EP4688857A1

EP4688857A1 - Methods of treating cancer with a lymphotoxin beta receptor agonist

Info

Publication number: EP4688857A1
Application number: EP24722916.4A
Authority: EP
Inventors: Frank Kuhnert; Disi AN
Original assignee: Regeneron Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc
Priority date: 2023-04-07
Filing date: 2024-04-04
Publication date: 2026-02-11
Also published as: KR20250169609A; AU2024253044A1; CN120882754A; MX2025011882A; WO2024211478A1; IL323162A; JP2026513920A

Abstract

This disclosure relates to methods of treating cancer by administering a lymphotoxin beta receptor (LTBR) agonist, such as in combination with adoptive cell therapy (ACT). The combination of LTBR agonist and ACT demonstrates unexpected synergistic anti-tumor efficacy in inducing potent and durable tumor control in subjects with cancer.

Description

METHODS OF TREATING CANCER WITH A LYMPHOTOXIN BETA RECEPTOR AGONIST

SEQUENCE LISTING

[0001] The sequence listing of the present application is submitted electronically as an ST.26 formatted xml file with a file name “SeqList-11483”, creation date of February 27, 2024, and a size of 2,682 bytes. This sequence listing submitted is part of the specification and is hereby incorporated by reference in its entirety.

FIELD

[0002] The present disclosure relates generally to methods of treating cancer with a lymphotoxin beta receptor agonist, such as in combination with adoptive cell therapy.

BACKGROUND

[0003] Manipulation of the immune system has become a critical resource in the fight against cancer. Immune cells move 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 lymph nodes through specialized blood vessels called high endothelial venules (HEV), which occur in regions between B and T cell zones. T cell zones contain CD4⁺ and CD8⁺ T cells and subsets of dendritic cells (DC). See Mueller et al., Nat. Rev. Immunol., 9:618-629 (2009). Tertiary lymphoid structures (TLS) are ectopic lymphoid formations that develop in inflamed, infected, or tumoral tissues. TLS contain HEV and B cell follicles surrounded by a T cell zone and are characterized by abundant chemokine expression. The presence of TLS and HEV in solid tumors is positively correlated with patient survival in many cancer types and may be predictive of better response to immune-checkpoint blockade. See Sautes-Fridman etal., Nat. Rev. Cancer., 19:307-325 (2019).

[0004] The lymphotoxin beta receptor (LTBR) plays a central role in the development and homeostasis of lymph nodes and secondary lymphoid organs by regulating the expression of several homeostatic lymphoid cytokines (e.g., CCL19, CCL21 , CXCL13) and adhesion molecules (ICAM-1 , VCAM-1 , MADCAM1) via the NF-kappa B pathway. See Schneider et al., Immunol. Rev., 202:49-66 (2004). LTBR is activated by two different trimeric ligands, LIGHT and lymphotoxin alphal beta2 (LTa1b2 or LTa1 [32). Activation of LTBR by its ligands leads to ectopic formation of tertiary lymphoid structures (TLS). See Schrama etal., Immunity 14:111-121 (2001). As presence of TLS in the tumor microenvironment typically correlates with immune infiltration and is also associated with better prognosis, treatment with LTBR agonists may induce anti-tumor immune responses and improve current cancer immunotherapies. See Tang et al., Cell. Mol. Immunol. 14:809-18 (2017).

[0005] Adoptive cell therapy (ACT) uses a subject’s own immune cells (or a donor’s immune cells) to treat diseases such as cancer. In general, ACT involves the transfer of genetically modified T lymphocytes into the subject. Some examples of ACT include the use of an engineered chimeric antigen receptor (CAR) or T cell receptor (TCR). In general, a CAR comprises a single chain fragment variable region of an antibody or a binding domain specific for a tumor associated antigen (TAA) coupled via a hinge and transmembrane regions to cytoplasmic domains of T cell signaling molecules. The most common lymphocyte activation moieties include a T cell costimulatory domain in tandem with a T cell effector function triggering moiety. CAR- mediated ACT allows CAR-grafted T cells to directly recognize and attack the TAAs on target tumor cells.

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

[0007] Due in part to the inherent complexity and patient-to-patient variability of live cell culture, ACT agents have tended to provide limited success with variable clinical activity. Thus, there is a need to improve anti-tumor activities of ACT.

SUMMARY

[0008] The disclosed technology addresses one or more of the foregoing needs. Applicant has shown herein that LTBR agonism led to the surprising effect of increasing the antitumor efficacy of administered adoptive cell therapy. Accordingly, in one aspect, the disclosed technology relates to a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a lymphotoxin beta receptor (LTBR) agonist in combination with a therapeutically effective amount of an adoptive cell therapy (ACT), wherein administration of the combination leads to increased efficacy and duration of anti-tumor response, as compared to a subject treated with the ACT as monotherapy. In another aspect, the disclosed technology relates to a method for increasing the efficacy of adoptive cell therapy (ACT), comprising: (a) selecting a subject with cancer; and (b) administering to the subject a therapeutically effective amount of an ACT in combination with a therapeutically effective amount of a lymphotoxin beta receptor (LTBR) agonist, wherein administration of the combination leads to increased efficacy and duration of anti-tumor response, as compared to a subject treated with the ACT as monotherapy.

[0009] In some embodiments of the disclosed methods, the LTBR agonist is an antibody. In some embodiments of the disclosed methods, the ACT comprises an immune cell selected from a T cell, a tumor-infiltrating lymphocyte, and a natural killer (NK) cell. In some embodiments, the immune cell comprises a modified T cell receptor (TCR) against a tumor- associated antigen (TAA), or a chimeric antigen receptor (CAR) against a TAA. In some embodiments, the TAA is selected from AFP, ALK, BAGE proteins, BCMA, BIRC5 (survivin), BIRC7, p-catenin, brc-abl, BRCA1 , BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1 , CYP1 B1 , EGFR, EGFRvlll, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1 , GAGE proteins, 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 , 0X40, p15, p53, PAP, PAX3, PAX5, PCTA-1 , PLAC1 , PRLR, PRAME, PSMA (F0LH1), RAGE proteins, Ras, RGS5, Rho, SART-1 , SART-3, STEAP1, STEAP2, TAG- 72, TGF-p, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1 , TRP-2, tyrosinase, and uroplakin-3.

[0010] In some embodiments, the disclosed methods further include administering an additional therapeutic agent or therapy to the subject. In some embodiments, the additional therapeutic agent or therapy is selected from radiation, surgery, a checkpoint inhibitor, a chemotherapeutic agent, a cancer vaccine, a vascular endothelial growth factor (VEGF) antagonist, an angiopoietin-2 (Ang2) inhibitor, a transforming growth factor beta (TGF|3) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an antibody to a tumor-specific antigen, Bacillus Calmette-Guerin vaccine, granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytotoxin, an interleukin 6 receptor (IL-6R) inhibitor, an interleukin 4 receptor (IL-4R) inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-12, IL-21 , IL-15, an antibody-drug conjugate, an anti-inflammatory drug, 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 receptor with collageneous structure (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.

[0011] In some embodiments of the disclosed methods, the cancer is selected from adrenal gland cancer, anal cancer, autonomic ganglial cancer, biliary tract cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cancer of the meninges, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, eye cancer, esophageal cancer, fallopian tube cancer, gastric cancer, genital tract cancers, head and neck cancer, kidney cancer, large intestinal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, peritoneal cancer, pituitary cancer, placental cancer, pleura cancer, prostate cancer, rectal cancer, renal cancer, salivary gland cancer, skin cancer, small intestinal cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive cancers, urinary tract cancer, uterine cancer, vaginal cancer, and vulva cancer. In some embodiments, the cancer expresses CXCL13, CCL19, CCL21 or LT alpha (LTa, LTa). In some embodiments, the cancer expresses LTa.

[0012] In some embodiments of the disclosed methods, administration of the combination produces a therapeutic effect selected from one or more of: increased tumor-specific HEV formation, increased dendritic cell and T cell infiltration, enhanced T cell activation in tumor microenvironment, increased expression of TLS-related chemokines, delay in tumor growth, reduction in tumor cell number, tumor regression, increase in survival, partial response, and complete response. In some embodiments of the disclosed methods, the therapeutically effective amount of the LTBR agonist comprises 0.005 mg/kg to 10 mg/kg of the subject’s body weight. In some embodiments of the disclosed methods, the therapeutically effective amount of the ACT comprises 1x10⁶ or more immune cells.

[0013] In some embodiments of the disclosed methods, the LTBR agonist and/or the ACT is administered in one or more doses to the subject. In some embodiments of the disclosed methods, the LTBR agonist and/or the ACT is administered intravascularly, subcutaneously, intraperitoneally, or intratumorally. In some embodiments of the disclosed methods, the LTBR agonist is administered before or after administration of the ACT. In some embodiments of the disclosed methods, the LTBR agonist is administered concurrently with administration of the ACT. In some embodiments of the disclosed methods, the LTBR agonist and the ACT are provided in separate compositions. In some embodiments of the disclosed methods, the LTBR agonist and the ACT are provided in a single composition.

[0014] In one aspect, the present disclosure provides methods for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a lymphotoxin beta receptor (LTBR) agonist in combination with a therapeutically effective amount of an CTLA4 inhibitor, wherein administration of the combination leads to increased efficacy and duration of anti-tumor response, as compared to a subject treated with the CTLA4 inhibitor as monotherapy. In some embodiments, the CTLA4 inhibitor is an antibody or antigen-binding fragment thereof that binds specifically to CTLA4. Examples of anti-CTLA4 antibodies that may be used include, but are not limited to, ipilimumab and REGN4659. As disclosed herein, combining anti-CTLA-4 antibody with LTBR agonist and/or LTa expression delayed tumor progression and promoted complete regression of tumors.

[0015] In another aspect, the disclosed technology relates to a method for increasing tumor-specific HEV formation in a subject in need thereof, the method comprising: (a) selecting a subject with cancer; and (b) administering to the subject a therapeutically effective amount of an ACT in combination with a therapeutically effective amount of a lymphotoxin beta receptor (LTBR) agonist, wherein administration of the combination leads to increased tumor-specific HEV formation and increased efficacy and duration of anti-tumor response, as compared to a subject treated with the ACT as monotherapy.

[0016] In another aspect, the disclosed technology relates to a method for increasing expression of TLS-related chemokines in a subject in need thereof, the method comprising: (a) selecting a subject with cancer; and (b) administering to the subject a therapeutically effective amount of an ACT in combination with a therapeutically effective amount of a lymphotoxin beta receptor (LTBR) agonist, wherein administration of the combination leads to increased expression of TLS-related chemokines and increased efficacy and duration of anti-tumor response, as compared to a subject treated with the ACT as monotherapy.

[0017] Other embodiments of the present disclosure will become apparent from the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a series of graphs showing expression analysis of multiple TLS related chemokines in MC38 tumors, as described in Example 1.

[0019] Figures 2A-2C show the effect of LTBR agonism on infiltration and activation of DC and T cells, as described in Example 2. Figure 2A is a series of graphs showing quantification of infiltration and activation of DC and T cells 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 with pre-treated BMDC and OT-I T cells. Bar graphs show mean values ± SEM and each dot represents one biological replicate. All experiments were performed with triplicate. Unpaired Student’s t-test (two-tailed) was used for the comparison of the two groups, p values <0.05 were considered significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001 ; ****: p < 0.0001). All statistical analyses were performed using GraphPad Prism software.

[0020] Figures 3A-3C show the augmentation of anti-tumor efficacy of T-cell based therapies when combined with LTBR agonism, 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 anti-tumor effect in the Colon 26 tumor model. Figure 3C is a series of graphs showing immune cell infiltration in the Colon26 tumor model.

[0021] Figure 4 is a schematic and graph showing the anti-tumor efficacy of LTBR agonism in combination with murine anti-hCD20 CAR T cell therapy in a MC38-hCD20 tumor model, as described in Example 4.

[0022] Figure 5 is a series of graphs showing enhanced tumor response to anti-CTLA- 4 treatment in TLS+ Colon26 tumors, as described in Example 6.

[0023] Figure 6 is a series of graphs showing enhanced tumor response to anti-CTLA- 4 treatment in the MC38-OVA tumor model, as described in Example 6.

[0024] Figure 7 shows an example model of multi-faceted immunomodulation of TME by LTBR agonism.

[0025] Figures 8A-8B are a series of graphs showing characterization of TA-HEC and TA-EC isolated from Colon26 tumors after 10 days of LTBR agonist or isotype control antibody treatment (n=6) by flow cytometry. Figure 8A shows the frequency of TA-HEC and MFI of PNAd. Figure 8B shows the endothelial inflammatory markers E-selectin, P-selectin, ICAM-1 and VCAM-1.

[0026] Figures 9A-9B are a series of graphs showing the expression of HEV marker MAdCAM-1 and endothelial inflammatory markers ICAM-1 , VCAM-1 and P-selectin (n=3) quantified by flow cytometry. Endothelial cells were activated by treatment with 1 pg/ml LTBR agonist or isotype control antibody and/or 10 ng/ml TNFa, 10 ng/ml IL-1 p or 10 pg/ml LPS for 18h in vitro. Mean ± SEM is 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.

[0027] Figure 10A is a heatmap showing RT-qPCR analysis of TLS-related chemokine expression in tumor lysates of 7-day LTBR agonist (n= 9) or isotype control antibody (n=7) treated MC38 tumors. The Z scores of log-transformed chemokine expression are plotted. Each column represents an individual tumor. Figure 10B is a series of graphs showing quantification of tumorinfiltrating CD45+ immune cells, T cells and DC of 7-day treated MC38 tumors (n=5) by flow cytometry. Figure 10C is a series of graphs showing RT -qPCR analysis of TL S-related chemokine expression in tumor lysates of 10-day LTBR agonist (n=12) or isotype control antibody (n=11) treated MC38 tumors. Figure 10D is a series of graphs showing quantification of tumor-infiltrating CD45+ immune cells, B, T, DC and NK cells of 10-day treated Colon26 tumors (n=15) by flow cytometry. Figures 10B-10D show mean ± SEM. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0028] Figures 11A-B are a series of graphs showing characterization of tumorinfiltrating DC and T cells in Colon26 tumors with 10-day LTBR agonist or isotype control antibody treatment by flow cytometry (n=15). Mean ± SEM is shown. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001 . Figure 11 A shows the frequency of CD40⁺ DC, CD83⁺ DC, and CD40⁺CD83⁺ DC. Figure 11 B shows the frequency of Ki67⁺, TCF1⁺PD1⁺ and SLAMF6'Tim3⁺ cells in CD4⁺ or CD8⁺ T cells. Figure 11C is a graph showing flow cytometry quantification of MHCI, MHCII, CD80, CD86, CD40, CD83 and CD8 expression in MutuDC1940 cells treated with PBS or 10 pg/ml LPS for 2h, followed by 10 pg/ml LTBR agonist or isotype control antibody treatment for 18h (n= 5). Mean ± SEM is shown. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0029] Figures 12A-12C are a schematic and graphs showing analysis of BMDC activation and licensing by LTBR agonism treatment in vitro (n=5). Figure 12A is a schematic of BMDC differentiation, treatment and co-culture with OT-I CD8⁺ T cells, followed by flow cytometry quantification. Figures 12B is a graph showing the percentage of MHCII⁺CD11c⁺ BMDC cells in the in vitro culture. Mean ± SEM is shown. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001. Figure 12C shows the frequency of CD83⁺ BMDC cells. Mean ± SEM is shown. *p < 0.05; **p < 0.01; ***p < 0.001 ; ****p < 0.0001. Figures 12D-E are a series of graphs showing characterization of tumor-infiltrating DC and T cells of MC38 tumors with 10-day LTBR agonist or isotype control antibody treatment by flow cytometry (n=5). Mean ± SEM is shown. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001. Figure 12D shows MFI of MHCII and CD40 in DC. Figure 12E shows frequency of Ki67⁺CD4⁺ T cells, IFNg⁺CD4⁺ T cells, Ki67⁺CD8⁺ T cells and IFNg⁺CD8⁺ T cells.

[0030] Figure 13A is a pair of graphs showing cell viability of Colon26 and MC38-OVA cells treated by 0, 10, 100, and 1000 ng/mL LTBR agonist antibody for 24h in vitro (n=3). Figure 13B is a graph showing that Colon26 tumor size changes in individual mice from Day 0 to Day 7 of LTBR agonist or isotype control antibody treatment in SRG mice (n=4-5); DO is the start day of treatment. Figure 13C is a graph showing Colon26 tumor size changes in individual mice from Day 0 to Day 13 of LTBR agonist treatment with and without CD8 T cell depletion via anti-CD8a antibody (n=8). Figure 13D is a graph showing Colon26 tumor size changes in individual mice from Day 0 to Day 11 of LTBR agonist treatment and/or PNAd blocking via MECA-79 antibody (n=8). Figure 13E-13F are series of graphs showing quantification of B and T cells in the tumors, peripheral blood, and lymph nodes of Colon26-tumor bearing mice with LTBR agonist and/or MECA-79 antibody treatment (n=7) by flow cytometry. Figure 13E shows percentages of CD45⁺ immune cells and CD19⁺ B cells in tumors, and frequency of CD19⁺ B cells among CD45⁺ cells in tumors, blood and lymph nodes. Figure 13F shows percentages of CD4⁺ and CD8⁺ T cells in tumors, and frequency of central memory (CM) cells (CD62L⁺CD44⁺) among CD4⁺ and CD8⁺ T cell subsets in tumors, blood, and lymph nodes. Figures 13A, 13E-13F: Mean ± SEM is shown. Figures 13A-13F: *p < 0.05; **p < 0.01; ***p < 0.001 ; ****p < 0.0001.

[0031] Figure 14A is a graph showing Colon26 tumor size changes in individual mice from Day 0 to Day 10 of LTBR agonist and/or anti-PD-1 antibody treatment (n=14). Data are pooled from two independent experiments. RR, response rate is defined by greater than 30% reduction of tumor size. Figure 14B is a series of graphs showing quantification of tumor-infiltrating CD45+ immune cells, CD4+ T cells, NK cells and CD103+ CD11 b- type 1 DC in Colon26 tumors treated with LTBR agonist and/or anti-PD-1 antibody (n=5) by flow cytometry. Mean ± SEM is shown. Figures 14A-14B: *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0032] Figure 15A is a graph showing MC3S-hCD20 tumor size changes in individual mice from Day 0 to Day 16 of LTBR agonist and/or hCD20 CAR T treatment (n= 9). DO is the start day of treatment. Figure 15B is a pair of graphs showing immunohistochemical analysis of numbers of CD4⁺ and CD8⁺ T cells in the tumors collected at study end (n=4-5). Figures 15A- 15B: *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0033] Figure 16 is a pair of graphs showing the number and area of B cell aggregate in chemokine-expressing Colon26 tumors with 10-day LTBR agonist or isotype control antibody treatment (n=10-12) quantified by immunostaining.

[0034] Figure 17 is a pair of graphs showing identification of B cell aggregates and TA- TLS in LTa-expressing Colon26 tumors with 10-day isotype control or LTBR agonist antibody treatment by immunofluorescent staining of B cells (B220+), T cells (CD3+) and HEV (PNAd+). Number and area of intratumoral B cell aggregates or TLS (n=4 for isotype control group and n=12 for LTBR agonist treatment group from two independent experiments are plotted. Mean ± SEM is shown.

[0035] Figure 18 is a series of graphs showing individual tumor growth curves of EV or LTa-expressing Colon26 tumors with 4 doses (indicated by arrows) of isotype control or LTBR agonist antibody treatment.

[0036] Figure 19A is a UMAP plot of all cells isolated from EV control or LTa- expressing Colon26 tumors with 7-day LTBR agonist or isotype control antibody treatment, colored by unsupervised clustering. Figure 19B is a UMAP plot of all cells isolated from EV control or LT a-expressing Colon26 tumors with 7-day LTBR agonist or isotype control antibody treatment, colored by the expression of LTBR. Figure 19C is a UMAP plot of EC from control or LTa- expressing Colon26 tumors with 7-day LTBR agonist or isotype control antibody treatment, colored by unsupervised clustering. Figure 19D is a chart showing the fraction of EC clusters in different treatment groups. Figure 19E is a UMAP plot of stromal cells colored by unsupervised clustering. Figure 19F is a chart showing the fraction of stromal cell clusters in different treatment groups.

[0037] Figure 20A is a chart showing tumor size changes of empty vector (EV) control or LTa-expressing Colon26 tumors in individual mice from Day O to Day 17 of LTBR agonist and/or anti-CTLA-4 antibody treatment (n=7-8). RR, response rate is defined by greater than 30% reduction of tumor size. Figure 20B is a chart showing tumor size changes of control MC38-OVA tumors in individual mice with LTBR agonist and/or anti-CTLA-4 antibody treatment (n=6-11). Figure 20C is a chart showing tumor size changes of LTa-expressing MC38-OVA tumors in individual mice with LTBR agonist and/or anti-CTLA-4 antibody treatment (n=13). RR, response rate is defined by greater than 30% reduction of tumor size. TF, tumor free ratio. Figures 20A- 20C: DO is the start day of anti-CTLA-4 treatment. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0038] Figures 21A-21C are a schematic and graphs showing analysis of OT-I CD8+ T cell activation in MC38-OVA tumor-bearing mice treated with FTY720. Figure 22A is a schematic of LTBR agonist, FTY720 administration 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 in OT-I CD8+ T cells within MC38-OVA tumors. Figure 22C is a graph showing the frequency of CD69+ proliferating cells in OT-I CD8+ T cells within MC38-OVA tumors. Mean ± SEM is shown (n=5-7). Statistical significance was assessed by oneway ANOVA with Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001 ; ****p < 0.0001.

[0039] Figures 22A-22H are a series of graphs showing characterization of T cells in MC38- OVA tumor-bearing mice with FTY720 treatment and adoptive transfer of OT-I CD8+ T cells. Figure 22A is a graph showing flow cytometry quantification of CD8+ T cells in blood. Figure 22B is a graph showing flow cytometry quantification of OT-I CD8+ T cells in blood. Figure 22C is a graph showing flow cytometry quantification of proliferating OT-I CD8+ T cells in blood. Figure 22D is a graph showing flow cytometry quantification of CD8+ T cells in tumor-draining lymph nodes. Figure 22E is a graph showing flow cytometry quantification of OT-I CD8+ T cells in tumordraining lymph nodes. Figure 22F is a graph showing flow cytometry quantification of proliferating OT-I CD8+ T cells in tumor-draining lymph nodes. Figure 22G is a graph showing flow cytometry quantification of OT-I CD8+ T cells in MC38-OVA tumors. Figure 22H is a graph showing flow cytometry quantification of proliferating OT-I CD8+ T cells in MC38-OVA tumors. Mean ± SEM is shown (n=5-7). Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0040] Figures 23A-23F are a series of graphs showing characterization of T DC in MC38-OVA tumor-bearing mice with FTY720 treatment and adoptive transfer of OT-I CD8+ T cells. Figure 23A is a graph showing flow cytometry quantification of DC in MC38-OVA tumors. Figure 23B is a graph showing flow cytometry quantification of CD40+ DC in MC38-OVA tumors. Figure 23C is a graph showing flow cytometry quantification of the percentage of CD40+ cells in DC in MC38- OVA tumors. Figure 23D is a graph showing flow cytometry quantification of DC in tumor-draining lymph nodes. Figure 23E is a graph showing flow cytometry quantification of CD40+ DC in tumordraining lymph nodes. Figure 23F is a graph showing flow cytometry quantification of the percentage of CD40+ cells in DC in tumor-draining lymph nodes. Mean ± SEM is shown (n=5-7). Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0041] Figure 24A is a graph showing the frequency of proliferating cells in OT-I CD8+ T cells in tumor-draining lymph nodes. Figure 24B is a graph showing the frequency of CD69+ proliferating cells in OT-I CD8+ T cells in tumor-draining lymph nodes. Mean ± SEM is shown (n=5-7). Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0042] Figure 25 is a graph showing quantification of numbers of CD45+ immune cells, CD19+ B cells, and CD3+ , CD4+, CD8+ T cells in peripheral blood of Colon26-tumor bearing mice with anti-CD8a or isotype control antibody treatment directly before LTBR agonist treatment by flow cytometry. Mean ± SEM is shown (n=16). Statistical significance was assessed by two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001. [0043] Figures 26A-B are a series of graphs showing the combination of LTBR agonism and intratumoral chemokine expression. Figure 26A is a graph showing Day 3 over Day 1 proliferation rate of engineered Colon26 cell lines transduced with empty vector (EV) control, Cxcl13 or Ccl19 in vitro (n=6). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons test. Figure 26B is a graph showing Day 3 over Day 1 proliferation rate of engineered Colon26 cell lines transduced with empty vector (EV) or LTa in vitro (n=8). Mean ± SEM is shown. Statistical significance was assessed by unpaired two-tailed Student’s t test. *p < 0.05; **p < 0.01 ;

***p < 0.001 ; ****p < 0.0001. [0044] Figures 27A-E are a series of graphs showing ICB treatment in colorectal tumor models with TA-TLS induced by combination of LTBR agonism and intratumoral LTa expression. Figure 27A shows tumor volume at start of anti-PD1 treatment for LTa-expressing Colon26 tumors treated with LTBR agonist and anti-PD-1. Figure 27B shows tumor size changes in individual mice from Day 0 to Day 19 of anti-PD1 treatment for LTa-expressing Colon26 tumors treated with LTBR agonist and anti-PD-1 . TF, tumor free mice. Figure 27C shows tumor volume at start of anti-CTLA- 4 treatment for empty vector (EV) or LT -expressing Colon26 tumors treated with LTBR agonist and anti-CTLA-4. Figure 27D shows tumor volume at start of anti-CTLA-4 treatment for MC38- OVA tumors treated with LTBR agonist and anti-CTLA-4. Figure 27E shows tumor volume at start of anti-CTLA-4 treatment for LTa-expressing MC38-OVA tumors treated with LTBR agonist and anti-CTLA-4. ISO, isotype control. Mean ± SEM is shown; *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

DETAILED DESCRIPTION

[0045] The present disclosure relates to methods of treating cancer by administering a lymphotoxin beta receptor (LTBR) agonist, such as in combination with adoptive cell therapy (ACT). As disclosed herein, the combination of LTBR agonist and ACT demonstrates unexpected synergistic anti-tumor efficacy in inducing potent and durable tumor control in subjects with cancer.

Methods for Treating Cancer

[0046] The present disclosure includes methods of treating cancer, wherein the method includes administering to a subject in need thereof a therapeutically effective amount of LTBR agonist in combination with a therapeutically effective amount of ACT. The present disclosure also includes methods of increasing the efficacy of ACT in treating cancer by selecting a subject with cancer and administering to the subject a therapeutically effective amount of LTBR agonist in combination with a therapeutically effective amount of ACT.

[0047] Applicant has shown herein that LTBR agonism promoted HEV formation and upregulated CXCL13, CCL19 and CCL21 chemokine expression, which increased T cell, B cell and DC tumor infiltration. LTBR agonism was also found to enhance DC-mediated T cell activation through direct effect on DC activation and maturation. Activated T and B cells expressing LTBR ligands may further amplify this process to achieve sustained antitumor immunity and potential TLS formation. Figure 7 shows a working model of the multi-faceted immunomodulation of TME by LTBR agonism, based on the results herein. LTBR agonism led to increased T cell and DC infiltration and activation in solid tumors, thus increasing the anti-tumor effect of ACT.

[0048] As used herein, the terms “treating,” “treat” or the like, mean to alleviate symptoms, eliminate the causation of symptoms either on a temporary or permanent basis, to delay or inhibit tumor growth, to reduce tumor cell load or tumor burden, to promote tumor regression, to cause tumor shrinkage, necrosis and/or disappearance, to prevent tumor recurrence, to prevent or inhibit metastasis, to inhibit metastatic tumor growth, and/or to increase duration of survival of the subject.

[0049] As used herein, the expression “a subject in need thereof refers to a human or non-human mammal that exhibits one or more symptoms or indications of cancer, and/or who has been diagnosed with cancer and who needs treatment for the same. The term “subject” includes subjects with primary or metastatic tumors (advanced malignancies). In certain embodiments, the expression “a subject in need thereof” includes a subject with a tumor that is resistant to or refractory to or is inadequately controlled by prior therapy (e.g., treatment with an anti-cancer agent). The expression also includes subjects with a tumor for which conventional anti-cancer therapy is inadvisable, for example, due to toxic side effects. For example, the expression includes subjects who have received one or more cycles of chemotherapy and have experienced toxic side effects.

[0050] As used herein, the term “tumor” or “cancer” refers to a disease characterized by the uncontrolled (and often rapid) growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0051] In some embodiments, the disclosed methods are used for treating or inhibiting the growth of a tumor, including but not limited to: adrenal gland cancer, anal cancer, autonomic ganglial cancer, biliary tract cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cancer of the meninges, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, eye cancer, esophageal cancer, fallopian tube cancer, gastric cancer, genital tract cancers, head and neck cancer, kidney cancer, large intestinal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, peritoneal cancer, pituitary cancer, placental cancer, pleura cancer, prostate cancer, rectal cancer, renal cancer, salivary gland cancer, skin cancer, small intestinal cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive cancers, urinary tract cancer, uterine cancer, vaginal cancer, and vulva cancer. The terms “tumor,” “cancer” and “malignancy” are interchangeably used herein.

[0052] In some embodiments, the cancer expresses CXCL13, CCL19, CCL21 or LTa. [0053] In some embodiments, the disclosed methods lead to increased efficacy and duration of anti-tumor response. Methods according to this aspect of the disclosure comprise selecting a subject with cancer and administering to the subject a therapeutically effective amount of a LTBR agonist in combination with a therapeutically effective amount of ACT. In certain embodiments, the methods provide for increased tumor inhibition, e.g., by about 20%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% as compared to a subject treated with the ACT as monotherapy.

[0054] In certain embodiments, the methods provide for increased duration of the antitumor response, e.g., by about 20%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70% or more than 80% as compared to a subject treated with the ACT as monotherapy. In certain embodiments, administration of the LTBR agonist in combination with ACT increases response and duration of response in a subject, e.g., by more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40% or more than 50% more than an untreated subject or a subject treated with ACT as monotherapy.

[0055] In certain embodiments, the disclosed methods lead to a delay in tumor growth and development, e.g., tumor growth may be delayed by about 3 days, more than 3 days, about 7 days, more than 7 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years as compared to an untreated subject or a subject treated with ACT monotherapy.

[0056] In certain embodiments, administration of any of the combinations disclosed herein prevents tumor recurrence and/or increases duration of survival of the subject, e.g., increases duration of survival by 1-5 days, by 5 days, by 10 days, by 15 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months more than the survival of an untreated subject or a subject treated with ACT as monotherapy or treated with ACT in combination with a non-LTBR agonist.

[0057] In certain embodiments, administration of the LTBR agonist in combination with ACT to a subject with a cancer leads to complete disappearance of all evidence of tumor cells (“complete response”). In certain embodiments, administration of the LTBR agonist in combination with ACT to a subject with a cancer leads to at least 30% or more decrease in tumor cells or tumor size (“partial response”). In certain embodiments, administration of the LTBR agonist in combination with ACT to a subject with a cancer leads to complete or partial disappearance of tumor cells/lesions including new measurable lesions. Tumor reduction can be measured by any methods known in the art, e.g., X-rays, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analyses.

[0058] In certain embodiments, administration of the LTBR agonist in combination with ACT to a subject with cancer leads to improved overall response rate, as compared to an untreated subject or a subject treated with ACT monotherapy.

[0059] In certain embodiments, administering to a subject with cancer therapeutically effective amounts of the disclosed ACT and LTBR agonist leads to increased overall survival (OS) or progression-free survival (PFS) of the subject as compared to a subject treated with ACT as monotherapy.

[0060] In certain embodiments, the PFS is increased by at least one 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 as compared to a subject treated with ACT as monotherapy.

[0061] In certain embodiments, the OS is increased by at least one 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 as compared to a subject treated with ACT as monotherapy.

[0062] In certain embodiments, additional treatment effects of the disclosed combination therapy may include increased tumor-specific HEV formation, increased dendritic cell and T cell infiltration, enhanced T cell activation in tumor microenvironment, and/or increased expression of TLS-related chemokines.

LTBR agonists

[0063] As used herein, “lymphoxin beta receptor,” “LTBR,” or “LT R” refers to a tumor necrosis factor receptor superfamily member 3 (TNFRSF3), a cell surface receptor for lymphotoxin involved in apoptosis and cytokine release. It is a member of the tumor necrosis factor receptor superfamily. LTBR is expressed on many different cell types including cells of epithelial and myeloid lineages.

[0064] As used herein, an “LTBR agonist” refers to as any substance that binds to LTBR and results in an increase of signaling resembling that induced by binding of a natural ligand. An LTBR agonist may have a variety of suitable forms including a natural ligand, a protein, a peptide, a peptidomimetic, a nucleic acid, a small molecule, or an antibody.

[0065] In some embodiments, the LTBR agonist may include a natural ligand such as LTaip2 and LIGHT. The expression of LTaip2 is primarily on activated lymphocytes, natural killer cells and lymphoid tissue-inducing cell. LIGHT has a similar expression pattern as LTa1[32 but is also expressed on immature dendritic cells.

[0066] In some embodiments, the LTBR agonist may include an antibody or antigenbinding fragment thereof that binds specifically to LTBR, such as human LTBR. Non-limiting examples of anti-LTBR agonizing antibodies include BHA10, CBE11 , and BS-1. See, e.g., Mackay et al., J. Immunol., 159:3299-3310 (1997); Hu et al., Carcinogenesis, 34:1105-1114 (2013), WO2018119118; US 7429644.

[0067] As used herein, an "agonist" refers to an agent that binds to a receptor and triggers a response in a cell. An agonist mimics the effect of an endogenous ligand, a hormone for example, and produces a physiological response similar to that produced by the endogenous ligand. A "partial agonist" refers to an agent that binds to a receptor and triggers a partial response in a cell. A partial agonist produces only a partial physiological response of the endogenous ligand.

[0068] As used herein, an “antibody” refers to an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (7.e. , “full antibody molecules”), as well as a multimer thereof (e.g., IgM) or antigenbinding fragments thereof. Each heavy chain is comprised of a heavy chain variable region (HCVR) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (LCVR) and a light chain constant region. The HCVR and LCVR regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each HCVR and LCVR is composed of three CDRs. The term “antibody” also includes antigen-binding fragments of full antibody molecules.

[0069] As used herein, an “antigen” refers to any substance that causes the immune system to produce antibodies or specific cell-mediated immune responses against it. A disease- associated antigen is any substance that is associated with any disease that causes the immune system to produce antibodies or a specific cell-mediated response against it.

[0070] As used herein, the “antigen-binding fragment” of an antibody, “antigen-binding portion” of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0071] 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 the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated CDR, such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR- grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.

[0072] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a _H domain associated with a V_L domain, the V_H and V_L domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V -V_H, V -V_L or V_L-V_L dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V_H or V_L domain.

Adoptive Cell Therapy (ACT)

[0073] As used herein, the term “adoptive cell therapy,” “ACT” or “adoptive immunotherapy” are used interchangeably and refer to the administration of a modified immune cell to a subject with cancer. An “immune cell” (also interchangeably referred to herein as an “immune effector cell”) refers to a cell that is part of a subject’s immune system and helps to fight cancer in the body of a subject. 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 may be autologous or heterologous to the subject undergoing therapy.

[0074] 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. A T cell can be a T helper (Th) cell, for example, a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4⁺ T cell) CD4⁺ T cell, a cytotoxic T cell (CTL; CD8⁺ T cell), a tumor-infiltrating cytotoxic T cell (TIL; CD8⁺ T cell), CD4⁺CD8⁺ T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells. Also included are “natural killer T (NKT) cells” or “NKT cells,” which refer to a specialized population of T cells that express a semi-invariant ab T cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. NKT cells include NK1.1⁺ and NK1.G, as well as CD4⁺, CD4, CD8⁺, and CD8 cells.

[0075] The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC l-like molecule CD Id. NKT cells can have either protective or deleterious effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are “gamma-delta T cells (yd T cells),” which refer to a specialized population that to a small subset of T cells possessing a distinct TCR on their surface, and unlike the majority of T cells in which the TCR is composed of two glycoprotein chains designated a- and b-TCR chains, the TCR in yd T cells is made up of a g- chain and a d-chain. yd T cells can play a role in immunosurveillance and immunoregulation and were found to be an important source of IL-17 and to induce robust CD8⁺ cytotoxic T cell response. Also included are “regulatory T cells” or “Tregs,” which refer to T cells that suppress an abnormal or excessive immune response and play a role in immune tolerance. Tregs are typically transcription factor Foxp3- positive CD4⁺ T cells and can also include transcription factor Foxp3 -negative regulatory T cells that are IL-10-producing CD4⁺ T cells.

[0076] T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL separation. In one embodiment, T cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets. [0077] The disclosed immune effector cells, such as T cells, can be genetically modified (forming modified immune cells) following isolation using known methods, or the immune cells can be activated and expanded, or differentiated in the case of progenitors, in vitro prior to being genetically modified. In some embodiments, immune effector cells, such as T cells, are genetically modified with the TCRs or CARs described herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a TCR or a CAR) and then may be activated and expanded in vitro. Techniques for activating and expanding T cells are known in the art and suitable for use with the disclosed technology. See, e.g., US 6,905,874; US 6,867,041; US 6,797,514; WO 2012079000; US 2016/0175358. TCR-expressing or CAR-expressing immune effector cells suitable for use in the disclosed methods may be prepared according to known techniques described in the art.

[0078] For use in the disclosed methods, the immune cells may be modified with a TCR or a CAR against a TAA. In other words, non-limiting examples of ACT for use in the disclosed methods include a modified TCR against a tumor-associated antigen (TAA), or a chimeric antigen receptor (CAR) against a TAA.

[0079] The TAA may be from any cancer including, but not limited to, adrenal gland cancer, anal cancer, autonomic ganglial cancer, biliary tract cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cancer of the meninges, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, eye cancer, esophageal cancer, fallopian tube cancer, gastric cancer, genital tract cancers, head and neck cancer, kidney cancer, large intestinal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, peritoneal cancer, pituitary cancer, placental cancer, pleura cancer, prostate cancer, rectal cancer, renal cancer, salivary gland cancer, skin cancer, small intestinal cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive cancers, urinary tract cancer, uterine cancer, vaginal cancer, and vulva cancer.

[0080] In certain embodiments, the TAA is selected from AFP, ALK, BAGE proteins, BCMA, BIRC5 (survivin), BIRC7, -catenin, brc-abl, BRCA1 , BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1 , CYP1 B1 , EGFR, EGFRvlll, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1 , FOLR1 , GAGE proteins (e.g., GAGE-1 , -2), 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 , 0X40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1 , PRLR, PRAME, PSMA (F0LH1), RAGE proteins, Ras, RGS5, Rho, SART-1 , SART-3, STEAP1, STEAP2, TAG-72, TGF-p, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1 , TRP-2, tyrosinase, and uroplakin-3.

[0081] As used herein, a “T cell receptor” refers to an isolated TCR polypeptide that binds specifically to a TAA, or a TCR expressed on an isolated immune cell (e.g., a T cell). TCRs bind to epitopes on small antigenic determinants (for example, comprised in a tumor associated antigen) on the surface of antigen-presenting cells that are associated with a major histocompatibility complex (MHC; in mice) or human leukocyte antigen (HLA; in humans) complex. TCR also refers to an immunoglobulin superfamily member having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor.

[0082] As used herein, the term “polypeptide” refers to any polymer preferably consisting essentially of any of the 20 natural amino acids regardless of its size. Although the term “protein” is often used in reference to relatively large proteins, and “peptide” is often used in reference to small polypeptides, use of these terms in the field often overlaps. The term “polypeptide” refers generally to proteins, polypeptides, and peptides unless otherwise noted. Peptides useful in accordance with the present disclosure will be generally between about 0.1 to 100 KD or greater up to about 1000 KD, preferably between about 0.1 , 0.2, 0.5, 1 , 2, 5, 10, 20, 30, and 50 KD as judged by standard molecule sizing techniques such as centrifugation or SDS- polyacrylamide gel electrophoresis.

[0083] A TCR can be found on the surface of a cell and generally is comprised of a heterodimer having a and p chains (also known as TCRa and TCRp, respectively), or y and 5 chains (also known as TCRy and TCR6, respectively). Like immunoglobulins, the extracellular portion of TCR chains (e.g., a-chain, p-chain) contain two immunoglobulin regions, a variable region (e.g., TCR variable a region or Va and TCR variable p region or VP; typically amino acids 1 to 116 based on Kabat numbering at the N-terminus), and one constant region (e.g., TCR constant domain a or Ca and typically amino acids 117 to 259 based on Kabat, TCR constant domain p or Cp, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. Also, like immunoglobulins, the variable domains contain CDRs separated by framework regions (FRs). In some embodiments, a TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex. The source of a TCR of the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit or other mammal. In some embodiments, the source of a TCR of the present disclosure is a mouse genetically engineered to produce TCRs comprising human alpha and beta chains (see, e.g., \J\IO 2016/164492).

[0084] As used herein, the terms “complementarity determining region” or “CDR” refer to the sequences of amino acids within antibody variable regions that confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1 , HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1 , LCDR2, and LCDR3). Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, the ABM definition, and the IMGT definition. See, e.g., 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). Public databases are also available for identifying CDR sequences within an antibody.

[0085] TCRa and TCRp polypeptides (and similarly TCRy and TCRS polypeptides) are linked to each other via a disulfide bond. Each of the two polypeptides that make up the TCR contains an extracellular domain comprising constant and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and the cytoplasmic tail also being a part of the constant region). The variable region of the TCR determines its antigen specificity, and similar to immunoglobulins, comprises three CDRs. The TCR is expressed on most T cells in the body and is known to be involved in recognition of MHC-restricted antigens. The TCR a chain includes a covalently linked Va and Ca region, whereas the p chain includes a V region covalently linked to a Cp region. The Va and p regions form a pocket or cleft that can bind an antigen in the context of a major histocompatibility complex (MHC) (or HI_A in humans).

[0086] The term “HLA” refers to the human leukocyte antigen (HLA) system or complex, which is a gene complex encoding the MHC proteins in humans. These cell-surface proteins are responsible for regulating the immune system in humans. HLAs corresponding to MHC class I (A, B, and C) present peptides from inside the cell. The term “HLA-A” refers to the group of human leukocyte antigens (HLA) that are coded for by the HLA-A locus. HLA-A is one of three major types of human MHC class I cell surface receptors. The receptor is a heterodimer and composed of a heavy a chain and a smaller p chain. The a chain is encoded by a variant HLA-A gene, and the p chain (p2-microglobulin) is an invariant p2 microglobulin molecule. The term “HLA-A2” (also referred to as “HLA-A2*01”) is one particular MHC class I allele group at the HLA-A locus; the a chain is encoded by the HLA-A*02 gene, and the p chain is encoded by the [32-microglobulin or B2M locus.

[0087] TCRs are detection molecules with exquisite specificity, and exhibit, like antibodies, an enormous diversity. The general structure of TCR molecules and techniques for making and using such molecules, including binding to a peptide: MHC, are described in PCT/US98/04274, PCT/US98/20263, WO 99/60120.

[0088] For example, non-human animals (e.g., rodents, e.g., mice or rats) can be genetically engineered to express a human or humanized TCR comprising a variable domain encoded by at least one human TCR variable region gene segment. See, e.g., WO 2016/164492. For example, the Veloci-T® mouse technology (Regeneron) provides a genetically modified mouse that allows for the production of fully human therapeutic TCRs against tumor and/or viral antigens, and can be used to produce TCRs suitable for use with the disclosed technology. Those of skill in the art, through standard mutagenesis techniques, in conjunction with the assays described herein, can obtain altered TCR sequences and test them for particular binding affinity and/or specificity. Useful mutagenesis techniques known in the art include, without limitation, de novo gene synthesis, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linkerscanning mutagenesis, and site-directed mutagenesis by PCR.

[0089] In some embodiments, methods for generating a TCR to a TAA may include immunizing a non-human animal (e.g., a rodent, e.g., a mouse or a rat), such as a genetically engineered non-human animal that comprises in its genome an un-rearranged human TCR variable gene locus, with a specified peptide from the TAA; allowing the animal to mount an immune response to the peptide; isolating from the animal a T cell reactive to the peptide; determining a nucleic acid sequence of a human TCR variable region expressed by the T cell; cloning the human TCR variable region into a nucleotide construct comprising a nucleic acid sequence of a human TCR constant region such that the human TCR variable region is operably linked to the human TCR constant region; and expressing from the construct a human T cell receptor specific for the peptide, respectively. The steps of isolating a T cell, determining a nucleic acid sequence of a human TCR variable region expressed by the T cell, cloning the human TCR variable region into a nucleotide construct comprising a nucleic acid sequence of a human TCR constant region, and expressing a human T cell receptor are performed using standard techniques known to those of skill the art.

[0090] As used herein, an HLA presented peptide (such as an HLA-A2 presented peptide) can refer to a peptide that is bound to a HLA protein, such as an HLA protein expressed on the surface of a cell. Thus, a TCR that binds to an HLA presented peptide binds to the peptide that is bound by the HLA, and optionally also binds to the HLA itself. Interaction with the HLA can confer specificity for binding to a peptide presented by a particular HLA. In some embodiments, the TCR may bind to an isolated HLA presented peptide. In some embodiments, the TCR may bind to an HLA presented peptide on the surface of a cell.

[0091] As used herein, a “chimeric antigen receptor” or “CAR” refers to an antigenbinding protein that includes an immunoglobulin antigen-binding domain (e.g., an immunoglobulin variable domain) and a TCR constant domain or a portion thereof, which can be administered to a subject as chimeric antigen receptor T-cell (CAR-T) therapy. As used herein, a “constant domain” of a TCR polypeptide includes a membrane-proximal TCR constant domain, and may also include a TCR transmembrane domain and/or a TCR cytoplasmic tail. For example, in some embodiments, the CAR is a dimer that includes a first polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCRp constant domain and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a K or A variable domain) linked to a TCRa constant domain. In some embodiments, the CAR is a dimer that includes a first polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCRa constant domain and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a K or A variable domain) linked to a TCRp constant domain.

[0092] As used herein, a “variable domain” refers to the variable region of an alpha chain or the variable region of a beta chain that is involved directly in binding the TCR to the antigen. As used herein, the term “constant domain” refers to the constant region of the alpha chain and the constant region of the beta chain that are not involved directly in binding of a TCR to an antigen, but exhibit various effector functions.

[0093] CARs are typically artificial, constructed hybrid proteins or polypeptides containing the antigen-binding domain of an scFv or other antibody agent linked to a T cell signaling domain. In the context of this disclosure, the CAR is directed to a tumor-associated antigen. Features of the CAR include its ability to redirect T cell specificity and reactivity against selected targets in a non-MHC-restricted manner using the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition provides CAR-expressing T cells with the ability to recognize antigens independent of antigen processing, thereby bypassing the major mechanism of tumor escape. As used in the ACT disclosed herein, immune cells can be manipulated to express the CAR in any known manner, including, for example, by transfection using RNA and DNA, both techniques being known in the art.

[0094] In some embodiments, TCR- or CAR-expressing immune effector cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment-effective amount. A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium may be supplemented with human serum albumin.

[0095] A therapeutically effective number of immune cells to be administered in the disclosed methods is typically greater than 10² cells, such as up to and including 10⁶, up to and including 10⁸, up to and including 10⁹ cells, or more than 10¹⁰ cells. The number and/or type of cells to be administered to a subject will depend upon the ultimate use for which the therapy is intended.

[0096] TCRs and CARs of the present disclosure may be recombinant, meaning that they may be created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology, which include, e.g., DNA splicing and transgenic expression. Recombinant TCRs or CARs may be expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

Combination Therapies

[0097] In some embodiments, the present disclosure includes administering to a subject with cancer a combination therapy comprising a therapeutically effective amount of a LTBR agonist and a therapeutically effective amount of an ACT. In some embodiments, the disclosed combination therapy exhibits a synergistic anti-tumor efficacy in various cancer types. In some embodiments, the disclosed combination therapy increases the efficacy of ACT administered to a subject with cancer as compared to a subject treated with the ACT as monotherapy, thereby more effectively treating the cancer.

[0098] With respect to pharmaceutical compositions, the disclosed LTBR agonist and/or ACT may be formulated with one or more pharmaceutically acceptable carriers, excipients and/or diluents. Pharmaceutical compositions comprising the disclosed LTBR agonist and/or ACT may be formulated for specific uses, such as for pharmaceutical uses in humans or for veterinary uses. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents and/or carriers used will depend upon the intended therapeutic use and desired mode of administration of the LTBR agonist and/or ACT.

[0099] A pharmaceutical composition of the present disclosure may contain either or both of the LTBR agonist and ACT, wherein the LTBR agonist and ACT are formulated as separate compositions or as a single composition. Such pharmaceutical compositions may be administered to a subject by a variety of routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically, or locally. In some embodiments, the pharmaceutical composition is administered to the subject intravenously or subcutaneously. Pharmaceutical compositions can be conveniently presented in unit dosage forms containing a predetermined amount of the disclosed LTBR agonist and/or ACT per dose.

[0100] In some embodiments, the disclosed methods further include administration of an additional therapeutic agent or therapy. In some embodiments, the additional therapeutic agent or therapy includes an immune checkpoint inhibitor (e.g., antibody) that targets an immune checkpoint receptor, such as but 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 activation gene 3 protein (LAG3), MHC class II-LAG3, 4-1 BB-4-1 BB ligand, 0X40-0X40 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, Butyrophilins, including BT L2, Siglec family, TIGIT and PVR family members, KTRs, ILTs and LI Rs, 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 (e.g., CTLA-4 or PD1 or PD-LI) and other immunomodulatory agents, such as interleukin-2 (IL-2), indoleamine 2,3-dioxygenase (IDO), IL-10, transforming growth factor- [3 (TGFp), CD39, CD73, Adenosine-CD39-CD73, and CXCR4-CXCL12.

[0101] Additional non-limiting examples of the additional therapeutic agent or therapy include radiation, surgery, a cancer vaccine, a CD47 inhibitor, an antagonist of another T cell coinhibitor or ligand (e.g., an antibody to CD-28, 2B4, LY108, LAIR1 , ICOS, CD160 or VISTA), a vascular endothelial growth factor (VEGF) antagonist [e.g., a “VEGF-Trap” such as aflibercept or other VEGF-inhibiting fusion protein as set forth in US 7,087,411 , or an anti-VEGF antibody or antigen binding fragment thereof (e.g., bevacizumab, or ranibizumab) or a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], an Ang2 inhibitor (e.g., nesvacumab), a transforming growth factor beta (TGFP) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor (e.g., erlotinib, cetuximab), an agonist to a co-stimulatory receptor (e.g., an agonist to glucocorticoid-induced TNFR-related protein), an antibody to a tumor-specific antigen (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), a vaccine (e.g., Bacillus Calmette-Guerin, a cancer vaccine), an adjuvant to increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), a cytotoxin, a chemotherapeutic agent (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), radiotherapy, an IL-6R inhibitor (e.g., sarilumab), an IL-4R inhibitor (e.g., dupilumab), an IL-10 inhibitor, a cytokine such as IL-2, IL-7, IL-21 , and IL-15, an antibody-drug conjugate (ADC) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC), an antiinflammatory drug (e.g., corticosteroids, and non-steroidal anti-inflammatory drugs), a dietary supplement such as anti-oxidants, and combinations thereof.

[0102] In some embodiments, the additional therapeutic agent or therapy comprises an anti-cancer drug. As used herein, an “anti-cancer drug” means any agent useful to treat cancer including, but not limited to, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (O,P'-(DDD)), biologies (e.g., antibodies and interferons) and radioactive agents. As used herein, “a cytotoxin or cytotoxic agent” also refers to a chemotherapeutic agent and means any agent that is detrimental to cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbiastine, coichicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

[0103] As used herein, a “therapeutic agent or therapy” refers to a molecule or compound or a procedure that confers some beneficial effect upon administration to a subject. The beneficial effect may include enablement of diagnostic determinations; amelioration of 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.

[0104] In some embodiments, the combined administration of the LTBR agonist and ACT with an additional therapeutic agent or therapy leads to improved anti-tumor efficacy, reduced side effects of one or both of the primary therapies, and/or reduced dosage of one or both of the primary therapies.

[0105] The present disclosure also provides kits comprising at least one LTBR agonist and at least one ACT (e.g., immune cells modified with an anti-TAA TCR or CAR). Kits typically include a label indicating the intended use of the contents of the kit and instructions for use. As used herein, the term “label” includes any writing, or recorded material supplied on, in or with the kit, or that otherwise accompanies the kit. In some embodiments, the present disclosure provides a kit for treating a subject afflicted with a cancer, wherein the kit includes: a therapeutically effective dosage of at least one LTBR agonist; a therapeutically effective dosage of at least one ACT; and (b) instructions for using the combination of dosages in any of the methods disclosed herein.

Administration Regimens

[0106] The present disclosure includes methods that comprise administering to a subject with cancer a combination of the LTBR agonist and/or the ACT at a dosing frequency that achieves a therapeutic response. In some embodiments, the LTBR agonist and/or ACT is administered to the subject in one or more doses so long as a therapeutic response is achieved.

[0107] In the disclosed methods, the ACT is administered to the subject in combination with the LTBR agonist. As used herein, the expression “in combination with” means that the 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 a selected therapy is administered to the subject at a different point in time, e.g., 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 the ACT (or LTBR agonist), followed by one or more secondary doses of the LTBR agonist (or ACT), optionally followed by one or more tertiary doses of the ACT (or LTBR agonist). For illustrative purposes, sequential administration may include administering to the subject an initial dose of the ACT (or LTBR agonist), followed by one or more secondary doses of the LTBR agonist (or ACT), and optionally followed by one or more tertiary doses of the LTBR agonist (or ACT).

Dosage

[0108] In general, the amount of LTBR agonist and/or ACT administered to a subject according to the methods of the present disclosure is a therapeutically effective amount. As used herein, “therapeutically effective amount” means an amount of the LTBR agonist in combination with the ACT that results in one or more of: (a) a reduction in the severity or duration of a symptom of a cancer; (b) enhanced inhibition of tumor growth, or an increase in tumor necrosis, tumor shrinkage and/or tumor disappearance; (c) delay in tumor growth and development; (d) inhibit or retard or stop tumor metastasis; (e) prevention of recurrence of tumor growth; (f) increase in survival of a subject with a cancer; (g) a reduction in the use or need for conventional anti-cancer therapy (e.g., reduced or eliminated use of chemotherapeutic or cytotoxic agents) as compared to an untreated subject or a subject treated with ACT as monotherapy; (h) increased tumorspecific HEV formation; (i) increased dendritic cell and T cell infiltration; (j) enhanced T cell activation in tumor microenvironment; and/or (k) increased expression of TLS-related chemokines.

[0109] In some embodiments, a therapeutically effective amount of the ACT may comprise immune effector cells expressing a modified TCR or CAR against a tumor-associated antigen administered in an amount of about 1x10⁶ or more, 5x10⁶ or more, 1x10⁷ or more, 5x10⁷ or more, 1x10⁸ or more, 5x10⁸ or more, 1x10⁹ or more, 5x10⁹ or more, or more cells.

[0110] In some embodiments, a therapeutically effective amount of the LTBR agonist may be from about 0.05 mg to about 600 mg of the LTBR agonist, such as 100 mg, 250 mg, or 350 mg. In some embodiments, the amount of the LTBR agonist administered to the subject comprises 0.005 mg/kg to 10 mg/kg, such as 1 mg/kg, 3 mg/kg or 5 mg/kg, of the subject’s body weight.

[0111] As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted. As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise. As used herein, the terms “and/or” or “/” means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0112] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 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 (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0113] The present disclosure merely illustrates the principles of the disclosed technology. Any examples set forth in this specification are not intended to be limiting and merely set forth some of the many possible embodiments for the appended claims. Those skilled in the art will readily recognize various modifications and changes that may be made without following the example embodiments and applications illustrated and described herein, and without departing from the true spirit and scope of the following claims. All references cited and/or discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

EXAMPLES

[0114] The disclosed technology is next described by means of the following examples. The use of these and other examples anywhere in the specification is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified form. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the claims, along with the full scope of equivalents to which the claims are entitled. Also, while efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, room temperature is about 25°C, and pressure is at or near atmospheric.

EXAMPLE 1 : Tumor-specific high endothelial venules (HEV) and tertiary lymphoid structures (TLS) induction by LTBR agonism

[0115] This example relates to LTBR agonism inducing to TLS including HEVs, T cells, B cells, and TLS-related chemokines. Single agent LTBR agonist mAb treatment induced tumorspecific MECA-79+ HEV formation in MC38-OVA tumors. Induced HEVs were associated with locally increased T cell infiltration. There was no HEV formation in normal organs and minimal effect shown by gene expression analysis.

[0116]

[0117] MC38-OVA cell line was generated by transducing Ovalbumin into MC38 mouse colorectal tumor cells. For MC38-OVA tumor model, 1 x 106 MC38-OVA mouse colorectal tumor cells were implanted subcutaneously in the right flank of C57BL/6 mice. When tumors reached 50-100 mm³, mice were randomized into treatment groups and injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11 , Abeam) mAb or isotype control (rat lgG2a, 2A3) mAb twice a week for a total of two doses. After a week of treatment, mice were sacrificed and dissected tumor samples were fixed in 10% neutral buffered formalin for 48 hours at 4°C before replacing with 70% ethanol, embedded in paraffin, and sectioned for immunofluorescent staining. High endothelial venules (HEV) were stained by MECA-79 antibody that recognizes HEV-specific PNAd epitope. T cells and endothelial cells were stained by anti-CD3 and anti-CD31 antibodies respectively.

[0118] MC38-OVA tumors of mice treated with LTBR agonist antibody showed HEV formation, while tumors of mice treated with isotype control antibody did not show HEV formation. No HEV formation occurred in normal organs, and there was minimal effect seen by geneexpression analysis. MC38-OVA tumors of mice treated with LTBR agonist antibody also showed increased T-cell infiltration relative to mice treated with isotype control.

[0119] In a second experiment, the Colon26 tumor model was used. 1 x 10⁶ Colon26 mouse colorectal tumor cells were implanted subcutaneously in the right flank of BALB/c mice. When tumors reached 50-100 mm³, mice were randomized into treatment groups and injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11) mAb or isotype control (rat lgG2a, 2A3) mAb twice a week for a total of three doses. After 10 days of treatment, mice were sacrificed and dissected tumor samples were fixed in 10% neutral buffered formalin for 48 hours at 4°C before replacing with 70% ethanol, embedded in paraffin, and sectioned for immunofluorescent staining. High endothelial venules (HEV) were stained by MECA-79 antibody that recognizes HEV-specific PNAd epitope. T cells and B cells were stained by anti-CD3 and anti-B220 respectively.

[0120] TLS associated with tumors show HEV formation and presence of B cells and T cells. Tumor-associated B cell aggregates or tertiary lymphoid structures (TLS) induced by LTBR agonist antibody treatment were categorized according to tumor-relative locations - adjacent, peripheral, or intra-tumoral. LTBR agonist antibody treatment induced TLS-like structures in -20% of treated Colon26 tumors, mostly at tumor peripheral regions.

[0121] Expression analysis of multiple TLS-related chemokines in MC38 tumors (Figure 1). For MC38 tumor model, 1 x 10⁶ MC38 mouse colorectal tumor cells were implanted subcutaneously in the right flank of C57BL/6 mice. When tumors reached 50-100 mm³, mice were randomized into treatment groups and injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11) mAb or isotype control (rat lgG2a, 2A3) mAb twice a week for a total of two doses.

[0122] After a week of treatment, mice were sacrificed, and RNA was extracted from dissected tumor samples using the TRIzolTM reagent (Life Technologies) and the MagMAX^TM-96 for Microarrays Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Pre-amplified cDNA from tumor lysates was made using the SuperScriptTM III First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. qPCR was then performed on a CFX96TM Real-Time System instrument (Biorad) by using the pre-amplified cDNA for the target genes. qPCR analysis was performed using SsoAdvanced Universal SYBR® Green Supermix (Biorad).

[0123] Relative gene expression was calculated as the fold change of target genes vs housekeeping gene Actb. Bar graphs show mean values ± SEM and each dot represents one tumor. All reactions were run in duplicate. Unpaired Student’s t-test (two-tailed) was used for the comparison of the two groups, p values <0.05 were considered significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001 ; ****: p < 0.0001). All statistical analyses were performed using GraphPad Prism software.

[0124] The target genes were mouse homologs of a published human 12-chemokine gene signature that is correlated with TLS presence and enhanced patient survival in colorectal cancer, melanoma and breast cancer patients. LTBR agonist mAb treatment significantly upregulated the expression of multiple TLS-related chemokines in MC38 tumors (Figure 1).

EXAMPLE 2: LTBR agonism promoted infiltration and activation of DCs and T cells

[0125] This example demonstrates quantitatively the effect of treatment with LTBR agonist antibodies in vivo and in vitro. LTBR agonism leads to infiltration and activation of DCs and T cells within tumors in vivo, compared to treatment with isotype control antibody. In vitro, LTBR agonist treatment directly upregulated activation and maturation markers in BMDCs and enhanced DC-mediated OT-I CD8 T cell activation in vitro.

[0126] Quantification of infiltration and activation of DCs and T cells in vivo (Figure 2A). For MC38 tumor model, 1 x ⁶ MC38 mouse colorectal tumor cells were implanted subcutaneously in the right flank of C57BL/6 mice. When tumors reached 50-100 mm3, mice were randomized into treatment groups and injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11) mAb or isotype control (rat lgG2a, 2A3) mAb twice a week for a total of three doses.

[0127] After 10 days of treatment, mice were sacrificed and tumors were dissected, minced, and incubated with enzymes A, D, and R (Tumor Dissociation Kit, Miltenyi) in combination with mechanical dissociation by gentleMACS Dissociator (Miltenyi). Single-cell suspensions were prepared from dissociated tumors, stained for 30 min at 4°C with antibody cocktails. The tumorinfiltrating immune cell data were acquired on LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo software. [0128] Immune cells were stained by anti-CD45 antibody. Myeloid cells were identified by CD45+CD11b+ staining. Dendritic cells were identified by CD45+CD11c+MHCII+F4/80- staining plus 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 plus the activation markers Ki67 and IFNg.

[0129] The tumor-infiltrating immune cells were quantified as percentage of total live cells. Bar graphs show mean values ± SEM, and each dot represents one tumor. ISO Bars labeled ISO indicate value for isotype treatment. Bars labeled LTBR indicate value for LTBR agonist treatment. Unpaired Student’s t-test (two-tailed) was used for the comparison of the two groups, p values <0.05 were considered significant (*: p < 0.05; **: p < 0.01 ; ***: p < 0.001 ; ****: p < 0.0001). All statistical analyses were performed using GraphPad Prism software.

[0130] LTBR agonist mAb treatment enhanced tumor-infiltration and activation of dendritic cells and T cells in MC38 tumors. Consistent results were observed in Colon26 tumor model (data not shown).

[0131] Bone marrow-derived DC (BMDC) assay (Figure 2B). To test direct effect of LTBR agonism on DC activation and DC-mediated T cell activation, antigen presentation assay was employed with bone marrow-derived DCs and primary CD8+ T cells isolated from spleen of OT-I mice. Briefly, bone marrow cells were isolated from femur and tibia of wild type C57BL/6 mice and differentiated by treatment of 200 ng/mL recombinant Human FLT3L proteins for 8 days in vitro in 6-well plates. The bone marrow-derived cells were then dissociated and plated in 96- well plates with 160 pg/mL anti-mouse LTBR agonist (5G11) mAb or isotype control (rat lgG2a, 2A3) mAb for 18h.

[0132] To assess DC activation, these treated cells were dissociated, prepared as single-cell suspensions, stained for 30 min at 4°C with antibody cocktails covering DC markers (CD11c+MHCII+) and DC activation and maturation markers (CD83, MHCI, MHCII, CD80, CD86, CD40, ICOSL). The data were acquired on LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo software.

[0133] The amount of bone marrow-derived DCs was quantified as percentage of CD11c+MHCII+ cells among the bone marrow-derived mixed culture. Percentage of CD83+ cells and median fluorescent intensity (MFI) of DC activation markers were measured in CD11c+MHCII+ DCs and shown as fold change between LTBR agonist vs isotype control treated DCs. [0134] LTBR agonist mAb treatment increased the percentage of DCs in the bone marrow-derived mixed culture and the percentage of CD83+ matured DC, and also upregulated the expression of DC activation markers in BMDCs.

[0135] Antigen presentation assay with pre-treated BMDC and OT-I T cells (Figure 2C). For antigen presentation assay, the isotype control or LTBR agonist mAb treated BMDCs were further incubated for 2h with 10 ng/mL OVA peptide (SIINFEKL), and co-cultured with primary OT-I CD8+ T cells by 1 :5 ratio for 3 days. The OT-I CD8+ T cells were isolated by EasySep™ Mouse CD8+ T Cell Isolation Kit (STEMCELL Technologies) from spleen of OT-I mice with transgenic T cell receptor designed to recognize OVA peptide SIINFEKL. In the antigen presentation assay, BMDCs can present the OVA peptide with MHCI molecule and activate OT-I SlINFEKL-specific CD8+ T cells.

[0136] After 3 days of co-culture, activation of CD8+ T cells was assessed by the percentage of CD44+ proliferating cells among CD8+ T cells and the median fluorescent intensity (MFI) of activation markers (CD44, TN Fa and GZMB) in CD44+ proliferating CD8+ cells. T cell proliferation was measured by CellTrace™ Far Red Cell Proliferation Kit.

[0137] Pre-treatment of BMDCs with LTBR agonist mAb increased the percentage of CD44+ proliferating CD8+ cells and enhanced the expression of activation (CD44) and cytotoxicity (TN Fa and GZMB) markers.

EXAMPLE 3: LTBR agonism augmented anti-tumor efficacy of PD-1 blockade

[0138] This example demonstrates the anti-tumor efficacy of PD-1 blockade. LTBR agonist treatment attenuated tumor growth in a CD8 T cell-dependent manner. Combination of LTBR agonist and anti-PD1 antibody treatment showed enhanced immune cell infiltration and anti-tumor effect in Colon26 tumor model.

[0139] CD8 T cell depletion in Colon26 tumor model (Figure 3A). For Colon26 tumor model, 1 x 106 Colon26 mouse colorectal tumor cells were implanted subcutaneously in the right flank of BALB/c mice on Day 0. To deplete CD8+ T cells, mice were dosed intraperitoneally with 10 mg/kg of anti-CD8a (cl.2.43; BioXCell) or isotype control (rat lgG2a, 2A3) mAb on Day -2, Day 0 and then twice a week until study end.

[0140] Mice were randomized into treatment groups and injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11) mAb or isotype control (rat lgG2a, 2A3) mAb on Day 8 and followed by twice weekly dosing for a total of four doses. CD8+ T cell depletion was confirmed by quantification of CD8+ T cells in blood by flow cytometry on Day 8 right before dosing of LTBR agonist mAb. [0141] Tumor volume was calculated from caliper measurements using the formula L x w x w x 0.5, where L is the longest dimension and W is the perpendicular dimension. The antitumor effect of LTBR agonist mAb treatment was dependent on the presence of CD8+ T cells (Figure 3A).

[0142] Anti-tumor effect in Colon 26 tumor model (Figure 3B). For Colon26 tumor model, 1 x 10⁶ Colon26 mouse colorectal tumor cells were implanted subcutaneously in the right flank of BALB/c mice. When tumors reached 50-100 mm³, mice were randomized into treatment groups and injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11) mAb and/or anti-mouse PD-1 (RPM1-14, BioXCell) mAb or isotype control (rat lgG2a, 2A3) mAb twice a week for a total of four doses.

[0143] Tumor volume was calculated from caliper measurements using the formula L x W x w x 0.5, where L is the longest dimension and W is the perpendicular dimension. As shown in Figure 3B, while single agent treatment of anti-PD-1 mAb or LTBR agonist mAb showed moderate anti-tumor effects, combination treatment with LTBR agonist mAb and anti-PD-1 mAb enhanced single agent anti-tumor efficacy and eradicated 3 out of 8 tumors on Day 28.

[0144] Immune cell infiltration in Colon26 tumor model (Figure 3C). In a repeat study of the combination treatment of LTBR agonist and anti-PD-1 in Colon26 tumor model, after 10 days of treatment, mice were sacrificed and tumors were dissected, minced and incubated with enzymes A, D, and R (Tumor Dissociation Kit, Miltenyi) in combination with mechanical dissociation by gentleMACS Dissociator (Miltenyi). Single-cell suspensions were prepared from dissociated tumors, stained for 30 min at 4°C with antibody cocktails. The tumor-infiltrating immune cell data were acquired on LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo software.

[0145] Immune cells were identified by anti-CD45 staining, CD4 and CD8 T cells by CD45+CD3+CD4+CD8- and CD45+CD3+CD4-CD8+ staining respectively. Dendritic cells were detected as CD45+CD11c+MHCII+F4/80-CD11 b+ type 2 DC and CD45+CD11c+MHCII+F4/80- CD11b-CD103+ type 1 DC. Natural killer (NK) cells were labeled by CD45+CD3-NKp46+ staining.

[0146] The tumor-infiltrating immune cells were quantified relative to the number of tumor cells. Bar graphs show mean values ± SEM, and each dot represents one tumor. Unpaired Student’s t-test (two-tailed) was used for the comparison of the two groups, p values <0.05 were considered significant (*: p < 0.05; **: p < 0.01 ; ***: p < 0.001 ; ****: p < 0.0001). All statistical analyses were performed using GraphPad Prism software. Combination treatment of anti-PD-1 mAb and LTBR agonist mAb significantly increased tumor infiltration of immune cells. EXAMPLE 4: LTBR agonism augmented anti-tumor efficacy of T cell therapy

[0147] LTBR agonism augmented anti-tumor efficacy of murine hCD20 CAR-T therapy in MC38-hCD20 tumor model.

[0148] A MC38-hCD20 cell line was generated by transducing human CD20 into MC38 mouse colorectal tumor cells. To generate murine hCD20 CAR T cells, murine CD3 T cells were isolated from spleen of wild type C57BL/6 mice, activated with IL-2 and CD3/CD28 beads in vitro, and transduced with retrovirus carrying a CAR sequence against hCD20 that contained CD3z and 4-1 BB intracellular signaling domains (SEQ ID NO: 1) (Table 1). Murine CAR T cells targeted to an irrelevant antigen were used as control (CTRL).

Table 1: Representative sequence

[0149] For the MC38-hCD20 tumor model, mice received lymphodepleting chemotherapy with 250 mg/kg cyclophosphamide intraperitoneally three days before tumor implantation (Day -3) and on Day 0, 1 * 10⁶ MC38-hCD20 cells were implanted subcutaneously in the right flank of C57BL/6 mice. 6 days after tumor implantation (Day 6), mice were randomized into four treatment groups: a control group was injected with 5 mg/kg of isotype control (rat lgG2a, 2A3) mAb intraperitoneally and 3 million CTRL CAR T cells intravenously; a LTBR agonist group was injected with 5 mg/kg of anti-mouse LTBR agonist (5G11) mAb intraperitoneally and 3 million CTRL CAR T cells intravenously; an anti-hCD20 CAR T group was injected with 5 mg/kg of isotype control mAb intraperitoneally and 3 million anti-hCD20 CAR T cells intravenously; a combo group was injected with 5 mg/kg of anti-mouse LTBR agonist mAb intraperitoneally and 3 million anti-hCD20 CAR T cells intravenously.

[0150] Anti-mouse LTBR agonist mAb and isotype control mAb were dosed twice a week for a total of four doses (Day 6, 9, 13, 16). Tumor volume was calculated from caliper measurements using the formula L x W x W x 0.5, where L is the longest dimension and W is the perpendicular dimension. Combination treatment of LTBR agonist and hCD20 CAR T showed significantly higher anti-tumor effect compared to either treatment alone (Figure 4).

EXAMPLE 5: Robust tumor-associated TLS induction by combining LTBR agonism with LTa expression

[0151] This example relates to robust induction of tumor-associated TLS by combining treatment with LTBR agonist antibodies with forced expression of cytokines/chemokines by Colon26 tumors. An in vivo tumor screen revealed that combination of LTBR agonism with CXCL13, CCL19, CCL21 or LTa local expression induced formation of TLS-like structures in Colon26 tumors. Combination of CXCL13 and CCL19/CCL21 expression with LTBR agonism induced B cell clusters and immature TLSs respectively. Combining LTBR agonism with LTa expression showed robust TLS induction.

[0152] In vivo screen in cytokine/chemokine-expressing Colon26 tumor model (Table 2). To perform an in vivo screen for TLS-inducing factors, a panel of Colon26 cell lines were generated by lentiviral transduction to express different TLS-related cytokines as shown in Table 2, left column. 1 x 10⁶ cytokine-expressing Colon26 cells were implanted subcutaneously in the right flank of BALB/c mice.

[0153] When tumors reached 50-100 mm³, mice were randomized into treatment groups and injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11) mAb or isotype control (rat lgG2a, 2A3) mAb twice a week for a total of three doses. After 10 days of treatment, mice were sacrificed and dissected tumor samples were fixed in 10% neutral buffered formalin for 48 hours at 4°C before replacing with 70% ethanol, embedded in paraffin, and sectioned for immunofluorescent staining. High endothelial venules (HEV) were stained by MECA-79 antibody that recognizes HEV-specific PNAd epitope. T cells, B cells and dendritic cells were stained by anti-CD3, anti-B220 and anti-CD11c antibodies respectively.

[0154] As summarized in Table 2, combination of LTBR agonism with CXCL13, CCL19, CCL21 or LTa local expression induced formation of B cell aggregates/TLS-like structures in Colon26 tumors. Table 2: Combination of LTBR agonism with CXCL13, CCL19, CCL21 or LTa local expression induced formation of B cell aggregates/TLS-like structures in Colon26 tumors

[0155] The combination of LTBR agonist treatment and CXCL13 expression induced TLS-like structures that are mostly intra-tumoral with dispersed morphology and without HEV. CCL21 and CCL19 expression showed similar effects, inducing immature TLS-like structures without a T cell zone. Notably, the combination of LTBR agonist and LTa expression induced robust TLS formation in both Colon26 (BALB/c) and MC38 (C57BL/6) tumor models. Activated T and B cells were identified by co-expression of Ki67 and CD3 or B220 respectively; PDPN+ Fibroblastic reticular cell (FRC) network and dendritic cells were found in the T cell zone; regulatory T cells were also observed in TLS as detected by CD4 and FOXP3 expression.

EXAMPLE 6: Tumor-associated TLS formation enhanced tumor response to anti-CTLA-4 treatment

[0156] This example relates to the combination of treatment with LTBR agonist and anti-CTLA-4 effect on growth of Colon26 tumors and MC38 tumors made to express LTa. Anti- CTLA-4 treatment promoted complete regression of TLS+ Colon26 tumors, and a combination of LTBR agonist and anti-CTLA-4 treatment showed a benefit in anti-tumor effect, especially in the LTa-expressing tumors in the MC38-OVA model.

[0157] TLS+ Colon26 tumors (Figure 5). Anti-CTLA-4 treatment promoted complete regression of TLS+ Colon26 tumors. For the TLS+ Colon26 model, 1 x 106 LTa-expressing Colon26 cells were implanted subcutaneously in the right flank of BALB/c mice on Day 0. All tumor-bearing mice were injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11) mAb on Day 10, 13, 17 and 21. Mice were randomized into treatment groups on Day 17 and injected intraperitoneally with 10 mg/kg of anti-PD-1(RPM1-14) mAb and/or 5 mg/kg of anti- CTLA-4 (9D9) mAb on Day 17, 21 , 25, and 28.

[0158] Tumor volume was calculated from caliper measurements using the formula L x W x w x 0.5, where L is the longest dimension and W is the perpendicular dimension. While anti-PD-1 mAb treatment eradicated tumors in 3 out of 7 mice, anti-CTLA-4 mAb treatment resulted in clearance of all TLS+ Colon26 tumors. A follow-up study showed that combining anti- CTLA-4 mAb with LTBR agonist and/or LTa expression promoted complete regression of large (~200 mm3) Colon26 tumors (Figure 5).

[0159] MC38-OVA tumor model (Figure 6). LTBR agonist and anti-CTLA-4 treatment showed combination benefit in anti-tumor effect, especially in the LTa-expressing tumors in MC38-OVA model. To further dissect the combination effect of anti-CTLA-4 mAb with LTBR agonist and LTa expression, the triple combo study was repeated in MC38-OVA model, which is more resistant to anti-CTLA-4 treatment.

[0160] MC38-OVA cells were transduced by lentivirus carrying empty vector (EV) or LTa to generate EV- and LTa-expressing MC38-OVA cell lines. For MC38-OVA tumor model, 1 x 10⁶ EV- or LTa-expressing MC38-OVA cells were implanted subcutaneously in the right flank of C57BL/6 mice. When tumors reached -100 mm³, mice were randomized into treatment groups and injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11) mAb or isotype control (rat lgG2a, 2A3) mAb and/or 5 mg/kg of anti-CTLA-4 (9D9) mAb twice a week for a total of four doses.

[0161] Tumor volume was calculated from caliper measurements using the formula L x W x w x 0.5, where L is the longest dimension and W is the perpendicular dimension. Anti- CTLA-4 mAb and LTBR agonist mAb treatment showed a combination effect and delayed tumor progression, especially in LTa-expressing TLS+ tumors (Figure 6). EXAMPLE 7: LTBR-mediated immunomodulation of the tumor microenvironment promotes anti-tumor responses

[0162] The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis in many cancer types and has been associated with better treatment responses to immune-checkpoint blockade (ICB). However, the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to anti-tumor responses remain unclear. Lymphotoxin beta receptor (LTBR) signaling is a critical regulator of lymph node organogenesis and when combined with antiangiogenic and ICB treatment can augment tumor-associated HEV formation. This example demonstrates that LTBR signaling modulates the tumor microenvironment via multiple mechanisms to promote antitumor T cell responses. Systemic activation of the LTBR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTBR agonism on DC activation and maturation and associated DC-mediated T cell activation. Single agent LTBR agonist treatment inhibited syngeneic tumor growth in a CD8 T cell- and HEV-dependent manner and enhanced anti-tumor effects of anti-PD-1 and CAR T therapies. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTBR agonism and lymphotoxin alpha (LTa) expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA-4 treatment. Collectively, these studies highlight crucial functions of LTBR signaling in modulating the tumor microenvironment and inform future therapeutic strategies to boost immune cell infiltration and activation in solid tumors.

Introduction

[0163] Immune-checkpoint blockade (ICB) has emerged as a promising strategy to activate anti-tumor cytotoxic T cells, highlighted by unprecedented patient responses in multiple cancer types. However, not all patients respond to ICB with lack of adequate intratumoral immune cell infiltration constituting one potential limiting factor. High endothelial venules (HEV) are specialized blood vessels that mediate lymphocyte trafficking into lymph nodes (LN) and Peyer's patches. High endothelial cells are characterized by high expression of adhesion molecules that are ligands for lymphocyte homing receptors including L-selectin (CD62L) (Gerard 2012). HEV can be specifically labeled by MECA-79 antibody staining of peripheral lymph node addressins (PNAd) in both mouse and human (Streeter 1988, Berg, 1991). The presence of PNAd⁺ HEV in human tumors is frequently associated with T and B cell infiltration, better overall survival and response to ICB treatment (Martinet 2011 , Martinet 2012, Asrir 2022, Hua 2022). Mouse tumor studies have demonstrated that tumor-associated HEV (TA-HEV) can function as major portals of lymphocyte infiltration into tumors (Asrir 2022). Tertiary lymphoid structures (TLS) are ectopic lymphoid formations that develop in inflamed or tumor tissues. TLS contain HEV and distinct B and T cell aggregates organized by Cxcl13⁺ follicular dendritic cells (FDC) and Ccl19⁺ fibroblastic reticular cells (FRC), thus structurally resembling secondary lymphoid tissues (Sautes-Fridman

2019). The presence of TLS in solid tumors is commonly associated with positive prognosis and better response to immunotherapy (Sautes-Fridman 2019, Cabrita 2020, Helmink 2020, Petitprez

2020). Tumor-associated TLS (TA-TLS) have been shown to serve as sites of B cell maturation toward anti-tumor antibody-producing plasma cells in renal cell cancer (Maylan 2022). Furthermore, TLS-associated mature dendritic cells (DC) correlated 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.

[0164] Lymphotoxin beta receptor (LTBR) signaling plays a critical role during LN organogenesis with LTBR knockout mice lacking LN and Peyer's patches (Futterer 1998). Mechanistically, LTBR signaling induces the expression of adhesion molecules and lymphoid chemokines in stromal cells of the LN anlage that promotes the formation of immune cell clusters, structural segregation and niche maturation (Onder 2018). Continued LTBR signaling is required for the expression of PNAd scaffolding proteins and core enzymes in high endothelial cells and thus crucial for homeostatic control of HEV differentiation and function (Browning 2005). TLS formation recapitulates LN development and relies 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 with enhanced tumor responses (Asrir 2022, Hua 2022, Allen 2017). Despite the significant roles of LTBR signaling in HEV and TLS formation, the mechanisms underlying LTBR agonism-mediated antitumor effects remain poorly understood. Anti-tumor immune responses induced by systemic LTBR activation via agonistic antibody treatment were investigated in syngeneic mouse tumor models. LTBR agonist treatment promoted tumor-specific HEV formation and upregulated TLS- related chemokine expression, leading to increased immune cell infiltration into tumors. LTBR agonism showed direct effect on DC licensing in vitro and enhanced DC and T cell activation and anti-tumor effect of immunotherapies in vivo. Finally, combining LTBR agonism with LT a expression significantly increased TA-TLS induction and augmented tumor response to anti- CTLA-4 treatment. Materials and Methods

[0165] Mice: 8-10-week-old female BALB/c and C57BL/6J wild-type mice, Prkdc^scid (BS.Cg-Pr dc^/SzJ) SCID mice, TNFRT^/_ mice (C57BUQ-Tnfrsf1a^tm1lmx/J) and OT-I mice (C57BL/6-Tg(TcraTcrb)1100Mjb/J) were purchased from Jackson Labs. Sirpa^hu/huRag2-^/’ll2rg-^/- SRG mice on a C57BL/6J background were generated using the VelociGene technology. All animals were maintained under pathogen-free conditions.

[0166] Cells and Cell lines: Cancer cell lines Colon26 (HCT876), MC38 parental (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. Pancreatic cancer cell line FC1242 was provided by David Tuveson, CSHL. Chemokine- or cytokine-expressing Colon26 and MC38-OVA cell lines used in in vivo screen for TA-TLS were generated by transduction of lentivirus carrying mouse transgene Cxcl9, Cxcl12, Cxcl13, Cell 9, Ccl21b, Vegfc, 1117a or Lta and Neomycin resistance gene linked by IRES sequence, followed by G418 selection. The LTa-expressing Colon26 cell lines used in scRNA-seq experiment were generated by transduction of lentivirus carrying mouse Lta and eGFP genes linked by IRES sequence. Colon26 and 4T1 cells were cultured in RPMI-1640 Medium supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin/Glutamine (Gibco). MC38 cells were cultured in DMEM supplemented with 10% FBS, 1% of MEM non- essential amino acids (NEAA, Gibco), 1 mM sodium pyruvate, 50 pM 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 by Cell Counting Kit 8 (Abeam).

[0167] 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% of 7.5% sodium bicarbonate solution, 50 pM 2-mercaptoethanol, and 1% Penicillin/Streptomycin/ Glutamine. Cells were cultured at 37°C in 5% CO₂.

[0168] T 0 generate murine hCD20 CAR T cells, CD3 T cells were isolated from spleens of wild type C57BL/6J mice using an untouched Mouse T-cell isolation kit (Invitrogen) before activation with anti-mouse CD3/CD28 Dynabeads (Invitrogen) and IL-2 (20 U/ml; Peprotech). After 16 hours, the T-cells were transduced via spin-infection on plates coated with Retronectin (Takara) with retrovirus encoding an anti-hCD20 CAR containing murine CD3z and 4-1 BB intracellular signaling domains. Murine CAR T cells that bind an irrelevant antigen were used as controls.

Mouse tumor studies

[0169] For tumor studies with LTBR agonist treatment, 1x10⁶ cells of MC38-OVA or transgenic variants, 5x10⁵ cells of B16-F10 or 5x10⁵ cells of F1242 with 50% Matrigel (Corning) were subcutaneously implanted into the right flanks of female C57BL/6 mice, 1x10⁶ cells of Colon26 or transgenic variants were subcutaneously implanted into the right flanks of female BALB/c mice and 5x10⁵ cells of 4T1 cells were injected into the mammary fat pad of female BALB/c mice. When tumors reached 50-100 mm³, animals were randomized into treatment groups and injected intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist (5G11, Enzo or Abeam) or isotype control (rat lgG2a, 2A3, Bio X Cell) antibodies twice a week for a total of two to four doses. For macrophage depletion, mice were treated intraperitoneally twice a week with 20 mg/kg of anti-CSF1 R (AFS98, BIO X Cell) or isotype control (2A3, Bio X Cell) antibodies on Day 5, 8, 12, 15 post tumor implantation, and 2 mg/kg of anti-mouse LTBR agonist or isotype control antibodies on Day 12 and Day 15 post tumor implantation. For cytokine blockade, mice were treated intraperitoneally with 25 mg/kg of anti-mouse TNFa antagonist (XT3.11 , Bio X cell) or isotype control (HRPN, Bio X Cell) antibodies, or 25 mg/kg of IL-1 trap (Kuhnert 2015) (Regeneron) or isotype control (mouse lgG2a, Regeneron) antibodies on Day 11 , 14 and 17 post tumor implantation, and 2 mg/kg of anti-mouse LTBR agonist or isotype control antibodies on Day 12 and Day 15 post tumor implantation. For blockade of VEGF and DII4 angiogenesis pathways, mice were injected intraperitoneally with 5 mg/kg of VEGF-trap (Daly 2013) (Aflibercept, Regeneron), anti-DII4 (Amend 2016) (Regeneron) or human Fc control antibodies on Day 14, 17 and 21 post tumor implantation, and 2 mg/kg of anti-mouse LTBR agonist or isotype control antibodies on Day 17 and 21 post tumor implantation. 10 mg/kg of anti-Ang2 (Brasel 2000) (Regeneron) or human Fc control antibodies were co-treated with 2 mg/kg of anti-mouse LTBR agonist or isotype control antibodies intraperitoneally twice a week for a total of two doses to block the pro-angiogenesis Ang2 pathway. For CD8 T cell depletion, mice were dosed intraperitoneally with 10 mg/kg of anti-CD8a (2.43, Bio X Cell) or isotype control (LTF-2, Bio X Cell) antibodies on Day -2 and Day 0 post tumor implantation and then twice a week until study end. Anti-mouse LTBR agonist or isotype control antibodies were injected intraperitoneally twice a week for a total of four doses since Day 8 post tumor implantation and blood was collected by retro-orbital bleeding right before LTBR agonist treatment to validate CD8 T cell depletion. To block PNAd- specific TA-HEV function, mice were dosed intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist or isotype control antibodies on Day 12, 15 and 19 post tumor implantation, and 10 mg/kg of MECA-79 (BioLegend Ultra-LEAF™ Purified) or rat IgM control (RTK2118, BioLegend Ultra- LEAF™ Purified) antibodies were dosed intraperitoneally on Day 13, 15, 17 and 19 post tumor implantation. For combination study of LTBR agonism and anti-PD-1 , tumor-bearing mice were co-treated with 2 mg/kg of anti-mouse LTBR agonist and/or 10 mg/kg of anti-PD-1 (RMP1-14, Bio X Cell) antibodies intraperitoneally twice a week for a total of three doses. To study the combination of LTBR agonism and CAR T cell therapy, C57BL/6J mice received lymphodepleting chemotherapy with 250 mg/kg cyclophosphamide (Sigma) intraperitoneally three days before subcutaneous implantation with 1x10⁶ MC38-hCD20 tumor cells into the right flank. On day 6 after tumor implantation, mice were injected intravenously with 3x10⁶ hCD20 or control CAR T cells. Starting on day 6, the mice were also dosed intraperitoneally with 5 mg/kg of anti-mouse LTBR agonist or isotype control antibodies twice a week for a total of four doses. For scRNA-seq study, 2x10⁶ cells of Colon26-GFP or Colon26-LTa-GFP were subcutaneously implanted into the right flanks of female BALB/c mice and 2 mg/kg of anti-mouse LTBR agonist or isotype control antibodies were dosed intraperitoneally twice a week. For combination study of LTBR agonism, anti-PD-1 and anti-CTLA-4 in Colon26-EV/LTa tumor model, all drugs were treated intraperitoneally twice a week for a total of four doses, but the ICB treatment i.e. , 10 mg/kg of anti- PD-1 or isotype control antibodies and/or 5 mg/kg of anti-CTLA-4 (9D9, InvivoGen) or isotype control (mouse lgG2a, Regeneron) antibodies, were dosed 4 days or 7 days after 2 mg/kg of antimouse LTBR agonist or isotype control antibody treatment to allow TA-TLS formation prior to ICB treatment. In detail, the ICB treatment started 7 days after LTBR agonist treatment in LTa- expressing tumors, and 4 days after LTBR agonist or isotype control antibody treatment in all the other conditions, in order to achieve similar tumor sizes among different groups at the start of ICB treatment. For combination study of LTBR agonism and anti-CTLA-4 in MC38-OVA-EV/LTa tumor model, mice with ~100 mm³ tumors were co-treated intraperitoneally with 2 mg/kg of anti-mouse LTBR agonist or isotype control antibodies and 5 mg/kg of anti-CTLA-4 or isotype control antibodies twice a week for a total of three doses. Tumor growth was assessed by measuring tumor volume with digital calipers. T umor volume was calculated using the formula L x W x W x 0.5, where L is the longest dimension and W is the smaller perpendicular dimension. Animals were euthanized by CO2 asphyxiation at an indicated timepoint for tissue dissections or when tumors grew to 2000mm³ or became ulcerated.

Immunostaining analysis

[0170] Tissues were fixed in 10% neutral buffered formalin for 48 hours at 4°C before replacing with 70% ethanol and embedded in paraffin. 4 pm-thick sections were collected using a microtome and baked at 65°C for 1 hour. Sections were deparaffinized, rehydrated and subjected to heat-induced antigen retrieval for 20 min at 95 °C in pH 6.0 citrate buffer or pH 9.0 Tris-based buffer (Vector Laboratories) before the blocking step. Sections were stained with primary antibodies overnight at 4°C, washed with PBS and stained with secondary antibodies for 1h at room temperature. Finally, slides were washed with PBS, stained with DAPI for 5 minutes and mounted using Fluoromount aqueous mounting medium (Sigma-Aldrich) for immunofluorescent staining. Opal 4-Color Anti-Rabbit IHC Kit (Akoya Biosciences) was used for multiplex immunofluorescent staining with multiple rabbit primary antibodies. For immunohistochemistry (IHC) staining, sections were incubated with 3% H2O2 (vol/vol, Sigma- Aldrich) for 10 min 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-conjugated MECA-79 (Invitrogen), anti-CD31 (ab28364, Abeam), AF647- conjugated anti-B220 (RA3-6B2, BioLegend), anti-CD3 (SP7, Abeam), anti-CD4 (EPR19514, Abeam), anti-CD8a (D4W2Z, Cell Signaling Technology), anti-Ki67 (D3B5, Cell Signaling Technology), anti-PDPN (RTD4E10, Abeam), anti-CD11c (D1V9Y, Cell Signaling Technology), anti-CXCL13 (EPR19259-147, Abeam) and anti-LYVE1 (NB600-1008, Novus Biologicals).

[0171] Fluorescent images were captured with a Zeiss Axio Imager 2 microscope (Zeiss) or scanned by a Zeiss Axioscan 7 microscope slide scanner with 20X magnification objective (Zeiss). IHC images were acquired by an Aperio AT2 slide scanner (Leica Biosystems). Image analysis and quantification were performed using Indica HALO software (Indica Labs) in scanned images of whole section. To quantify the number and area of TLS, aggregates containing B220⁺ B cells and CD3⁺ T cells and larger than 7000 pm² in size were identified as TLSs by HALO AI^TM Deep Learning Classifier function and normalized to the non-necrotic tumor area.

Flow cytometry

[0172] Tumors were excised, mechanically minced, dissociated to single cell suspensions using the mouse Tumor Dissociation Kit according to the manufacturer’s protocol (Miltenyi), and resuspended in FACS buffer containing PBS supplemented with 2% FBS, 2 mM EDTA (VWR) and 0.1% sodium azide (VWR). Cell cultures in vitro were washed with PBS, dissociated by TrypLE™ Express Enzyme (Gibco) and resuspended in FACS buffer. Dead cell discrimination was performed using the Zombie Violet™ Fixable Viability Kit (BioLegend). Prior to antibody staining, cells were incubated with TrueStain Mouse FC-block (BioLegend). Surface antigens were stained first by 30 min incubation with antibody cocktails in FACS buffer at 4°C. For intracellular staining, cells were fixed and permeabilized 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, except where 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-CD8a (53-6.7, BD Biosciences), anti-CD19 (1 D3, BD Biosciences), anti-CD11b (M1/70, BD Biosciences), anti- CD11c (N418), anti-MHCll (M5/114.15.2), anti-MHCI (M1/42), anti-NKp46 (29A1.4), anti-CD49b (HMa2), 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 Scientific), 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-TNFa (MP6-XT22), and BUV395 Streptavidin (BD Biosciences). Data were acquired on LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo.

Gene expression analysis by qPCR

[0173] Tumors were excised, collected in TRI reagent (Invitrogen) with 5 mm Stainless Steel Beads (Qiagen) and dissociated using TissueLyser II with 2 min at 29 Hz (Qiagen). RNA was extracted using the MagMAX^TM-96 for Microarrays Total RNA Isolation Kit (Invitrogen) and cDNA was generated using the SuperScript IV First-Strand Synthesis System (Invitrogen) according to the manufacturer’s instructions. qPCR was then performed on a CFX96 Real-Time PCR detection System instrument (Bio-Rad) with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and qPCR primers designed for genes of interest. p-Actin was used as housekeeping gene to calculate relative gene expression.

LTBR activation in EC and DC cell lines in vitro

[0174] For EC activation, 96-well plates were coated with 10 pg/mL polyclonal goat anti-Rat IgG F(ab')₂ fragments (Jackson ImmunoResearch Labs) in PBS overnight at4°C, washed with PBS, blocked with 2% BSA (Sigma-Aldrich) for 1h at room temperature, incubated with 1 pg/mL LTBR agonist (5G11 , Enzo or Abeam) or isotype control (rat lgG2a, 2A3, Bio X Cell) antibodies for 1h and washed with PBS. 1x10⁴ cells of SVEC4-10 or b.End3 were then seeded per well in complete medium supplemented with PBS, 10 ng/mL of mouse TNFa (R&D Systems), 10 ng/mL of mouse I L-p (R&D Systems) or 10 pg/mL of LPS (Sigma-Aldrich) and incubated for 18h before collection for flow cytometry analysis. [0175] For DC activation, 12-well plates were coated with 10 pg/mL LTBR agonist or isotype control antibodies in 0.2% gelatin (Sigma-Aldrich), or 0.2% gelatin alone overnight at 4°C and washed with PBS. MutuDC1940 cells were treated with 10 pg/mL LPS or PBS for 2h before dissociation. Then 3x10⁵ DC cells were seeded per well with complete medium and incubated for 18h before collection for flow cytometry analysis.

BMDC differentiation and co-culture with OT-I T cells

[0176] Bone marrow cells were isolated from femur and tibia of wild-type C57BL/6J mice as described (Amend 2016). Isolated bone marrow cells were resuspended with 10% ammonium chloride solution (STEMCELL Technologies) in PBS at room temperature for 2 min to lyse red blood cells and washed with BMDC medium consisting of McCoy's 5A (Modified) Medium (Gibco) supplemented with 10% FBS, 1% of MEM NEAA, 1 mM sodium pyruvate, 50 pM 2-mercaptoethanol, and 1% Penicillin/Streptomycin. For DC differentiation, bone marrow cells were cultured in BMDC medium containing 200 ng/mL human FLT3L (BioLegend) for 8 days at 1x10⁶ cells per mL in 6-well plates (Brasel 2000). Cells were cultured at 37°C with 5% CO2. After 8 days, BMDC were harvested from the cultures by vigorously pipetting to collect the non-adherent and loosely adherent cells, and cultured in BMDC medium with 160 pg/mL LTBR agonist (5G11) or isotype control antibodies for 18h. BMDC were seeded in 96-well plates at 1x10⁵ cells per well for flow cytometry analysis and 3x10⁴ cells per well for co-culture with OT-I T cells.

[0177] For BMDC-T cell co-culture, the LTBR agonist or isotype control antibody treated BMDC were pulsed with 10 ng/mL OVA257-264 (AnaSpec) or scrambled peptides (AnaSpec) for 2h at 37°C and washed with T cell medium consisting of RPMI-1640 Medium supplemented with 10% FBS, 50 pM 2-mercaptoethanol, and 1% Penicillin/Streptomycin/Glutamine. OT-I CD8 T cells were isolated from spleen 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). 1.5x10⁵ OT-I cells were seeded per well with T cell medium to achieve 1 :5 DC:T cell ratio. After 3-day co-culture, T cells were collected for flow cytometry analysis.

Adoptive transfer of OT-I T cells

[0178] MC38-OVA tumor bearing mice were treated with 2 mg/kg LTBR agonist (5G11) or isotype control antibodies intraperitoneally twice a week 7 days after tumor implantation. 1.5 mg/kg FTY720 (Cayman Chemical) or DMSO with 2% [3-hydroxypropyl-cyclodextrin (Sigma- Aldrich) in PBS was dosed intraperitoneally after 5-day LTBR agonist treatment and every two days later. OT-I CD8+ T Cells were isolated from lymph nodes and spleen of OT-I mice as described before and labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific). Two days after FTY720 treatment, 2.5x10⁶ OT-I CD8+ T Cells were dosed in each animal by retro-orbital injection, and blood, tumor-draining lymph nodes and tumor tissues were collected 5 days later for flow cytometry analysis.

Single cell RNA-seq

[0179] For scRNA-seq of Colon26-GFP/LTa-GFP tumors with 7-day LTBR agonist or isotype control antibody treatment, study design and preparation of tumor single cell suspension were described above. After staining with anti-CD45 and anti-CD31 antibodies for 30 min at 4°C, the tumor dissociated cells were sorted by FACS into endothelial cells (GFP’CD45’CD31⁺), stromal cells (GFP’CD45’CD31 and immune cells ((GFP’CD45⁺CD3T), and mixed for loading. Dead cells were discriminated by DAPI staining at sorting. Single cells suspended in PBS with 0.04% BSA were loaded, 10K cells per lane or as many cells as were available, on a Chromium X Controller (10X Genomics). RNA-seq and V(D)J libraries were prepared using 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 Illumina NovaSeq 6000 for RNA-seq libraries (Read 1 26-bp for UMI and cell barcode, Read 2 80-bp for transcript read, with 10-bp I7 and 10-bp i5 reads). For RNA-seq libraries, Cell Ranger Single Cell Software Suite (10X Genomics, 6.1.1) was used to perform sample de-multiplexing, alignment, filtering, and UMI counting. The mouse GRCm38 genome assembly was used for the alignment.

Single cell RNA-seq datasets analysis

[0180] The initial filtered scRNA-seq data obtained from Cell Ranger software was utilized. Droplets with more than 10% mitochondrial reads, or with less than 200 genes expressed (defined as UMI > 0) were excluded from downstream analysis due to higher likelihood to contain dying cells or being empty droplet, respectively. Following initial filtering, a library-size correction method was applied to normalize the raw counts using the “normalize_total” function in scanpy (Wolf 2018), with parameter “target_sum” = 10,000. The 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, “min_disp” - 0.5. Subsequently, the effects of the total count per droplet and the percentage of mitochondrial gene count were removed using the scanpy. pp.regress_out” function. Principal component analysis was then performed with the “scanpy. tl. pea” function with parameters “scd_solver = ‘arpack’”. Dimensionality reduction on the data set was performed using the “scanpy. tl.umap” function with parameters “n_neighbors” = 15 and “n_pcs = 20”, as well as clustering using function “scanpy. tl.leiden” with parameter “resolution = 1.4”. Cluster-specific marker genes were identified using the scnapy.tl.rank_gene_groups” function with default parameters, and major cell types were annotated based on the expression of Ptprc (CD45) and Pecaml (CD31). The major immune cell types within the CD45⁺ clusters were identified by Cd3e (T cells), Cd19 (B cells), Cd14/Fcgr4 (Monocytes/Microphages/DC). Through the clustering, clusters were identified with low number of genes expressed (< 1000 in all cells in the cluster, likely dying cells) or clusters with multiple markers expressed (likely doublets) to be removed. UMAP and Leiden cluster is performed again on the remaining droplets to generate the final clusters using parameters “resolution = 0.4”. The choice of resolution was based on whether the cluster definition can separate the cell population of interest.

[0181] To identify sub-populations within the endothelial cells, the Pecam1⁺ endothelial cells were re-processed using the workflow described in the previous section, and clustered with Leiden algorithm using parameter “resolution - 0.2”.

[0182] In the CD31'CD45' clusters, the clusters that were Bmp2⁺/Bmp4⁺/eGFP⁺ were removed (Figure S8A, cluster 6, 7, 12), which are cancer cells that passed through the depletion process due to low eGFP expression. The remaining stromal cells were re-processed and clustered with Leiden algorithm using parameter “resolution = 0.3”. Within the CD45⁺CD3⁺ T cell clusters, the cells were re-processed and Leiden-clustered with “resolution = 0.8.” Clusters expressing Cd8a were identified and re-process, UMAP, and clustering was performed again to identify subtypes of CD8⁺ T cells.

[0183] All UMAP figures with gene expression were generated using the CP1 OK values from “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, then for each gene we normalized the cluster mean expression value by calculating the z-score across clusters.

[0184] The progenitor and exhaustion scores of CD8⁺ T cells were calculated using the “scanpy.tl.score_genes” function with parameters “ctrl_size” = 100, “n_bins” = 24, and gene set definitions based on Pai et al., 2023 (Pai 2023). The significance of score differences between clusters were calculated by two-sample t-test, and the p values were multiple-test corrected using Benjamini-Hochberg correction.

Quantification and statistical analysis

[0185] Sample sizes were empirically chosen to ensure adequate statistical power and were in line with standards in the field for techniques utilized in this study. Multiple independent experiments were performed when feasible. Statistical significance was determined by unpaired two-tailed Student’s t-test or one- and two-way analysis of variance (ANOVA) with multiple comparison analysis for experiments with more than 2 groups. Statistical details for each experiment can be found in the figure legends. Bar graphs show mean values ± SEM. p values <0.05 were considered significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).

RESULTS

Systemic LTBR activation via agonist antibody induced tumor-specific HEV and TLS formation

[0186] To test the effects of systemic LTBR activation on HEV induction, mice bearing syngeneic subcutaneous colorectal tumors were treated with the LTBR agonist antibody 5G11 twice weekly at 2 mg/kg. TA-HEV, identified by PNAd immunostaining with MECA-79 antibody, were readily detected in Colon26 and MC38-OVA tumors 7 days after LTBR agonist treatment but not in isotype control antibody treated tumors. LTBR agonism-induced TA-HEV exhibited the typical “plump” morphology of LN high endothelial cells (HEC). LTBR agonist antibody treatment similarly induced the formation of TA-HEV in additional murine tumor types, including subcutaneous B16-F10 melanoma, FC1242 pancreatic and orthotopically implanted 4T1 mammary tumors.

[0187] 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 the immunostaining data, LTBR agonist treatment significantly increased the frequency of tumor-associated high endothelial cells (TA- HEC) and PNAd expression levels, while the overall CD31⁺ endothelial cell content was unchanged (Figure 8A). The expression of inflammatory endothelial makers P-selectin, E-selectin and ICAM-1 was significantly increased in PNAd⁺ HEC relative to PNAd' EC (Figure 8B). Lymphocyte immunostaining revealed the presence of TA-TLS, characterized by organized lymphoid aggregates with discrete B cell and T cell zones, in about 10% of LTBR agonist treated Colon26 tumors. TA-TLS were formed mainly at the periphery of tumors and commonly associated with HEV.

[0188] LTBR shows widespread expression in normal tissues, both in parenchymal and stromal compartments (Consortium 2020, Eraslan 2022, MacParland 2018). To assess 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 by MECA-79 immunostaining for HEV induction. Strikingly, ectopic HEV formation was not detected in any of the normal organs including lymphoid tissues. Furthermore, histological analysis of LN from LTBR agonist treated mice showed normal tissue architecture and HEV distribution. Overall, systemic LTBR activation via agonist antibody induced tumor-specific HEV with occasional TLS formation. Redundant proinflammatory signals contribute to HEC differentiation

[0189] The mechanism of the tumor-specific HEV induction by systemic activation of LTBR signaling was examined. Immune cell contribution to TA-HEV formation was tested in immunodeficient mice. TA-HEV were observed in colorectal tumor-bearing Sirpa^hu/huRag2-^/’ll2rg-^/- (SRG) and Prkdc^scid (SCID) mice at comparable levels to immune-competent hosts, indicating that T, B, natural killer (NK) and innate lymphoid cells are not required for LTBR agonism-induced TA-HEV formation. Depletion of macrophages by anti-CSF1 R antibody treatment did not affect TA-HEV formation, indicating that Fc receptor-mediated clustering of the LTBR agonist antibodies was not critical for TA-HEV formation. Similarly, neutralization of the inflammatory cytokines TNFa or IL-10 with blocking antibodies did not impact LTBR agonist-induced TA-HEV formation. Lastly, LTct3-TNFR1 signaling was reported to drive TA-HEV formation in intraperitoneal tumor models (Peske 2015). When tested in TNFRT^/_ mice, LTBR agonist treatment showed similar HEV- inducing activity in MC38 tumors as in wildtype mice.

[0190] In vitro assays were established to assess the induction of HEV marker MAdCAM-1 and endothelial cell adhesion molecules to further dissect the roles of LTBR agonism and proinflammatory signaling in endothelial cell activation (Berg (1993). Single agent treatment with the LTBR agonist antibody or proinflammatory factors TNFa, IL-1 p and LPS significantly increased the percentage of cells expressing MAdCAM-1 in LN-derived endothelial SVEC4-10 cells (O’Connell 1990) (Figure 9A). Importantly, combination of LTBR agonism with any of the three proinflammatory signals significantly increased the frequency of MAdCAM-1 expression (Figure 9A). The LTBR agonist also showed significant additive activities with TNFa, IL-1 and LPS upregulating the expression of endothelial inflammatory markers ICAM-1 and VCAM-1 in SVEC4-10 cells (Figure 9A). Similar combination effects for the induction of ICAM-1 , VCAM-1 and P-selectin were observed in brain endothelial b.End3 cells (Omidi 2003) (Figure 9E). Collectively, these data suggest that redundant inflammatory signals contribute to HEC differentiation, explaining the lack of effect of selective TNFa and IL-10 blockers on LTBR agonist- mediated HEV formation in tumors in vivo.

[0191] VEGF receptor blockade with anti-VEGFR2 antibody DC101 has been shown to promote intratumoral HEV formation when combined with anti-PD-L1 antibody (Allen 2017). Thus, the intersection of LTBR agonism-induced TA-HEV formation with the activity of known tumor angiogenesis pathways was investigated. Blockade of VEGF, DII4-Notch and Ang2-Tie2 signaling by VEGF-Trap, anti-DII4 and anti-Ang2 antibody treatment did not impact LTBR agonist- mediated HEV formation in Colon26 tumors, suggesting that TA-HEC differentiation occurred independently of major tumor angiogenesis pathways. LTBR agonist treatment upregulated TLS-related chemokine expression and increased immune cell infiltration into tumors

[0192] A chemokine gene signature has been described to correlate with TA-TLS presence and better survival in colorectal carcinoma, melanoma and breast cancer patients (Coppola 2011, Messina 2012, Prabhakaran 2017). The expression of mouse homologs of the human TLS-related chemokines in MC38 and Colon26 tumors was assessed by qPCR analysis. LTBR agonist treatment was associated with the general 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 showed that the frequency of tumor infiltrating CD8⁺ T cells and DC was significantly augmented in LTBR agonist treated MC38 tumors (Figure 10B). Consistently, intratumoral expression of Cxcl13 and Ccl19 were significantly upregulated in Colon26 tumors with LTBR agonist treatment (Figure 10C). LTBR agonism significantly increased the frequency of tumor-infiltrating CD45⁺ immune cells, particularly B cells, CD4⁺ T cells, DC and NK cells, in Colon26 tumors (Fig. 10D). Overall, LTBR agonist treatment upregulated TLS-related chemokine expression and broadly increased intratumoral immune cell infiltration.

LTBR agonism promotes DC licensing and DC-mediated T cell activation in the tumor microenvironment

[0193] LTBR signaling has been implicated in DC homeostasis and licensing (Kabashima 2005, Summers deLuca 2012, Summers deLuca 2011). The effect of LTBR agonism on tumorinfiltrating DC in Colon26 tumors was evaluated. Flow cytometry analysis showed that the frequency of CD40⁺ activated DC and CD40⁺CD83⁺ mature DC was significantly increased by LTBR agonist treatment (Figure 11A). Corresponding to the enhanced DC activation, a significantly larger proportion of Ki67⁺ CD4 and CD8⁺ T cells were observed in tumors treated with the LTBR agonist (Figure 11 B). LTBR agonism also increased the frequency of TCF1⁺PD-1 ⁺ stem-like cells and reduced the frequency of SLAMF6’Tim3⁺PD1 ⁺ exhausted cells in the CD8⁺ T cell population (Figure 11B). Similarly, LTBR agonist treatment elevated DC expression of MHC class II and CD40 (Fig. 12D), and enhanced CD4⁺ and CD8⁺ T cell activation in MC38 tumors (Fig. 12E).

[0194] To investigate if LTBR agonism can directly impact DC function, in vitro models of DC activation and licensing were utilized. The effects of LTBR agonism in the murine DC cell line MutuDC1940 model were tested (Fuertes Marraco 2012). MutuDC1940 cells were pretreated with LPS or PBS for 2h and then cultured with coated LTBR agonist or isotype control antibodies for 18h. The expression of molecules involved in DC licensing, antigen presentation and co- stimulation machinery were analyzed by flow cytometry. LTBR agonist treatment significantly upregulated the expression of MHC class I, CD80 and CD83 to similar levels as in LPS stimulated DC and doubled CD40 expression (Figurel 1C). LTBR agonism also significantly increased MHC class II and CD8 expression (Figurel 1C).

[0195] DC activation in response to LTBR agonism in mouse bone marrow-derived DC (BMDC) was analyzed (Figure 12A). 18-hour LTBR agonist treatment resulted in a significantly increased number of BMDC, suggesting enhanced DC differentiation and/or expansion (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 MHC class I, MHC class II, CD80, CD86, ICOSL and particularly CD40 in CD11c⁺MHC ll⁺ BMDC (Figure 12B). To further assess LTBR agonism effects on DC licensing, LTBR agonist treated BMDCs were pulsed with OVA257-264 peptide for 2h and then co-cultured with OT-I CD8 T cells for 3 days to assess T cell activation (Figure 12A). Co-culture with LTBR agonist-treated BMDC significantly increased the percentage of CD44⁺ proliferating OT-I T cells and enhanced the expression of effector and cytotoxicity markers CD44, TNFa and GzmB (Figure 12C).

[0196] LTBR agonism effects on tumor-specific T cell priming and activation in the tumor microenvironment was tested. To decouple LN from intratumoral T cell priming, lymphocyte egress from secondary lymphoid organs was inhibited via FTY720 treatment in MC38-OVA tumorbearing mice, followed by adoptive transfer of labeled OT-I CD8+ T cells (Fig. 21A). As expected, FTY720 treatment significantly decreased the frequency of OT-I T cells in peripheral blood and tumor-draining LN (Fig. 22A-F). Intratumoral T cell frequencies were reduced by FTY720 treatment; this effect was less pronounced in animals treated with the LTBR agonist (Fig. 22G- H). Importantly, the frequency of proliferating and CD69+ proliferating OT-I T cells was significantly higher in tumors treated with the LTBR agonist (Fig. 21 B-C), indicating in situ cell priming and activation within the tumor microenvironment. Consistent with this result and the data in the MC38 and Colon26 tumor models (Fig. 11A and Fig. 12D), an increase in activated DC in the LTBR agonist treated MC38-OVA tumors was observed (Fig.23A-C). LTBR agonism also increased the frequency of CD69+ proliferating OT-I T cells in the tumor-draining LN (Figs. 23D- F, 24A-B), indicating a systemic effect of LTBR agonism to promote tumor-specific T cell activation. Overall, these data highlight the LTBR signaling function of directly promoting DC licensing and resultant DC-mediated T cell activation in the tumor microenvironment.

CD8+ T cell- and HEV-dependent anti-tumor effect of LTBR agonism

[0197] The effects of LTBR agonist treatment on Colon26 tumor growth were assessed. Single agent LTBR agonist treatment significantly delayed Colon 26 tumor growth relative to isotype control treatment (Fig. 13C). CD8+ T cell depletion studies via anti-CD8a antibody treatment demonstrated that LTBR agonist-mediated anti-tumor effects were CD8+ T cell dependent (Figures 13C and 25). Consistent with previous data, CD8+ T cell depletion did not impact intratumoral HEV formation. Thus, LTBR agonist treatment showed CD8 T cell-dependent antitumor effect in the syngeneic Colon26 tumor model.

[0198] Increased immune cell infiltration attributable to HEV activity has been implicated in better outcomes in cancer patients (Martinet 2011 , Asrir 2022, Hua 2022, Karpathiou 2021 , Park 2021 , Sawada 2022). However, direct evidence linking TA-HEV function and antitumor effects is still missing. To address this mechanism, high-dose MECA-79 antibody treatment (M’Rini 2003) was utilized to interrupt PNAd/L-selectin dependent HEC-immune cell interactions and immune cell infiltration into tumors. The anti-tumor activity of LTBR agonist antibody treatment was neutralized by MECA-79 antibody treatment (Figure 13D). Correspondingly, MECA-79 antibody treatment significantly reduced tumor infiltration of B cells (Figure13E) and central memory (CD44⁺L-selectin⁺) CD4 and CD8 T cells (Figure 13F). LTBR agonism-associated anti-tumor functions are critically dependent on HEV-mediated promotion of immune cell infiltration.

LTBR agonist treatment augmented anti-tumor efficacy of T cell-based immunotherapies

[0199] Given the effects of LTBR agonism on immune cell infiltration and DC licensing, it was investigated if LTBR agonism could enhance tumor responses to T cell-based immunotherapies. The activity of LTBR agonist and anti-PD-1 combination treatment in Colon26 tumors was tested. Single agent anti-PD-1 treatment of established Colon26 tumors did not show significant activity, whereas the LTBR agonist antibody consistently promoted moderate but significant anti-tumor effect (Figure 14A). The combination of anti-PD-1 with LTBR agonist significantly reduced tumor growth with 5/14 mice exhibiting a greater than 30% decrease in tumor size, including two tumor-free mice after 10-day treatment (Figure 14A). Flow cytometry analysis showed that the combination treatment of anti-PD-1 and LTBR agonist increased the frequency of tumor-infiltrating immune cells especially CD103⁺ migratory type 1 DC (Figure 14B).

Combination of LTBR agonism with murine CAR T cell therapy

[0200] The effect of combining LTBR agonism with murine CAR T cell therapy was assessed. For this, the MC38 cell line was engineered to express human CD20 (hCD20) to enable targeting by hCD20 CAR T cells. MC38-hCD20 tumor-bearing mice were treated with LTBR agonist antibody and hCD20 CAR T cells on Day 6 post tumor implantation. While single treatment groups did not show anti-tumor effect, the combination of LTBR agonist and hCD20 CAR T therapy significantly reduced tumor growth (Figure 15A). Immunohistochemistry analysis at the end of study showed higher number of CD4 and CD8 T cells in the LTBR agonist containing treatment groups (Figure 15B). Overall, these data highlight the potential of LTBR agonism to augment anti-tumor efficacies of T cell-based immunotherapies.

In vivo tumor cytokine screen identified mediators of intratumoral TLS formation

[0201] LTBR agonist antibody treatment induced the formation of TA-TLS in -10% of Colon26 tumors. To assess if TA-TLS formation frequency could be increased and to establish a robust pre-clinical TA-TLS model, Colon26 tumors were engineered to express different TLS- associated cytokines. The expression of cytokines was validated by qPCR and flow cytometry and no impact on in vitro tumor cell proliferation was observed (Figure 26A-B). Cytokineexpressing Colon26 tumors were screened for their in vivo activity to induce TA-TLS by immunofluorescent analysis (Table 2). The frequency of intratumoral B cell aggregate formation was dramatically increased by combining 10-day LTBR agonist treatment (2 mg/kg) with intratumoral expression of Cxcl13 (64%, 7/11 tumors, Fig. 16), Ccl19 (100%, 12/12 tumors, Fig. 16), Ccl21 (100% 3/3 tumors, Table 2) and LTa (100% 12/12 tumors, Fig. 6C), respectively. Intratumoral B cell aggregates induced by the combination of Cxcl13 expression and LTBR agonism predominantly exhibited a dispersed phenotype that lacked HEV, whereas the majority of B cell aggregates induced by combination of Cell 9 or Ccl21 expression and LTBR agonist treatment were found in tumor peripheral regions with compact morphology and associated HEV. Notably, T cell aggregates were not induced in tumors expressing Cxcl13, Ccl19 or Ccl21 combined with LTBR agonist treatment. Among the cytokines examined, LTa appeared to be the most potent inducer of TA-HEV and B cell aggregates (Table 2). B cell aggregates were detected in most LTa-expressing tumors and typically associated with HEV, but they were relatively small in size and lacked discrete T cell zones (Fig. 17). Remarkably, the combination of LTa expression and LTBR agonist treatment efficiently induced TA-TLS with discrete B cell and T cell zones that were invariably associated with HEV (Fig. 17). Ki67+ activated B cells and T cells were readily detected in these TA-TLS. Characterization of stromal components in the TA-TLS identified FDC- like CD4-CD8-Cxcl13+ cells in the B cell zone and a PDPN+ FRC-like cell network with associated CD11c+ DC in the T cell zone. LYVE1+ lymphatic vessels were also observed in the vicinity of TA-TLS. Notably, LTa associated TA-TLS formation promoted long-term Colon26 tumor control even after cessation of LTBR agonist treatment (Fig. 18). Combinatorial activity of LTBR agonism and LTa expression to induce TA-TLS was also observed in MC38-OVA tumors (Table 3). Overall, the combination of LTa expression and LTBR agonism uniquely induced the formation of intratumoral TA-TLS. Table 3: Summary of in vivo tumor screen for HEV and B cell aggregate in MC38-OVA tumors engineered to express different TLS-related cytokines with LTBR agonist or isotype control antibody treatment (n=3-4)

LTBR agonism mediates the expansion of HEV-associated postcapillary venule compartment

[0202] To obtain insights into molecular and cellular changes associated with the induction of TA-TLS by combination of LTa expression and LTBR agonist treatment, single-cell transcriptional profiling focusing on tumor-associated endothelial cells (TA-EC), stromal cells and immune cells was performed. Colon26 cells were engineered to express GFP to facilitate tumor cell depletion. Tumor-associated endothelial, stromal and immune cells were isolated on day 7 post LTBR agonist treatment by fluorescence-activated cell sorting (FACS) based on GFP-CD45’ CD31⁺, GFP’CD45’CD3T and GFP’CD45⁺CD3T marker expression, respectively. Identities of the different cell types were confirmed by unsupervised clustering on uniform manifold approximation and projection (UMAP) (Figure19A). LTBR was widely expressed by EC, stromal cells, myeloid cells as well as remained tumor cells (Figure19B).

[0203] TA-EC could be subclustered into 8 groups (E1-8) using marker genes from published EC single-cell RNA sequencing (scRNA-seq) datasets (Hua 2022, Goveia 2020, Kalucka 2020): arterial EC, capillary arterial EC, tip/stalk cells, capillary EC, postcapillary venule (PCV) EC, PCV/venous EC, lymphatic EC and mitotic EC (Figure 19C). PCV marker Ackrl (DARC) is expressed in both LN HEV and TA-HEV (Asrir 2022, Hua 2022). Most of the GlycamT TA-HEC were embedded in the Ackr1⁺ PCV cluster. Additionally, the HEV signature enzymes Chst4 and Fut7 were expressed in a pattern similar to Glycaml indicating functional HEC differentiation. LTBR agonism, but not LTa expression, increased the frequency of PCV EC by 50% relative to control (Figure 19D). Concomitant to the increase in PCV EC, the tip/stalk cell compartment showed a corresponding decrease in LTBR agonist treated tumors (Figure 19D). In addition, combination of LTa expression and LTBR agonism decreased the fraction of mitotic EC and increased the proportion of lymphatic EC (Figure 19D). The TLS-associated chemokine Ccl21 was specifically expressed by the Lyve1⁺ lymphatic EC. Overall, LTBR agonism promoted the expansion of the HEV-associated PCV compartment more potently than LTa while the combination of LTa expression and LTBR activation specifically expanded the Ccl21 -expressing lymphatic EC pool.

LTBR agonism and LTa signaling cooperatively suppressed pro-tumoral CAF phenotypes

[0204] Single-cell transcriptomic analysis identified 5 fibroblast clusters (F1-5) in the GFP'CD45'CD3T tumor-associated stromal compartment (Figure 19E). Cluster F1 was characterized by high expression of pan-tissue adventitial fibroblast marker Pi 16 (Buechler 2021), while clusters F2 and F3 expressed high level of pan-tissue parenchymal fibroblast marker Col15a1 (Buechler 2021). Pi16^h'9^h CAF and Col15a1^h'9^h CAF broadly aligned with the transcriptional features of the pan-tissue Pi16⁺ and Col15a1⁺ fibroblast subsets, respectively. LTBR agonist treatment as well as LTa expression increased the percentage of F1 Pi 16^h'9^h CAF but decreased the proportion of F2/F3 Col15a1^h'9^h CAF (Figure 19F). Combination of LTBR agonism and LTa expression further amplified these trends resulting in 3.4-fold higher frequency of Pi 16^h'9^h CAF and 3.1-fold lower frequency of Col15a1^h'9^h CAF in the combination group relative to control (Figure 19F). Col15a1^h'9^h CAF expressed higher levels of tumor-promoting factors Tgfbl, Vegfa, Cxcl14 (Sjoberg 2016) and Bgn (Zheng 2023). Notably, Cxcl13⁺BAFF⁺ FDC-like cells and CCL19⁺ADH1⁺ FRC-like cells (Grout 2022) were found predominantly in the treatment- enriched Pi16^h'9^h CAF populations. Overall, these data suggested that LTBR agonism and LTa signaling cooperatively shifted tumor fibroblast populations from a Col15a1^h'9^h pro-tumoral phenotype to a Pi 16^h'9^h TLS-promoting phenotype.

[0205] The effects of LTBR agonism and LTa expression on composition and transcriptional profiles of tumor-infiltrating CD8⁺ T cells were assessed. Consistent with the increased frequency of TCF1⁺PD1⁺ CD8⁺ T cells and decreased frequency of SLAMF6- Tim3⁺PD1⁺ CD8⁺ T cells measured by flow cytometry (Figure 11 B), CD8⁺ T cell analysis showed that LTBR agonist treatment and LTa expression significantly increased the progenitor signature score in effector and proliferating CD8⁺ T cells and decreased the exhaustion signature score in naive, effector and proliferating CD8⁺ T cells (Pai 2023). Overall, the single-cell transcriptional analysis further defined the effects of LTBR agonism on multiple tumor-associated cellular compartments, including EC, CAF and T cells that are consistent with the promotion of anti-tumor immunity.

TA-TLS formation enhanced tumor response to anti-CTLA-4 treatment

[0206] To test the impact of TLS on tumor responses to ICB therapies, anti-PD-1 antibody treatment was applied in the LTBR agonist-treated LTa-expressing Colon26 tumors (Figure 27A). Anti-PD-1 treatment moderately augmented the depth of treatment response of Colon26-LTa tumors that were pretreated with the LTBR agonist (Figure 27B). Next, the effects of combining CTLA-4 blockade with LTBR agonism and LTa expression was tested (Figure 27C). Single agent anti-CTLA-4 treatment provided potent tumor control in wild type Colon26 tumors with 5/8 mice showing at least a 30% reduction in tumor volume (Figure 20A). Combining CTLA- 4 blockade with either LTBR agonism or tumoral LTa expression further enhanced the anti-tumor efficacy achieving deep tumor responses in nearly all mice (Figure 20A).

[0207] Compared to Colon26, the MC38-OVA tumor model is more resistant to CTLA- 4 blockade with modest anti-tumor effect of single agent anti-CTLA-4 treatment and consistent combination benefit of anti-CTLA-4 and LTBR agonist treatment (Figures 20B and 28D). In LTa- expressing MC38-OVA tumors, CTLA-4 blockade significantly delayed tumor progression (Figure 20C). Combination of LTa expression and LTBR agonism, which induced TA-TLS formation in MC38-OVA tumors (Table 3), augmented tumor responses to CTLA-4 blockade with 5/13 mice showing a greater than 30% decrease in tumor size, including three tumor-free mice (Figure 20C).

DISCUSSION

[0208] Systemic activation of the LTBR pathway modulated the tumor microenvironment via multiple mechanisms to promote anti-tumor immunity. Single agent LTBR agonist antibody treatment induced the tumor-specific formation of HEV across different syngeneic mouse tumor models representing different histologies. Strikingly, HEV were not detected in normal organs and moreover, while LTBR signaling is required for LN HEV homeostasis (Browning 2008), LN from LTBR agonist treated mice displayed overall normal tissue architecture and HEV distribution. Additionally, liver function testing by measuring blood alanine transaminase (ALT) and aspartate transaminase (AST) levels in mice treated with LTBR agonist monoclonal antibodies, notwithstanding LTBR expression in hepatocytes (MacParland 2018), did not detect significant changes, again consistent with tumor-specific effects of systemic LTBR activation.

[0209] As previously described (Asrir 2022), intratumoral HEV induced by LTBR exhibited the characteristic plump morphology of LN HEC and the upregulation of cell adhesion molecules. Mechanistically, these tumor-specific effects occurred independent of the major tumor-infiltrating immune cells, as well as the major angiogenic signaling pathways (VEGF, Notch and Tie2), consistent with a transdifferentiation mechanism of HEV formation (Hua 2022). Rather, a combination of in vitro and in vivo studies highlighted that redundant inflammatory signaling appeared to facilitate the tumor-specific HEV formation. Simultaneous in vivo blockade of multiple inflammatory signaling pathways might be required to identify the relevant factors in the tumor microenvironment. A previous study demonstrated that combined anti-PD-1 and anti-angiogenic treatment can induce TA-HEV formation (Allen 2017). Such a combination effect was not observed; these differences could represent tumor model specific features. Indeed, the driving signals of TA-HEV formation (LTaiP₂-LTBR vs. LTa₃-TNFR1) and the involved immune cell types (CD4, CD8 T cells or NK cells) were found to be tumor model dependent (Asrir 2022, Hua 2022, Peske 2015).

[0210] LN HEV are specialized blood vessels that mediate lymphocyte entry from blood. Consistent with this function, immune cell infiltration (B, T, NK and DC cells) was significantly increased in LTBR agonist treated tumors. Interestingly, blockade of the L-selection/PNAd interactions reversed LTBR-mediated effects on immune cell infiltration and tumor growth, highlighting the relevance of HEV-mediated immune cell trafficking to promote anti-tumor immunity. In addition to the effects on immune cell infiltration, LTBR agonism was associated with increased activation of intratumoral DC and T cells. LTBR agonist treatment significantly increased the frequency of activated CD40+CD83+ DC in Colon26 tumors and elevated DC CD40 and MHCII expression in MC38 tumors. Further, in vitro studies with BMDC revealed a direct effect of LTBR agonism on DC licensing and DC-mediated T cell activation. Finally, OT-I CD8+ T cell adoptive transfer studies in FTY720 treated MC38-OVA tumor-bearing mice demonstrated that systemic LTBR activation enhanced tumor-specific T cell activation within the tumor microenvironment.

[0211] The effects of LTBR agonism on immune cell infiltration and activation highlight the potential for therapeutic combination approaches with T cell- and DC-based immunotherapies. For proof of concept, LTBR agonist treatment was combined with PD-1 or CTLA-4 ICB as well as hCD20-directed CAR-T cell therapies and moderate yet significant combination anti-tumor benefits were observed. While encouraging, these results leave room for improvement, and additional combination approaches with novel classes of immunotherapies, such as costimulatory antibodies, immunocytokines, oncolytic viruses or cancer vaccines, warrant further investigation (Murciano-Goroff 2020). Reported direct cytotoxic effects against colorectal tumor models (Lukashev 2006) may contribute to the anti-tumor activity of the LTBR agonist antibody treatment, consistent with the moderate tumor growth inhibition observed in LTBR agonist treated Colon26 tumors in immunodeficient SRG mice. [0212] LTBR-mediated formation of TA-HEV was associated with the presence of organized lymphoid aggregates consisting of discrete B cell and T cell zones in about 10% of Colon 26 tumors. To test if the frequency of TA-TLS induction could be increased, a selection of TLS-related chemokines/cytokines was screened for TLS induction activity in the Colon26 and MC38-OVA tumor models. The combination of LTBR agonism and tumoral LTa expression most robustly induced TA-TLS formation in both tumor models. LTBR plus LTa associated TLS were characterized by discrete B cell and T cell zones, associated with HEV, FDC and FRC-like stromal compartments and lymphatic vessels. Although LTa signaling alone induced intratumoral HEV formation and B cell aggregates as reported before (Schrama 2001), combination with LTBR agonism was found to be essential for efficient TLS induction and tumor control. Further characterization of TA-TLS by scRNA-seq highlighted the specific expansion of HEC-containing PCV population, Ccl21-expressing lymphatic EC and the CAF compartment enriched with FDC/FRC-like cells, as well as the shifting of CD8 T cells from exhaustion toward progenitor phenotype. TLS can function as sites of antigen presentation and adaptive immune activation within tumors (Aoyama 2021). While additional studies are necessary to further assess TLS functionality, it was demonstrated that TA-TLS contained Ki67+ activated B and T cells and Colon26 tumors in the LTBR/LTa combination group exhibited prolonged tumor control after cessation of LTBR agonist treatment. Additionally, TA-TLS formation enhanced Colon26 and MC38-OCA response to ICB, particularly to anti-CTLA-4 treatment. To what extent the Treg depleting function of anti-CTLA-4 plays a role in this activity will be subject to further investigation (Economides 2003). Considering the growing clinical evidence of TA-TLS correlation with better treatment response to ICB (Sautes-Fridman 2019, Cabrita 2020, Helmink 2020, Petitprez 2020), this new inducible TA-TLS model represents a valuable tool for mechanistic studies on TA-TLS neogenesis and associated anti-tumor strategies, and complements recently described intratumoral TLS-like models (Rodriguez 2021 , Ramachandran 2023, Economides 2003, Holash 2002).

[0213] Collectively, this study provides new insights into the mechanisms of LTBR mediated tumor-specific HEV formation and immunomodulation of the tumor microenvironment. Further, it describes a new mouse model of tumor-associated TLS formation based on combined activation of LTBR and TNFR signaling. Of note, the presence of TLS in many human tumor types has been associated with better prognosis and improved ICB response. Lastly, it lays the foundation for treatment strategies aiming to promote adaptive immune responses within the tumor site. 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 U S A, 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.

[0214] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

We claim:

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

2. A method for increasing the efficacy of adoptive cell therapy (ACT), comprising:

(a) selecting a subject with cancer; and

(b) administering to the subject a therapeutically effective amount of an ACT in combination with a therapeutically effective amount of a lymphotoxin beta receptor (LTBR) agonist, wherein administration of the combination leads to increased efficacy and duration of anti-tumor response, as compared to a subject treated with the ACT as monotherapy.

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

4. The method of any one of claims 1-3, wherein the ACT comprises an immune cell selected from a T cell, a tumor-infiltrating lymphocyte, and a natural killer (NK) cell.

5. The method of claim 4, wherein the immune cell comprises a modified T cell receptor (TCR) against a tumor-associated antigen (TAA), or a chimeric antigen receptor (CAR) against a TAA.

6. The method of claim 5, wherein the TAA is selected from AFP, ALK, BAGE proteins, BCMA, BIRC5 (survivin), BIRC7, [3-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1 , CYP1 B1 , EGFR, EGFRvlll, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1 , FOLR1 , GAGE proteins, 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 , 0X40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1 , PRLR, PRAME, PSMA (F0LH1), RAGE proteins, Ras, RGS5, Rho, SART-1 , SART-3, STEAP1 , STEAP2, TAG-72, TGF- , TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1 , TRP- 2, tyrosinase, and uroplakin-3.

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

8. The method of claim 7, wherein the additional therapeutic agent or therapy is selected from radiation, surgery, a checkpoint inhibitor, a chemotherapeutic agent, a cancer vaccine, a vascular endothelial growth factor (VEGF) antagonist, an angiopoietin-2 (Ang2) inhibitor, a transforming growth factor beta (TGF ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an antibody to a tumor-specific antigen, Bacillus Calmette-Guerin vaccine, granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytotoxin, an interleukin 6 receptor (IL-6R) inhibitor, an interleukin 4 receptor (IL-4R) inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-12, IL-21, IL-15, an antibody-drug conjugate, an anti-inflammatory drug, and combinations thereof.

9. The method of 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 with collageneous structure (MARCO), phosphatidylserine (PS), TIGHT, VISTA, and VTCN1.

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

11. The method of any one of claims 1-10, wherein the cancer is selected from adrenal gland cancer, anal cancer, autonomic ganglial cancer, biliary tract cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cancer of the meninges, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, eye cancer, esophageal cancer, fallopian tube cancer, gastric cancer, genital tract cancers, head and neck cancer, kidney cancer, large intestinal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, peritoneal cancer, pituitary cancer, placental cancer, pleura cancer, prostate cancer, rectal cancer, renal cancer, salivary gland cancer, skin cancer, small intestinal cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive cancers, urinary tract cancer, uterine cancer, vaginal cancer, and vulva cancer.

12. The method of any one of claims 1-11 , wherein the cancer expresses CXCL13, CCL19, CCL21 or LTa.

13. The method of any one of claims 1-12, wherein the cancer expresses LTa.

14. The method of any one of claims 1-13, wherein administration of the combination produces a therapeutic effect selected from one or more of: increased tumor-specific HEV formation, increased dendritic cell and T cell infiltration, enhanced T cell activation in tumor microenvironment, increased expression of TLS-related chemokines, delay in tumor growth, reduction in tumor cell number, tumor regression, increase in survival, partial response, and complete response.

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

16. The method of any one of claims 1-15, wherein the therapeutically effective amount of the ACT comprises 1x10⁶ or more immune cells.

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

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

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

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

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

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

23. A method for increasing tumor-specific HEV formation in a subject in need thereof, the method comprising:

(a) selecting a subject with cancer; and

(b) administering to the subject a therapeutically effective amount of an ACT in combination with a therapeutically effective amount of a lymphotoxin beta receptor (LTBR) agonist, wherein administration of the combination leads to increased tumor-specific HEV formation and increased efficacy and duration of anti-tumor response, as compared to a subject treated with the ACT as monotherapy.

24. A method for increasing expression of TLS-related chemokines in a subject in need thereof, the method comprising:

(a) selecting a subject with cancer; and

(b) administering to the subject a therapeutically effective amount of an ACT in combination with a therapeutically effective amount of a lymphotoxin beta receptor (LTBR) agonist, wherein administration of the combination leads to increased expression of TLS-related chemokines and increased efficacy and duration of anti-tumor response, as compared to a subject treated with the ACT as monotherapy.