressing chemokines or cytokines, used for in vivo screening of TA-TLS, were generated by transducing lentiviruses carrying mouse transgenes Cxcl9, Cxcl12, Cxcl13, Ccl19, Ccl21b, Vegfc, Il17a, or Lta, and neomycin resistance genes linked by IRES sequences, followed by selection of G418. LTα-expressing Colon26 cell lines used for scRNA-seq experiments were generated by transducing lentiviruses carrying mouse Lta and eGFP genes linked by IRES sequences. Colon26 and 4T1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin / glutamine (Gibco). MC38 cells were cultured in DMEM supplemented with 10% FBS, 1% MEM non-essential amino acids (NEAA, Gibco), 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 1% penicillin / streptomycin / glutamine. B16-F10 and FC1242 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin / glutamine. Cells were cultured at 37°C in 5% CO2. Cell proliferation was measured using Cell Counting Kit 8 (Abcam).

[0143] Endothelial cell lines SVEC4-10 (HCT1247), b.End3 (HCT669), and dendritic cell line MutuDC1940 (HCT936) were obtained from the Regeneron cell bank. SVEC4-10 and b.End3 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin / glutamine. MutuDC1940 cells were cultured in complete IMDM supplemented with 10% FBS, 4 mM GlutaMAX, 1% 7.5% sodium bicarbonate solution, 50 μM 2-mercaptoethanol, and 1% penicillin / streptomycin / glutamine. Cells were cultured at 37°C in 5% CO2.

[0144] To generate mouse hCD20 CAR T cells, CD3 T cells were isolated from the spleen of wild-type C57BL / 6J mice using an untouched mouse T-cell isolation kit (Invitrogen) and subsequently activated with anti-mouse CD3 / CD28 Dynabeads (Invitrogen) and IL-2 (20 U / ml; Peprotech). After 16 hours, the T cells were transduced by spin infection on plates coated with Retronectin (Takara) using a retrovirus encoding anti-hCD20 CAR containing mouse CD3z and 4-1BB intracellular signaling domains. Mouse CAR T cells binding to unrelated antigens were used as a control.

[0145] Mouse tumor research In tumor studies using LTBR agonist treatment, 1 × 10⁶ was used in conjunction with 50% Matrigel (Corning). 6 MC38-OVA or transgenic variant of individual cells, 5 × 10 5 B16-F10 of individual cells, or 5 × 10 5 Individual F1242 cells were subcutaneously transplanted into the right flank of female C57BL / 6 mice, resulting in 1 × 10⁶ cells. 6 Individual Colon26 or transgenic variant cells were subcutaneously transplanted into the right flank of female BALB / c mice, resulting in 5 × 10⁶ cells. 5Individual 4T1 cells were injected into the mammary fat pad of female BALB / c mice. The tumors were 50-100 mm in size. 3Upon reaching a certain stage, animals were randomized to a treatment group and intraperitoneally injected 2 mg / kg of anti-mouse LTBR agonist (5G11, Enzo, or Abcam) or isotype control (rat IgG2a, 2A3, Bio X Cell) antibody twice a week for a total of 2-4 doses. For macrophage depletion, mice were intraperitoneally treated twice a week with anti-CSF1R (AFS98, Bio X Cell) or isotype control (2A3, Bio X Cell) antibody 20 mg / kg on days 5, 8, 12, and 15 post-tumor transplantation, and with anti-mouse LTBR agonist or isotype control antibody 2 mg / kg on days 12 and 15 post-tumor transplantation. To block cytokines, mice were intraperitoneally treated with 25 mg / kg of anti-mouse TNFα antagonist (XT3.11, Bio X Cell) or isotype control (HRPN, Bio X Cell) antibody, or 25 mg / kg of IL-1 trap (Kuhnert 2015) (Regeneron) or isotype control (mouse IgG2a, Regeneron) antibody on days 11, 14, and 17 post-tumor transplantation, and with 2 mg / kg of anti-mouse LTBR agonist or isotype control antibody on days 12 and 15 post-tumor transplantation. To block the VEGF and Dll4 angiogenic pathways, mice were intraperitoneally injected with VEGF-trap (Daly 2013) (aflibercept, Regeneron), anti-Dll4 (Amend 2016) (Regeneron), or human Fc control antibody 5 mg / kg on days 14, 17, and 21 post-tumor transplantation, and with anti-mouse LTBR agonist or isotype control antibody 2 mg / kg on days 17 and 21 post-tumor transplantation. The pro-angiogenic Ang2 pathway was blocked by co-administration of anti-Ang2 (Brasel 2000) (Regeneron) or human Fc control antibody 10 mg / kg with anti-mouse LTBR agonist or isotype control antibody 2 mg / kg in a total of two doses, twice a week. To deplete CD8 T cells, mice were intraperitoneally administered 10 mg / kg of anti-CD8α (2.43, Bio X Cell) or isotype control (LTF-2, Bio X Cell) antibody on day 2 and day 0 after tumor transplantation, and then administered twice a week until the end of the study.Anti-mouse LTBR agonists or isotype control antibodies were administered intraperitoneally twice a week for a total of four doses starting on day 8 post-tumor transplantation. Blood was collected posterior orbitally and immediately treated with the LTBR agonist to verify CD8 T cell depletion. To block PNAd-specific TA-HEV function, mice were intraperitoneally administered 2 mg / kg of anti-mouse LTBR agonist or isotype control antibody on days 12, 15, and 19 post-tumor transplantation, and 10 mg / kg of MECA-79 (purified by BioLegend Ultra-LEAF®) or rat IgM control (RTK2118, purified by BioLegend Ultra-LEAF®) antibody on days 13, 15, 17, and 19 post-tumor transplantation. For the combined study of LTBR agonism and anti-PD-1, tumor-bearing mice were concurrently treated intraperitoneally twice a week with a total of three doses of anti-mouse LTBR agonist 2 mg / kg and / or anti-PD-1 (RMP1-14, Bio X Cell) antibody 10 mg / kg. To study the combination of LTBR agonism and CAR T-cell therapy, 1 × 10⁶ cells were administered to the right flank. 6 Three days prior to subcutaneous transplantation of individual MC38-hCD20 tumor cells, C57BL / 6J mice were subjected to lymphocyte depletion chemotherapy by intraperitoneal administration of cyclophosphamide 250 mg / kg (Sigma). Six days after tumor transplantation, the mice received 3 × 10⁶ doses. 6 Individual hCD20 or control CAR T cells were intravenously injected. Starting on day 6, mice were also intraperitoneally administered 5 mg / kg of anti-mouse LTBR agonist or isotype control antibody twice a week for a total of four doses. For scRNA-seq studies, 2 × 10⁶ mice were administered. 6Individual cells of Colon26-GFP or Colon26-LTα-GFP were subcutaneously transplanted into the right flank of female BALB / c mice, and 2 mg / kg of an anti-mouse LTBR agonist or isotype control antibody was administered intraperitoneally twice a week. For combined studies of LTBR agonism, anti-PD-1, and anti-CTLA-4 in the Colon26-EV / LTα tumor model, all drugs were administered intraperitoneally twice a week in a total of four doses. ICB treatment, i.e., 10 mg / kg of anti-PD-1 or isotype control antibody and / or 5 mg / kg of anti-CTLA-4 (9D9, InvivoGen) or isotype control (mouse IgG2a, Regeneron) antibody, was administered 4 or 7 days after treatment with the anti-mouse LTBR agonist or isotype control antibody to enable TA-TLS formation before ICB treatment. In detail, to achieve similar tumor sizes among different groups at the start of ICB treatment, ICB treatment was initiated 7 days after LTBR agonist treatment in LTα-expressing tumors, and 4 days after LTBR agonist or isotype control antibody treatment in all other conditions. For the combined study of LTBR agonism and anti-CTLA-4 in the MC38-OVA-EV / LTα tumor model, approximately 100 mm 3 Mice with tumors were concurrently treated intraperitoneally twice a week with a total of three doses: 2 mg / kg of an anti-mouse LTBR agonist or isotype control antibody, and 5 mg / kg of an anti-CTLA-4 or isotype control antibody. Tumor growth was assessed by measuring tumor volume using a digital caliper. Tumor volume was calculated using the formula L × W × W × 0.5, where L is the longest dimension and W is the smaller vertical dimension. Animals were shown for histological dissection, or when the tumor was 2000 mm 3 They were euthanized by CO2 asphyxiation when they grew larger or when ulcers developed.

[0146] Immunostaining analysis The tissue was fixed in 10% neutral buffered formalin at 4°C for 48 hours, then replaced with 70% ethanol and embedded in paraffin. 4 μm thick sections were collected using a microtome and heated at 65°C for 1 hour. The sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval for 20 minutes at 95°C in citrate buffer at pH 6.0 or Tris buffer at pH 9.0 (Vector Laboratories) before the blocking step. The sections were stained with primary antibody overnight at 4°C, washed with PBS, and stained with secondary antibody at room temperature for 1 hour. Finally, the slides were washed with PBS, stained with DAPI for 5 minutes, and mounted using Fluoromount aqueous mounting medium (Sigma-Aldrich) for immunofluorescence staining. For multiplex immunofluorescence staining using multiple rabbit primary antibodies, the Opal 4-Color Anti-Rabbit IHC Kit (Akoya Biosciences) was used. For immunohistochemical (IHC) staining, sections were incubated with 3% H2O2 (vol / vol, Sigma-Aldrich) for 10 minutes before the blocking step, stained with PowerVision Poly-HRP anti-Rabbit IHC Detection Systems (Leica Biosystems), counterstained with hematoxylin (Agilent), and mounted using Epredia Cytoseal Mountant (Thermo Fisher Scientific). The following primary antibodies were used: AF488 conjugate MECA-79 (Invitrogen), anti-CD31 (ab28364, Abcam), AF647 conjugate anti-B220 (RA3-6B2, BioLegend), anti-CD3 (SP7, Abcam), anti-CD4 (EPR19514, Abcam), anti-CD8α (D4W2Z, Cell Signaling Technology), anti-Ki67 (D3B5, Cell Signaling Technology), anti-PDPN (RTD4E10, Abcam), anti-CD11c (D1V9Y, Cell Signaling Technology), anti-CXCL13 (EPR19259-147, Abcam), and anti-LYVE1 (NB600-1008, Novus Biologicals).

[0147] Fluorescent images were taken using a Zeiss Axio Imager 2 microscope (Zeiss) or scanned by a Zeiss Axioscan 7 microscope slide scanner (Zeiss) equipped with a 20x objective lens. IHC images were acquired by an Aperio AT2 slide scanner (Leica Biosystems). Image analysis and quantification were performed using Indica HALO software (Indica Labs) on scanned images of whole sections. To quantify the number and area of TLSs, B220 + B cells and CD3 + T cells were included, and aggregates larger than 7000 μm 2 in size were identified as TLSs by the HALO AI™ deep learning classification function and normalized to the non-necrotic tumor area.

[0148] Flow cytometry The tumors were excised, mechanically chopped, and dissociated into single-cell suspensions using a mouse tumor dissociation kit according to the manufacturer's protocol (Miltenyi). These suspensions were then resuspended in FACS buffer containing PBS supplemented with 2% FBS, 2 mM EDTA (VWR), and 0.1% sodium azid (VWR). Cell cultures were washed in vitro with PBS, dissociated with TrypLE® Express Enzyme (Gibco), and resuspended in FACS buffer. Dead cell identification was performed using Zombie Violet® Fixable Viability Kit (BioLegend). Cells were incubated with TrueStain Mouse FC-block (BioLegend) before antibody staining. Surface antigens were stained by first incubating an antibody cocktail in FACS buffer at 4°C for 30 minutes. For intracellular staining, cells were fixed and permeabilized, and then intracellular antigens were stained using the eBioscience Foxp3 / transcription factor staining buffer set according to the manufacturer's protocol (Thermo Fisher Scientific).The following anti-mouse antibodies were used (BioLegend unless otherwise indicated): BV785 anti-CD31 (390), AF488 anti-PNAd (MECA-79, Invitrogen), PE anti-E-selectin (10E9.6, BD Biosciences), APC anti-P-selectin (RB40.34, BD Biosciences), BV510 anti-ICAM-1 (YN1 / 1.7.4, BD Biosciences), PE / Cy7 anti-VCAM-1 (429, BD Biosciences), BUV496 anti-CD45 (30-F11, BD Biosciences), BV510 anti-CD90.2 (30-H12, BD Biosciences), PE anti-CD3 (17A2), BV605 anti-CD4 (RM4-5), anti-CD8α (53-6.7, BD Biosciences), anti-CD19 (1D3, BD Biosciences), anti-CD11b (M1 / 70, BD Biosciences), anti-CD11c (N418), anti-MHCII (M5 / 114.15.2), anti-MHCI (M1 / 42), anti-NKp46 (29A1.4), anti-CD49b (HMα2), anti-F4 / 80 (BM8), anti-CD103 (2E7), anti-CD40 (3 / 23), anti-CD44 (IM7) ), anti-CD62L (MEL-14), PE anti-CD69, BV650 anti-CD80 (16-10A1), anti-CD83 (Michel-19), anti-CD86 (GL-1), anti-GzmB (GB11), anti-ICOSL (HK5.3), anti-IFNg (XMG1.2), anti-Ki67 (SolA15, Thermo Fisher The drugs used were anti-MAdCAM-1 (MECA-367), anti-PD-1 (RMP1-30), anti-SLAMF6 (330-AJ), anti-TCF1 (C63D9, Cell Signaling Technology), anti-Tim-3 (RMT3-23), anti-TNFα (MP6-XT22), and BUV395 streptavidin (BD Biosciences). Data were acquired using LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo.

[0149] Gene expression analysis using qPCR The tumor was excised and recovered in TRI reagent (Invitrogen) containing 5 mm stainless steel beads (Qiagen), and dissociated using TissueLyser II (Qiagen) at 29 Hz for 2 minutes. RNA was extracted using MagMAX™-96 (Invitrogen) for microarray total RNA isolation kits, and cDNA was constructed using SuperScript IV First-Strand Synthesis System (Invitrogen) according to the manufacturer's protocol. Subsequently, qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and qPCR primers designed for the gene of interest on a CFX96 real-time PCR detection system instrument (Bio-Rad). Relative gene expression was calculated using β-actin as a housekeeping gene.

[0150] LTBR activation in vitro in EC and DC cell lines For EC activation, 96-well plates were coated overnight at 4°C with 10 μg / mL of polyclonal goat anti-rat IgG F(ab')2 fragment in PBS (Jackson ImmunoResearch Labs), washed with PBS, blocked with 2% BSA (Sigma-Aldrich) at room temperature for 1 hour, incubated with 1 μg / mL of LTBR agonist (5G11, Enzo, or Abcam) or isotype control (rat IgG2a, 2A3, Bio X Cell) antibody for 1 hour, and washed with PBS. Subsequently, 1 × 10⁶ cells per well were placed in complete medium supplemented with PBS, mouse TNFα 10 ng / mL (R&D Systems), mouse IL-β 10 ng / mL (R&D Systems), or LPS 10 μg / mL (Sigma-Aldrich). 4 Individual SVEC4-10 or b.End3 cells were seeded, incubated for 18 hours, and then harvested for flow cytometry analysis.

[0151] For DC activation, 12-well plates were coated overnight at 4°C with 10 μg / mL of LTBR agonist or isotype control antibody in 0.2% gelatin (Sigma-Aldrich), or with 0.2% gelatin alone, and washed with PBS. MutuDC1940 cells were treated with LPS or PBS 10 μg / mL for 2 hours before dissociation. Then, 3 × 10⁶ cells were collected per well. 5 Individual DC cells were seeded in complete medium, incubated for 18 hours, and then harvested for flow cytometry analysis.

[0152] BMDC differentiation and co-culture with OT-I T cells As described (Amend 2016), myeloid cells were isolated from the femur and tibia of wild-type C57BL / 6J mice. The isolated myeloid cells were resuspended in 10% ammonium chloride solution in PBS (STEMCELL Technologies) at room temperature for 2 minutes to lyse the erythrocytes, and washed in BMDC medium consisting of McCoy's 5A (modified) medium (Gibco) supplemented with 10% FBS, 1% MEM NEAA, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 1% penicillin / streptomycin. For DC differentiation, myeloid cells were divided into 1 × 10⁶ cells per 1 mL in a 6-well plate. 6 Cells were cultured for 8 days in BMDC medium containing human FLT3L (BioLegend) 200 ng / mL (Brasel 2000). Cells were cultured at 37°C in 5% CO2. After 8 days, BMDC was collected from the culture by vigorous pipetting to collect non-adherent and loosely adherent cells, which were then cultured for 18 hours in BMDC medium containing LTBR agonist (5G11) 160 μg / mL or isotype control antibody. For flow cytometry analysis, 1 × 10⁶ cells were collected per well. 5 For co-culture of individual cells and OT-I T cells, use 3 × 10⁶ cells per well. 4 Individual BMDC cells were seeded into a 96-well plate.

[0153] For BMDC-T cell co-culture, BMDCs treated with LTBR agonist or isotype control antibody were pulsed with OVA 257-264 10 ng / mL (AnaSpec) or scrambled peptide (AnaSpec) for 2 hours at 37 °C and washed with T cell medium consisting of RPMI-1640 medium supplemented with 10% FBS, 50 μM 2-mercaptoethanol, and 1% penicillin / streptomycin / glutamine. OT-I CD8 T cells were isolated from the spleens of OT-I mice (C57BL / 6-Tg(TcraTcrb)1100Mjb / J) using the EasySep™ Mouse CD8+ T Cell Isolation Kit (STEMCELL Technologies) and labeled with CellTrace™ Far Red Cell Proliferation Kit (Thermo Fisher Scientific). 1.5 × 10 5 OT-I cells per well were seeded in T cell medium to achieve a 1:5 DC:T cell ratio. After 3 days of co-culture, T cells were harvested for flow cytometry analysis.

[0154] Adoptive transfer of OT-I T cells MC38-OVA bearing tumor mice were treated intraperitoneally twice a week with LTBR agonist (5G11) or isotype control antibody 2 mg / kg, 7 days after tumor implantation. Five days after LTBR agonist treatment, FTY720 1.5 mg / kg (Cayman Chemical) or DMSO containing 2% β-hydroxypropyl-cyclodextrin (Sigma-Aldrich) in PBS was administered intraperitoneally and then every 2 days. OT-I CD8+ T cells were isolated from the lymph nodes and spleens of OT-I mice as previously described and labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific). Two days after FTY720 treatment, 2.5 × 10 6 OT-I CD8+ T cells per animal were administered by retro-orbital injection and blood, tumor perfused lymph nodes and tumor tissue were collected 5 days later for flow cytometry analysis.

[0155] Single-cell RNA-seq For scRNA-seq of Colon26-GFP / LTα-GFP tumors treated with LTBR agonists or isotype control antibodies for 7 days, the study design and preparation of tumor single-cell suspensions have been previously described. After staining with anti-CD45 and anti-CD31 antibodies at 4°C for 30 minutes, tumor dissociated cells were identified by FACS as endothelial cells (GFP). - CD45 - CD31 + ), interstitial cells (GFP - CD45 - CD31 - ), and immune cells ((GFP) - CD45 + CD31 - Cells were sorted and mixed for loading. Dead cells were identified during sorting by DAPI staining. Single cells suspended in PBS containing 0.04% BSA were loaded onto a Chromium X Controller (10X Genomics) with 10K cells per lane or the maximum number of cells available. RNA-seq and V(D)J libraries were prepared using the Chromium Next GEM Single Cell 5' Kit, v2 (10X Genomics). After amplification, cDNA was split into separate RNA-seq and V(D)J aliquots. Paired-end sequencing was performed on the RNA-seq library using an Illumina NovaSeq 6000 (read 1: 26 bp for UMI and cell barcode, read 2: 80 bp for transcript reads, including 10 bp i7 and 10 bp i5 reads). For the RNA-seq library, Cell Ranger Single Cell Software Suite (10X Genomics, 6.1.1) was used to perform sample demultiplexing, alignment, filtering, and UMI counting. A mouse GRCm38 genome assembly was used for alignment.

[0156] Single-cell RNA-seq dataset analysis Initial filtered scRNA-seq data obtained from Cell Ranger software was used. Droplets with more than 10% mitochondrial reads or fewer than 200 expressed genes (defined as UMI>0) were excluded from downstream analysis, as they were likely to contain dead cells or be empty droplets, respectively. After initial filtering, a library size correction method was applied to normalize to raw counts using the "normalize_total" function of scanpy (Wolf 2018) with the parameter "target_sum" = 10,000. Highly variable genes were selected for downstream clustering analysis using the "scanpy.pp.highly_variable_genes" function with the parameters "min_mean" = 0.02, "max_mean" = 4, and "min_disp" = 0.5. Subsequently, the "scanpy.pp.regress_out" function was used to remove the influence of the percentage of total counts and mitochondrial gene counts per droplet. Next, principal component analysis was performed using the `scanpy.tl.pca` function with the parameter `scd_solver=`arpack`. Dimensionality reduction of the dataset was performed using the `scanpy.tl.umap` function with the parameters `n_neighbors`=15 and `n_pcs=20`, and clustering was performed using the `scanpy.tl.leiden` function with the parameter `resolution=1.4`. Cluster-specific marker genes were identified using the `scanpy.tl.rank_gene_groups` function with default parameters, and major cell types were annotated based on the expression of Ptprc(CD45) and Pecam1(CD31). CD45 +The dominant immune cell types within each cluster were identified as Cd3e (T cells), Cd19 (B cells), and Cd14 / Fcgr4 (monocytes / macrophages / dendritic cells). Clustering identified clusters with a low number of expressed genes (less than 1000 in all cells within the cluster, likely indicating dead cells) or clusters expressing multiple markers that should be removed (likely indicating duplication). UMAP and Leiden clustering were performed again on the remaining droplets to generate the final clusters using the parameter "resolution=0.4". The resolution selection was based on whether the cluster definition could separate the desired cell population.

[0157] To identify a subgroup within endothelial cells, Pecam1 + Endothelial cells were reprocessed using the workflow described in a previous section and clustered using the Leiden algorithm with the parameter "resolution=0.2".

[0158] CD31 - CD45 - In the cluster, Bmp2 + / Bmp4 + / eGFP + The clusters that were present were removed (Figure S8A, clusters 6, 7, and 12), which are cancer cells that have undergone the depletion process due to low eGFP expression. The remaining stromal cells were reprocessed and clustered using the Leiden algorithm with the parameter "resolution=0.3". CD45 + CD3 + Within the T cell cluster, the cells were reprocessed and clustered by Leiden with a resolution of 0.8. Clusters expressing Cd8a were identified, reprocessed, and subjected to UMAP and clustering again. + We identified subtypes of T cells.

[0159] All UMAP plots, including those containing gene expression, were generated using the CP10K value from the `scanpy.pp.normalize_total` function with `target_sum` = 10,000. For heatmaps of marker gene expression in each cluster, the mean Log(CP10K+1) value was calculated across all cells in the cluster, and then, for each gene, the inventors normalized the cluster-average expression value by calculating the z-score across clusters.

[0160] CD8 + T cell precursor and depletion scores were calculated using the `scanpy.tl.score_genes` function with parameters `ctrl_size` = 100 and `n_bins` = 24, and the gene set definition was based on Pai et al., 2023 (Pai 2023). The significance of score differences between clusters was calculated by a two-sample t-test, and the p-value was corrected for multiple tests using the Benjamini-Hochberg correction.

[0161] Quantitative and statistical analysis Sample size was empirically selected to ensure sufficient statistical power, following the standards of the technical field used in this study. Where feasible, multiple independent experiments were conducted. Statistical significance was determined by unpaired two-tailed Student's t-tests, or by one-way and two-way analysis of variance (ANOVA) with multiple comparison analysis for experiments with more than two groups. Statistical details for each experiment can be found in the legend of the figures. Bar graphs show the mean ± SEM. A p-value < 0.05 was considered significant. * :p<0.05; ** :p<0.01; *** :p<0.001; **** (p<0.0001).

[0162] result Systemic LTBR activation by agonist antibodies induced tumor-specific HEV and TLS formation. To test the effect of systemic LTBR activation on HEV induction, syngeneic mice with subcutaneous colorectal tumors were treated with the LTBR agonist antibody 5G11 at 2 mg / kg twice weekly. TA-HEVs, identified by PNAd immunostaining using MECA-79 antibody, were readily detected in Colon26 and MC38-OVA tumors 7 days after LTBR agonist treatment, but not in tumors treated with isotype control antibodies. LTBR agonism-induced TA-HEVs exhibited the typical "swollen" morphology of LN hyperendothelial cells (HECs). LTBR agonist antibody treatment similarly induced TA-HEV formation in further mouse tumor types, including subcutaneous B16-F10 melanoma, FC1242 pancreatic tumor, and orthotopically transplanted 4T1 mammary gland tumors.

[0163] To further characterize the endothelial cell phenotype induced by LTBR agonism, flow cytometry analysis of tumor endothelial cells isolated from LTBR agonist-treated Colon26 tumors was performed. Consistent with immunohistochemical data, LTBR agonist treatment significantly increased the frequency of tumor-associated hyperendothelial cells (TA-HECs) and PNAd expression levels, but overall CD31 + Endothelial cell content did not change (Figure 8A). Expression of the inflammatory endothelial markers P-selectin, E-selectin, and ICAM-1 was observed in PNAd - Compared to EC, PNAd + HEC was significantly increased (Figure 8B). Lymphocyte immunohistochemistry revealed the presence of TA-TLS, characterized by organized lymphocyte aggregates with distinct B-cell and T-cell zones, in approximately 10% of LTBR agonist-treated Colon 26 tumors. TA-TLS formed mainly in the periphery of the tumor and frequently associated with HEV.

[0164] LTBR exhibits widespread expression in normal tissues, both in parenchymal and stromal compartments (Consortium 2020, Eraslan 2022, MacParland 2018). To evaluate the effects of systemic LTBR activation on normal organs, brain, thymus, lung, liver, spleen, kidney, and intestine from LTBR agonist-treated MC38-OVA tumor-bearing mice were analyzed for HEV induction by MECA-79 immunostaining. Notably, no ectopic HEV formation was detected in any normal organ, including lymphoid tissue. Furthermore, histological analysis of LNs from LTBR agonist-treated mice showed normal tissue structure and HEV distribution. Overall, systemic LTBR activation by agonist antibodies induced tumor-specific HEVs, occasionally accompanied by TLS formation.

[0165] Excessive pro-inflammatory signals contribute to HEC differentiation. We investigated the mechanism of tumor-specific HEV induction by systemic activation of LTBR signaling. We examined the contribution of immune cells to TA-HEV formation in immunodeficient mice. TA-HEVs were identified in Sirpa, a patient with colorectal tumors. hu / hu Rag2 - / - Il2rg - / - (SRG) and Prkdc scid In (SCID) mice, levels were observed at levels comparable to those in immune hosts, indicating that T cells, B cells, natural killer (NK) cells, and innate lymphoid cells are not required for LTBR agonist-induced TA-HEV formation. Macrophage depletion by anti-CSF1R antibody treatment did not affect TA-HEV formation, indicating that Fc receptor-mediated clustering of LTBR agonist antibodies is not essential for TA-HEV formation. Similarly, neutralization of inflammatory cytokines TNFα or IL-1β by blocking antibodies did not affect LTBR agonist-induced TA-HEV formation. Finally, LT α3 -TNFR1 signaling has been reported to drive TA-HEV formation in intraperitoneal tumor models (Peske 2015). TNFR1 - / -When tested in mice, LTBR agonist treatment showed HEV-inducing activity in MC38 tumors, similar to wild-type mice.

[0166] To evaluate the induction of the HEV marker MAdCAM-1 and endothelial cell adhesion molecules, in vitro assays were established to further elucidate the roles of LTBR agonism and pro-inflammatory signaling in endothelial cell activation (Berg (1993)). Monotherapy with LTBR agonist antibodies or pro-inflammatory factors such as TNFα, IL-1β, and LPS significantly increased the percentage of cells expressing MAdCAM-1 in LN-derived endothelial SVEC4-10 cells (O'Connell). (1990) (Figure 9A). Importantly, the combination of LTBR agonism and any of the three pro-inflammatory signals significantly increased the frequency of MAdCAM-1 expression (Figure 9A). LTBR agonists also showed significant additive activity with TNFα, IL-1β, and LPS, which upregulate the expression of endothelial inflammation markers ICAM-1 and VCAM-1 in SVEC4-10 cells (Figure 9A). Similar combined effects on the induction of ICAM-1, VCAM-1, and P-selectin were observed in brain endothelial b.End3 cells (Omidi 2003) (Figure 9E). In summary, these data suggest that excessive inflammatory signaling contributes to HEC differentiation and explain the lack of effect of selective TNFα and IL-1β blockers on LTBR agonist-mediated HEV formation in tumors in vivo.

[0167] Blocking the VEGF receptor with the anti-VEGFR2 antibody DC101 has been shown to promote intratumoral HEV formation when combined with an anti-PD-L1 antibody (Allen 2017). Therefore, we investigated the intersection of LTBR agonism-induced TA-HEV formation and the activity of known tumor angiogenic pathways. Blocking of VEGF, Dll4-Notch, and Ang2-Tie2 signaling by VEGF-Trap, anti-Dll4 antibody, and anti-Ang2 antibody treatment did not affect LTBR agonist-mediated HEV formation in Colon26 tumors, suggesting that TA-HEC differentiation occurred independently of major tumor angiogenic pathways.

[0168] LTBR agonist treatment upregulated TLS-related chemokine expression and increased immune cell infiltration into tumors. Chemokine gene signatures have been reported to correlate with the presence of TA-TLS and favorable survival rates in patients with colorectal cancer, melanoma, and breast cancer (Coppola 2011, Messina 2012, Prabhakaran 2017). Expression of mouse homologs of human TLS-related chemokines in MC38 and Colon26 tumors was evaluated by qPCR analysis. LTBR agonist treatment was associated with overall upregulation of chemokine expression in MC38 tumors, significantly increasing the expression of Ccl4, Cxcl10, Cxcl13, Ccl19, and Ccl21 in the tumor microenvironment (Figure 10A). Flow cytometry analysis revealed tumor-infiltrating CD8 + T cell and DC frequencies were significantly increased in MC38 tumors treated with LTBR agonists (Figure 10B). Consistently, intratumoral expression of Cxcl13 and Ccl19 was significantly upregulated in Colon26 tumors treated with LTBR agonists (Figure 10C). LTBR agonism was observed in tumor-infiltrating CD45 in Colon26 tumors. + Immune cells, especially B cells, CD4 + The frequencies of T cells, DCs, and NK cells were significantly increased (Figure 10D). Overall, LTBR agonist treatment upregulated the expression of TLS-related chemokines and broadly increased immune cell infiltration into tumors.

[0169] LTBR agonism promotes DC licensing and DC-mediated T cell activation in the tumor microenvironment. LTBR signaling is involved in DC homeostasis and licensing (Kabashima 2005, Summers deLuca 2012, Summers deLuca 2011). The effect of LTBR agonism on tumor-infiltrating DCs in Colon26 tumors was evaluated. Flow cytometry analysis revealed that CD40 + Activated DC and CD40 + CD83 + The frequency of mature DCs was shown to be significantly increased by LTBR agonist treatment (Figure 11A). Corresponding to the enhanced DC activation, a significantly higher proportion of Ki67 was observed. + CD4 and CD8 + T cells were observed in tumors treated with LTBR agonists (Figure 11B). LTBR agonism also affects CD8 + In the T cell population, TCF1 + PD-1 + Increase the frequency of stem-like cells, SLAMF6 - Tim3 + PD1 + The frequency of depleted cells was reduced (Figure 11B). Similarly, LTBR agonist treatment increased the DC expression of MHC class II and CD40 (Figure 12D), and CD4 in MC38 tumors. + and CD8 + This enhanced T cell activation (Figure 12E).

[0170] To investigate whether LTBR agonism directly affects DC function, in vitro models of DC activation and licensing were utilized. The effect of LTBR agonism was tested in the mouse DC cell line MutuDC1940 model (Fuertes Marraco 2012). MutuDC1940 cells were pretreated with LPS or PBS for 2 hours and then cultured with coated LTBR agonist or isotype control antibody for 18 hours. Expression of molecules involved in DC licensing, antigen presentation, and co-stimulatory machinery was analyzed by flow cytometry. LTBR agonist treatment significantly upregulated the expression of MHC class I, CD80, and CD83 to levels similar to LPS-stimulated DCs, and CD40 expression increased twofold (Figure 11C). LTBR agonism also significantly increased the expression of MHC class II and CD8 (Figure 11C).

[0171] DC activation in response to LTBR agonism in mouse bone marrow-derived DCs (BMDCs) was analyzed (Figure 12A). Treatment with LTBR agonist for 18 hours significantly increased the number of BMDCs, suggesting enhanced DC differentiation and / or proliferation (Figure 12B). Consistent with the MutuDC1940 data, treatment with LTBR agonist antibody significantly increased the proportion of CD83 + cells (Figure 12C) and upregulated the expression of CD11c + MHC II + in BMDCs, as well as MHC class I, MHC class II, CD80, CD86, ICOS-L, and particularly CD40 (Figure 12B). To further evaluate the effect of LTBR agonism on DC licensing, LTBR agonist-treated BMDCs were pulsed with OVA 257-264 peptide for 2 hours and then co-cultured with OT-I CD8 T cells for 3 days to evaluate T cell activation (Figure 12A). Co-culture with LTBR agonist-treated BMDCs significantly increased the percentage of CD44 + proliferating OT-I T cells and enhanced the expression of the effector and cytotoxic markers CD44, TNF-α, and GzmB (Figure 12C).

[0172] The effects of LTBR agonism on priming and activation of tumor-specific T cells in the tumor microenvironment were investigated. To isolate LNs from intratumor T cell priming, lymphocyte outflow from secondary lymphoid organs was inhibited in MC38-OVA tumor-bearing mice by FTY720 treatment, followed by adoptive transfer of labeled OT-I CD8+ T cells (Figure 21A). As expected, FTY720 treatment significantly reduced the frequency of OT-I T cells in peripheral blood and tumor perfusion LNs (Figures 22A-F). The frequency of intratumor T cells was reduced by FTY720 treatment; this effect was significantly lower in animals treated with LTBR agonists (Figures 22G-H). Importantly, proliferation and CD69 + The frequency of proliferating OT-I T cells was significantly higher in tumors treated with LTBR agonists (Figure 21B-C), indicating in situ T cell priming and activation within the tumor microenvironment. Consistent with these results and data from MC38 and Colon26 tumor models (Figure 11A and Figure 12D), an increase in activated DCs was observed in LTBR agonist-treated MC38-OVA tumors (Figure 23A-C). LTBR agonism also increased the frequency of CD69+ proliferating OT-I T cells in tumor-perfused LNs (Figure 23D-F, 24A-B), demonstrating a systemic effect of LTBR agonism promoting tumor-specific T cell activation. Overall, these data highlight the function of LTBR signaling in directly promoting DC licensing within the tumor microenvironment and consequently activating DC-mediated T cells.

[0173] CD8+ T cell and HEV-dependent antitumor effects of LTBR agonism The effect of LTBR agonist treatment on Colon26 tumor growth was evaluated. Treatment with the LTBR agonist alone significantly delayed Colon26 tumor growth compared to isotype control treatment (Figure 13C). CD8+ T cell depletion studies with anti-CD8α antibody treatment demonstrated that the LTBR agonist-mediated antitumor effect is CD8+ T cell-dependent (Figures 13C and 25). Consistent with previous data, CD8+ T cell depletion did not affect intratumoral HEV formation. Thus, LTBR agonist treatment showed a CD8 T cell-dependent antitumor effect in the syngeneic Colon26 tumor model.

[0174] Increased immune cell infiltration due to HEV activation has been implicated in the favorable outcome of cancer patients (Martinet 2011, Asrir 2022, Hua 2022, Karpathiou 2021, Park 2021, Sawada 2022). However, evidence directly linking the function and antitumor effect of TA-HEV remains elusive. To address this mechanism, high-dose MECA-79 antibody treatment (M’Rini 2003) was utilized to disrupt PNAd / L-selectin-dependent HEC-immune cell interactions and immune cell infiltration into the tumor. The antitumor activity of LTBR agonist antibody treatment was neutralized by MECA-79 antibody treatment (Figure 13D). Correspondingly, MECA-79 antibody treatment significantly reduced the tumor infiltration of B cells (Figure 13E), as well as central memory (CD44 + L-selectin+) CD4 and CD8 T cells (Figure 13F). The antitumor function associated with LTBR agonism is critically dependent on the HEV-mediated promotion of immune cell infiltration.

[0175] LTBR agonist treatment enhanced the antitumor efficacy of T cell-based immunotherapy Considering the effects of LTBR agonism on immune cell infiltration and dendritic cell licensing, we investigated whether LTBR agonism could enhance the tumor response to T-cell-based immunotherapy. The activity of combined treatment with an LTBR agonist and anti-PD-1 was tested in Colon26 tumors. Monotherapy with anti-PD-1 in established Colon26 tumors did not show significant activity, while the LTBR agonist antibody consistently promoted a moderate but significant antitumor effect (Figure 14A). The combination of anti-PD-1 and an LTBR agonist significantly reduced tumor growth, showing a reduction of over 30% in tumor size in 5 out of 14 mice, including 2 tumor-free mice, after 10 days of treatment (Figure 14A). Flow cytometry analysis showed that combined treatment with anti-PD-1 and an LTBR agonist enhanced tumor-infiltrating immune cells, particularly CD103. + It was shown to increase the frequency of migratory type 1 DCs (Figure 14B).

[0176] Combination of LTBR agonism and mouse CAR T cell therapy The effects of combining LTBR agonism with mouse CAR T cell therapy were evaluated. For this purpose, MC38 cell lines were modified to express human CD20 (hCD20) to enable targeting by hCD20 CAR T cells. MC38-hCD20 tumor-bearing mice were treated with an LTBR agonist antibody and hCD20 CAR T cells 6 days after tumor transplantation. While the monotherapy group showed no antitumor effect, the combination of LTBR agonist and hCD20 CAR T cell therapy significantly reduced tumor growth (Figure 15A). Immunohistochemical analysis at the end of the study showed an increase in the number of CD4 and CD8 T cells in the LTBR agonist-treated group (Figure 15B). Overall, these data highlight the potential of LTBR agonism to enhance the antitumor efficacy of T cell-based immunotherapy.

[0177] In vivo tumor cytokine screening identified mediators of intratumoral TLS formation. Treatment with LTBR agonist antibodies induced TA-TLS formation in approximately 10% of Colon26 tumors. To evaluate whether the frequency of TA-TLS formation could be increased and to establish a robust preclinical TA-TLS model, Colon26 tumors were manipulated to express different TLS-related cytokines. Cytokine expression was validated by qPCR and flow cytometry, and no effect on tumor cell proliferation in vitro was observed (Figure 26A-B). The cytokine-expressing Colon26 tumors were screened for in vivo activity in inducing TA-TLS by immunofluorescence analysis (Table 2). The frequency of intratumoral B-cell aggregate formation was dramatically increased by combining 10 days of LTBR agonist treatment (2 mg / kg) with intratumoral expression of Cxcl13 (64%, 7 / 11 tumors, Figure 16), Ccl19 (100%, 12 / 12 tumors, Figure 16), Ccl21 (100%, 3 / 3 tumors, Table 2), and LTα (100%, 12 / 12 tumors, Figure 6C), respectively. Intratumoral B-cell aggregates induced by the combination of Cxcl13 expression and LTBR agonism mainly exhibited a dispersed phenotype lacking HEV, while the majority of B-cell aggregates induced by the combination of Ccl19 or Ccl21 expression and LTBR agonist treatment were found in a dense morphology in the peritumoral region and were associated with HEV. Notably, T cell aggregates were not induced in tumors expressing Cxcl13, Ccl19, or Ccl21 in combination with LTBR agonist treatment. Among the cytokines examined, LTα appeared to be the most potent inducer of TA-HEV and B cell aggregates (Table 2). B cell aggregates were detected in the majority of LTα-expressing tumors, typically associated with HEVs, but they were relatively small in size and lacked distinct T cell zones (Figure 17). Surprisingly, the combination of LTα expression and LTBR agonist treatment effectively induced TA-TLSs with distinct B cell and T cell zones consistently associated with HEVs (Figure 17). Ki67+ activated B cells and T cells were readily detectable in these TA-TLSs.Characterization of trast components of TA-TLS identified FDC-like CD4-CD8-Cxcl13+ cells in the B-cell zone and a PDPN+ FRC-like cell network along with associated CD11c+ DCs in the T-cell zone. LYVE1+ lymphatic vessels were also observed in proximity to TA-TLS. Notably, LTα-associated TA-TLS formation promoted long-term control of Colon 26 tumors even after discontinuation of LTBR agonist treatment (Figure 18). The combined activity of LTBR agonism and LTα expression inducing TA-TLS was also observed in MC38-OVA tumors (Table 3). Overall, the combination of LTα expression and LTBR agonism specifically induced intratumoral TA-TLS formation.

[0178] [Table 3]

[0179] LTBR agonism mediates dilation of post-HEV-associated capillary venules. To gain insights into the molecular and cellular changes associated with the induction of TA-TLS by combining LTα expression and LTBR agonist treatment, single-cell transcriptional profiling was performed focusing on tumor-associated endothelial cells (TA-ECs), stromal cells, and immune cells. Colon26 cells were engineered to express GFP to facilitate tumor cell depletion. Tumor-associated endothelial cells, stromal cells, and immune cells were each engineered to express GFP. - CD45 - CD31 + GFP - CD45 - CD31 - , and GFP - CD45 + CD31 - Cells were isolated 7 days after LTBR agonist treatment by fluorescence-activated cell sorting (FACS) based on marker expression. Identity of different cell types was confirmed by unsupervised clustering using homogeneous manifold approximation and projection (UMAP) (Figure 19A). LTBR was widely expressed in ECs, stromal cells, myeloid cells, and residual tumor cells (Figure 19B).

[0180] TA-ECs can be subclustered into eight groups (E1-8) using marker genes from publicly available EC single-cell RNA sequencing (scRNA-seq) datasets (Hua 2022, Goveia 2020, Kalucka 2020): arterial ECs, capillary arterial ECs, end / stalk cells, capillary ECs, post-capillary venule (PCV) ECs, PCV / venous ECs, lymphoid ECs, and mitotic ECs (Figure 19C). The PCV marker Ackr1 (DARC) is expressed in both LN HEVs and TA-HEVs (Asrir 2022, Hua 2022). Glycam1 + Most TA-HECs use Ackr1 + It was embedded in the PCV cluster. Furthermore, the HEV signature enzymes Chst4 and Fut7 were expressed in a similar pattern to Glycam1, indicating functional HEC differentiation. LTBR agonism, rather than LTα expression, increased the frequency of PCV ECs by 50% compared to controls (Figure 19D). In parallel with the increase in PCV ECs, LTBR agonist-treated tumors showed a corresponding decrease in stipe / stalk cell compartments (Figure 19D). In addition, the combination of LTα expression and LTBR agonism reduced the proportion of mitotic ECs and increased the proportion of lymphoid ECs (Figure 19D). The TLS-related chemokine Ccl21 was linked to Lyve1 + It was specifically expressed by lymphoid ECs. Overall, LTBR agonism promoted expansion of HEV-associated PCV compartments more potently than LTα, but the combination of LTα expression and LTBR activation specifically expanded the lymphoid EC pool expressing Ccl21.

[0181] LTBR agonism and LTα signaling coordinately suppressed the tumor-promoting CAF phenotype. Single-cell transcriptome analysis revealed that GFP - CD45 - CD31 -Five fibroblast clusters (F1-F5) were identified in the tumor-associated stromal compartment (Figure 19E). Cluster F1 was characterized by high expression of the general tissue outer membrane fibroblast marker Pi16 (Buechler 2021), while clusters F2 and F3 expressed high levels of the general tissue parenchymal fibroblast marker Col15a1 (Buechler 2021). high CAF and Col15a1 high CAF is a pan-organizational Pi16. + and Col15a1 + The transcriptional characteristics broadly matched those of the fibroblast subset. LTBR agonist treatment and LTα expression were associated with F1 Pi16 high The CAF percentage was increased, but F2 / F3 Col15a1 high The proportion of CAFs was reduced (Figure 19F). The combination of LTBR agonism and LTα expression further amplified these trends, and Pi16 was observed in the combination group compared to the control. high The frequency of CAF increased 3.4 times, Col15a1 high The frequency of CAF decreased 3.1 times (Figure 19F). Col15a1 high CAF expressed tumor-promoting factors Tgfb1, Vegfa, Cxcl14 (Sjoberg 2016), and Bgn (Zheng 2023) at higher levels. In particular, Cxcl13 + BAFF + FDC-like cells and CCL19 + ADH1 + FRC-like cells (Grout 2022) were enriched with Pi16 by treatment. high It was found to be primarily present in the CAF population. Overall, these data suggest that LTBR agonism and LTα signaling affect the tumor fibroblast population, Col15a1 high From tumor-promoting phenotype to Pi16 high This suggests a coordinated transition to the TLS-enhanced phenotype.

[0182] Tumor-infiltrated CD8 +The effects of LTBR agonism and LTα expression on T cell composition and transcriptional profiles were evaluated. TCF1 was measured by flow cytometry. + PD1 + CD8 + Increased frequency of T cells and SLAMF6 - Tim3 + PD1 + CD8 + Consistent with the decreased frequency of T cells (Figure 11B), CD8 + T cell analysis revealed that LTBR agonist treatment and LTα expression were associated with effectors and proliferating CD8 cells. + Significantly increased precursor signature score in T cells, naive, effector, and proliferating CD8 + It was shown to reduce the depletion signature score in T cells (Pai 2023). Overall, single-cell transcriptional analysis further defined the effects of LTBR agonism on multiple tumor-associated cell compartments, including EC, CAF, and T cells, which is consistent with the enhancement of antitumor immunity.

[0183] TA-TLS formation enhanced the tumor response to anti-CTLA-4 treatment. To test the effect of TLS on the tumor response to ICB therapy, anti-PD-1 antibody treatment was applied to LTBR agonist-treated Colon26 tumors expressing LTα (Figure 27A). Anti-PD-1 treatment moderately increased the depth of the treatment response in Colon26-LTα tumors pre-treated with LTBR agonists (Figure 27B). Next, the effects of combining CTLA-4 blockade with LTBR agonism and LTα expression were tested (Figure 27C). Monotherapy with anti-CTLA-4 resulted in potent tumor control in wild-type Colon26 tumors, showing at least a 30% reduction in tumor volume in 5 out of 8 mice (Figure 20A). Combining CTLA-4 blockade with either LTBR agonism or tumor LTα expression further enhanced the antitumor efficacy, achieving a deep tumor response in almost all mice (Figure 20A).

[0184] Compared to Colon26, the MC38-OVA tumor model exhibited greater resistance to CTLA-4 blockade, and the antitumor effect of monotherapy with anti-CTLA-4 was mild, consistent with the combined benefit of anti-CTLA-4 and LTBR agonist treatment (Figures 20B and 28D). In LTα-expressing MC38-OVA tumors, CTLA-4 blockade significantly delayed tumor progression (Figure 20C). The combination of LTα expression that induced TA-TLS formation in MC38-OVA tumors and LTBR agonism (Table 3) increased the tumor response to CTLA-4 blockade, showing a more than 30% reduction in tumor size in 5 out of 13 mice, including 3 tumor-free mice (Figure 20C).

[0185] Consideration Systemic activation of the LTBR pathway modulated the tumor microenvironment through multiple mechanisms, promoting anti-tumor immunity. Treatment with a single LTBR agonist antibody induced tumor-specific HEV formation across different syngeneic mouse tumor models exhibiting different tissue structures. Surprisingly, HEVs were not detected in normal organs, and furthermore, although LTBR signaling is required for LN HEV homeostasis (Browning 2008), LNs from mice treated with LTBR agonists showed overall normal tissue structure and HEV distribution. In addition, liver function tests, including measurements of serum alanine transaminase (ALT) and aspartate transaminase (AST) levels, in mice treated with LTBR agonist monoclonal antibodies did not detect significant changes despite LTBR expression in hepatocytes (MacParland 2018), which is also consistent with the tumor-specific effects of systemic LTBR activation.

[0186] As previously described (Asrir 2022), LTBR-induced intratumor HEVs exhibited the characteristic swollen morphology of LN HECs and upregulation of cell adhesion molecules. Mechanistically, these tumor-specific effects occurred independently of major tumor-infiltrating immune cells and major angiogenic signaling pathways (VEGF, Notch, and Tie2), consistent with the transdifferentiation mechanism of HEV formation (Hua 2022). Rather, a combination of in vitro and in vivo studies highlighted that excessive inflammatory signaling appears to promote tumor-specific HEV formation. Simultaneous in vivo blockade of multiple inflammatory signaling pathways may require identifying relevant factors in the tumor microenvironment. Previous studies have demonstrated that combined anti-PD-1 and anti-angiogenic treatments can induce TA-HEV formation (Allen 2017). Such combined effects were not observed; these differences may represent tumor model-specific characteristics. In fact, the driving signals for TA-HEV formation (LTα1β2-LTBR vs. LTα3-TNFR1) and the involved immune cell types (CD4, CD8 T cells, or NK cells) have been found to be tumor model-dependent (Asrir 2022, Hua 2022, Peske 2015).

[0187] LN HEVs are specialized blood vessels that mediate lymphocyte invasion from the blood. Consistent with this function, LTBR agonist-treated tumors showed a significant increase in immune cell infiltration (B cells, T cells, NK cells, and DC cells). Interestingly, blocking the L-selectivity / PNAd interaction reversed the LTBR-mediated effects on immune cell infiltration and tumor growth, highlighting the relevance of HEV-mediated immune cell transport to promote anti-tumor immunity. In addition to its effects on immune cell infiltration, LTBR agonism was associated with increased activation of intratumoral DCs and T cells. LTBR agonist treatment significantly increased the frequency of activated CD40+CD83+ DCs in Colon26 tumors and increased DC CD40 and MHCII expression in MC38 tumors. Furthermore, in vitro studies using BMDCs revealed the direct effects of LTBR agonism on DC licensing and DC-mediated T cell activation. Finally, adoptive transfer studies of OT-I CD8+ T cells in FTY720-treated MC38-OVA tumor-bearing mice demonstrated that systemic LTBR activation enhances tumor-specific T cell activation within the tumor microenvironment.

[0188] The effects of LTBR agonism on immune cell infiltration and activation highlight the potential of therapeutic combination approaches with T cell and DC-based immunotherapies. For proof of concept, combining LTBR agonist treatment with PD-1 or CTLA-4 ICBs, as well as hCD20-targeted CAR-T cell therapy, resulted in moderate but significant antitumor benefits. While promising, these results leave room for improvement, and further investigation is needed into additional combination approaches with novel classes of immunotherapies, such as costimulatory antibodies, immune cytokines, oncolytic viruses, or cancer vaccines (Murciano-Goroff 2020). The reported direct cytotoxic effects on colorectal tumor models (Lukashev 2006) may contribute to the antitumor activity of LTBR agonist antibody treatment, consistent with the moderate tumor growth inhibition observed in LTBR agonist-treated Colon26 tumors in immunodeficient SRG mice.

[0189] LTBR-mediated formation of TA-HEVs was associated with the presence of organized lymphoid aggregates consisting of distinct B-cell and T-cell zones in approximately 10% of Colon26 tumors. To test whether the frequency of TA-TLS induction could be increased, a selection of TLS-related chemokines / cytokines were screened for TLS induction activity in Colon26 and MC38-OVA tumor models. The combination of LTBR agonism and tumor LTα expression most potently induced TA-TLS formation in both tumor models. LTBR and LTα-mediated TLSs were characterized by distinct B-cell and T-cell zones associated with HEVs, FDCs, and FRC-like stromal compartments, as well as lymphatic vessels. LTα signaling alone induced intratumoral HEV formation and B-cell aggregation, as previously reported (Schrama 2001), but combination with LTBR agonism was found essential for efficient TLS induction and tumor control. Further characterization of TA-TLS by scRNA-seq highlighted specific expansion of CAF compartments enriched with HEC-containing PCV populations, Ccl21-expressing lymphoid ECs, and FDC / FRC-like cells, as well as the transition from CD8 T cell depletion to the precursor phenotype. TLS can function as a site for antigen presentation and activation of adaptive immunity within tumors (Aoyama 2021). While further research is needed to further evaluate the functionality of TLS, TA-TLS is associated with Ki67 +It has been demonstrated that Colon26 tumors in the LTBR / LTα combination group, which contain activated B and T cells, exhibit sustained tumor control even after discontinuation of LTBR agonist treatment. Furthermore, TA-TLS formation enhanced the responses of Colon26 and MC38-OCA to ICB, particularly anti-CTLA-4 treatment. The extent to which the Treg depletion function of anti-CTLA-4 contributes to this activity will require further investigation (Economides 2003). Considering the increasing clinical evidence of the correlation between TA-TLS and a favorable treatment response to ICB (Sautes-Fridman 2019, Cabrita 2020, Helmink 2020, Petitprez 2020), this new inducible TA-TLS model serves as a useful tool for mechanistic studies on TA-TLS neogenesis and related antitumor strategies, complementing the recently described intratumoral TLS-like models (Rodriguez 2021, Ramachandran 2023, Economides 2003, Holash 2002).

[0190] In summary, this study provides new insights into the mechanism of LTBR-mediated tumor-specific HEV formation and the immune regulation of the tumor microenvironment. Furthermore, it describes a new mouse model of tumor-associated TLS formation based on the combined activation of LTBR and TNFR signaling. Notably, the presence of TLS in many human tumor types is associated with a favorable prognosis and improved ICB response. Finally, it lays the foundation for treatment strategies aimed at promoting adaptive immune responses within the tumor site.

[0191] References Girard et al., Nat Rev Immunol, 2012. 12(11): p. 762-73. Streeter et al., J Cell Biol, 1988. 107(5): p. 1853-62. Berg et al., J Cell Biol, 1991. 114(2): p. 343-9. Martinet et al., Cancer Res, 2011. 71(17): p. 5678-87. Martinet et al., Oncoimmunology, 2012. 1(6): p. 829-839. Asrir et al., Cancer Cell, 2022. 40(3): p. 318-334 e9. Hua et al., Cancer Cell, 2022. 40(12): p. 1600-1618 e10. Sautes-Fridman et al., Nat Rev Cancer, 2019. 19(6): p. 307-325. Cabrita et al., Nature, 2020. 577(7791): p. 561-565. Helmink et al., Nature, 2020. 577(7791): p. 549-555. Petitprez et al., Nature, 2020. 577(7791): p. 556-560. Meylan et al., Immunity, 2022. 55(3): p. 527-541 e5. Goc et al., Cancer Res, 2014. 74(3): p. 705-15. Futterer et al., Immunity, 1998. 9(1): p. 59-70. Onder et al., Trends Immunol, 2018. 39(10): p. 775-787. Browning et al., Immunity, 2005. 23(5): p. 539-50. Browning, Immunol Rev, 2008. 223: p. 202-20. Rodriguez et al., Cell Rep, 2021. 36(3): p. 109422. Ramachandran et al., Cancer Cell, 2023. 41(6): p. 1134-1151 e10. Allen et al., Sci Transl Med, 2017. 9(385). Consortium, Science, 2020. 369(6509): p. 1318-1330. Eraslan et al., Science, 2022. 376(6594): p. eabl4290. MacParland et al., Nat Commun, 2018. 9(1): p. 4383. Peske et al., Nat Commun, 2015. 6: p. 7114. Berg et al., Nature, 1993. 366(6456): p. 695-8. O'Connell et al., J Immunol, 1990. 144(2): p. 521-5. Omidi et al., Brain Res, 2003. 990(1-2): p. 95-112. Coppola et al., Am J Pathol, 2011. 179(1): p. 37-45. Messina et al., Sci Rep, 2012. 2: p. 765. Prabhakaran et al., Breast Cancer Res, 2017. 19(1): p. 71. Fuertes Marraco et al., Front Immunol, 2012. 3: p. 331. Browning et al., J Exp Med, 1996. 183(3): p. 867-78. Lukashev et al., Cancer Res, 2006. 66(19): p. 9617-24. Karpathiou et al., Pathol Res Pract, 2021. 220: p. 153392. Park et al., J Immunother Cancer, 2021. 9(10). Sawada et al., Cancer Immunol Res, 2022. 10(4): p. 468-481. M'Rini et al., J Exp Med, 2003. 198(9): p. 1301-12. Goveia et al., Cancer Cell, 2020. 37(3): p. 421. Kalucka et al., Cell, 2020. 180(4): p. 764-779 e20. Buechler et al., Nature, 2021. 593(7860): p. 575-579. Sjoberg et al., Br J Cancer, 2016. 114(10): p. 1117-24. Zheng et al., Clin Transl Med, 2023. 13(2): p. e1189. Grout et al., Cancer Discov, 2022. 12(11): p. 2606-2625. Pai et al., Cancer Cell, 2023. 41(4): p. 776-790 e7. Kabashima et al., Immunity, 2005. 22(4): p. 439-50. Summers deLuca et al., Nat Rev Immunol, 2012. 12(5): p. 339-51. Summers deLuca, L., et al., Proc Natl Acad Sci U S A, 2011. 108(5): p. 2046-51. Murciano-Goroff et al., Cell Res, 2020. 30(6): p. 507-519. Aoyama et al., Front Immunol, 2021. 12: p. 675538. Economides et al., Nat Med, 2003. 9(1): p. 47-52. Holash et al., Proc Natl Acad Sci USA, 2002. 99(17): p. 11393-8. Kuhnert et al., Cancer Res, 2015. 75(19): p. 4086-96. Daly et al., Cancer Res, 2013. 73(1): p. 108-18. Amend et al. J Vis Exp, 2016(110). Brasel et al., Blood, 2000. 96(9): p. 3029-39. Wolf et al., Genome Biol, 2018. 19(1): p. 15.

[0192] This disclosure is not limited in scope by any specific embodiment described herein. In fact, various modifications of this disclosure, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included in the accompanying claims.

Claims

1. A method for treating cancer, comprising administering a therapeutically effective dose of a lymphotoxin beta receptor (LTBR) agonist in combination with a therapeutically effective dose of adoptive cell therapy (ACT) to a subject in need thereof, wherein the combination administration results in an increased efficacy and duration of antitumor response compared to a subject treated with ACT as monotherapy.

2. A method for increasing the efficacy of adoptive cell therapy (ACT), (a) Selecting subjects who have cancer; (b) Administering a therapeutically effective dose of ACT to the subject in combination with a therapeutically effective dose of a lymphotoxin beta receptor (LTBR) agonist. Includes, A method wherein combination administration results in increased efficacy and duration of the antitumor response compared to subjects treated with ACT as monotherapy.

3. The method according to claim 1 or 2, wherein the LTBR agonist is an antibody or an antigen-binding fragment thereof that specifically binds to LTBR.

4. The method according to any one of claims 1 to 3, wherein the ACT comprises immune cells selected from T cells, tumor-infiltrating lymphocytes, and natural killer (NK) cells.

5. The method according to claim 4, wherein the immune cells include a modified T cell receptor (TCR) for tumor-associated antigens (TAAs), or a chimeric antigen receptor (CAR) for TAAs.

6. The aforementioned TAAs include AFP, ALK, BAGE protein, BCMA, BIRC5 (Survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, and CYP1B.

1. EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, Glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE Proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, Mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA- 1. The method according to claim 5, selected from PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-Nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

7. The method according to any one of claims 1 to 6, further comprising administering an additional therapeutic agent or treatment to the subject.

8. The method according to claim 7, wherein the additional therapeutic agent or treatment is selected from radiation, surgery, checkpoint inhibitors, chemotherapeutic agents, cancer vaccines, vascular endothelial growth factor (VEGF) antagonists, angiopoietin-2 (Ang2) inhibitors, transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens, Bacillus Calmette-Guélain vaccine, granulocyte-macrophage colony-stimulating factor (GM-CSF), cytotoxins, interleukin-6 receptor (IL-6R) inhibitors, interleukin-4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-12, IL-21, IL-15, antibody-drug conjugates, anti-inflammatory drugs, and combinations thereof.

9. The method according to claim 8, wherein the checkpoint inhibitor is selected from inhibitors of PD-1, PDL-1, PDL-2, LAG-3, CTLA-4, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD80, CD86, CD160, CD276, GAL9, HAVCR2, IDO1, IDO2, KIR, LAIR1, macrophage receptor having a collagen structure (MARCO), phosphatidylserine (PS), TIGHT, VISTA, and VTCN1.

10. The method according to claim 8 or 9, wherein the checkpoint inhibitor is an inhibitor of PD-1, PDL-1, PDL-2, LAG-3, or CTLA-4.

11. The method according to any one of claims 1 to 10, wherein the cancer is selected from adrenal cancer, anal cancer, autonomic ganglion cancer, biliary tract cancer, bladder cancer, hematological cancer, bone cancer, brain cancer, breast cancer, meningeal cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, eye cancer, esophageal cancer, fallopian tube cancer, stomach cancer, reproductive organ cancer, head and neck cancer, kidney cancer, colorectal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, peritoneal cancer, pituitary cancer, placental cancer, pleural cancer, prostate cancer, rectal cancer, kidney cancer, salivary gland cancer, skin cancer, small intestine cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, upper respiratory tract and gastrointestinal cancer, urinary tract cancer, uterine cancer, vaginal cancer, and vulvar cancer.

12. The method according to any one of claims 1 to 11, wherein the cancer expresses CXCL13, CCL19, CCL21, or LTα.

13. The method according to any one of claims 1 to 12, wherein the cancer expresses LTα.

14. The method according to any one of claims 1 to 13, wherein administration of the combination produces one or more therapeutic effects selected from among increased tumor-specific HEV formation, increased dendritic cell and T cell infiltration, enhanced T cell activation in the tumor microenvironment, increased expression of TLS-related chemokines, delayed tumor growth, decreased tumor cell count, tumor regression, extended survival, partial response, and complete response.

15. The method according to any one of claims 1 to 14, wherein the therapeutically effective dose of the LTBR agonist comprises 0.005 mg / kg to 10 mg / kg of body weight of the subject.

16. The therapeutically effective dose of ACT is 1 × 10 ６ The method according to any one of claims 1 to 15, comprising one or more immune cells.

17. The method according to any one of claims 1 to 16, wherein the LTBR agonist and / or the ACT is administered to the subject in one or more doses.

18. The method according to any one of claims 1 to 17, wherein the LTBR agonist and / or the ACT is administered intravascularly, subcutaneously, intraperitoneally, or intratumorally.

19. The method according to any one of claims 1 to 18, wherein the LTBR agonist is administered before or after the administration of the ACT.

20. The method according to any one of claims 1 to 18, wherein the LTBR agonist is administered simultaneously with the administration of the ACT.

21. The method according to any one of claims 1 to 20, wherein the LTBR agonist and the ACT are provided in separate compositions.

22. The method according to any one of claims 1 to 18, wherein the LTBR agonist and the ACT are provided in a single composition.

23. A method for increasing tumor-specific HEV formation in subjects requiring it, (a) Selecting subjects who have cancer; (b) Administering a therapeutically effective dose of ACT to the subject in combination with a therapeutically effective dose of a lymphotoxin beta receptor (LTBR) agonist. Includes, A method by which combination administration results in increased tumor-specific HEV formation, as well as increased efficacy and duration of the antitumor response, compared to subjects treated with ACT as monotherapy.

24. A method for increasing the expression of TLS-related chemokines in subjects that require it, (a) Selecting subjects who have cancer; (b) Administering a therapeutically effective dose of ACT to the subject in combination with a therapeutically effective dose of a lymphotoxin beta receptor (LTBR) agonist. Includes, A method by which combination administration results in increased expression of TLS-related chemokines and increased efficacy and duration of the antitumor response compared to subjects treated with ACT as monotherapy.

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