WO2025217270A1 - METHODS OF TREATMENT USING TGFb-RECEPTOR 1 INHIBITION AND LTBR AGONISM - Google Patents

Abstract

Methods of treating a patient in need thereof are described herein. In some embodiments, the method comprises disposing a TGFb-receptor 1 inhibitor within a biological compartment of the patient, and disposing a lymphotoxin beta receptor (LTBR) agonist within the biological compartment of the patient. In some instances, the patient has cancer. In some cases, the cancer is pancreatic cancer, or more specifically pancreatic ductal adenocarcinoma. In tertiary lymphoid structures (TLS)-resistant tumors, the disposing of the TGFb-receptor 1 inhibitor within the biological compartment of the patient results in a reprogramming of fibroblast phenotype and overcomes resistance to generation of TLS in tumors of the patient.

Description

METHODS OF TREATMENT USING TGFb-RECEPTOR 1 INHIBITION AND LTBR

AGONISM

RELATED APPLICATION DATA

[0001] The present application claims priority to United States Provisional Patent Application Number 63/631,641 filed April 09, 2024 which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 CA286017 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] An electronic sequence listing (069596-00087 SL.xml; size 20.0 KB; date of creation April 3, 2025) submitted herewith is incorporated by reference in its entirety.

FIELD

[0004] The present application relates to methods of treating patients in need thereof using TGFb-receptor 1 inhibitors and lymphotoxin beta receptor (LTBR) agonists.

BACKGROUND

[0005] Pancreatic ductal adenocarcinoma (PDAC) is considered an immunologically cold cancer type. This perception is due to its rapid progression, strong presence of immunosuppressive cells and lack of response to current immunotherapies. Further, PDAC has a 5-year survival of less than 13% while in the top five causes of cancer-related deaths worldwide. Presently, the general treatment involves surgical resection followed by chemotherapy, though only around 10% to 20% of patients have resectable tumors. In other cases, tumors are often more advanced, metastatic, and/or non-resectable, with systemic chemotherapy offered as a typical treatment. Thus, there is an urgent need to develop improved therapies and novel biomarkers that will improve survival. SUMMARY

[0006] In an aspect of the disclosure, there is presented a method of treating a condition of a patient in need thereof. The method includes disposing a TGFb-receptor 1 inhibitor within a biological compartment of the patient and disposing a lymphotoxin beta receptor (LTBR) agonist within the biological compartment of the patient. In some instances, the step of disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient is carried out before the step of disposing the LTBR agonist within the biological compartment of the patient. In some instances, the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient alleviates TGFb-mediated suppression of lymphocyte recruitment to reticular fibroblasts. In some instances, the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient overcomes resistance to a therapeutic effect of the LTBR agonist. In some instances, the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient overcomes resistance to generation of tertiary lymphoid structures in tumors of the patient.

[0007] In some instances, the condition is a cancer. In some cases, the cancer is pancreatic cancer. In some cases, the pancreatic cancer is pancreatic ductal adenocarcinoma. In some instances, the patient has a myofibroblastic cancer associated fibroblast (myCAF) phenotype. In some instances, the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient reprograms the mCAF phenotype to a reticular cancer associated fibroblast (rCAF) phenotype. In some instances, the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient reverses suppression of an rCAF phenotype.

[0008] In some instances, the method further includes the step of disposing at least one chemotherapeutic compound within the biological compartment of the patient. In some cases, the at least one chemotherapeutic compound is a combination of gemcitabine and nab-paclitaxel. [0009] In another aspect of the present disclosure, there is provided a composition for treating a condition of a patient in need thereof. The composition includes a lymphotoxin beta receptor (LTBR) agonist and a TGFb-receptor 1 inhibitor. In some instances, the LTBR agonist is an anti-LTBR monoclonal antibody. In some instances, the composition is formulated for oral delivery or intravenous delivery. In some instances, the LTBR agonist and the TGFb-receptor 1 inhibitor are admixed for simultaneous administration. [0010] In yet another aspect of the present disclosure, there is provided a method of inducing lymphocyte recruitment to a tumor. The method includes disposing a TGFb-receptor 1 inhibitor within a biological compartment of a patient and disposing a lymphotoxin beta receptor (LTBR) agonist within the biological compartment of the patient.

[0011] In yet another aspect of the present disclosure, there is provided a method of generating tertiary lymphoid structures (TLS) in a TLS-resistant tumor. The method includes disposing a TGFb-receptor 1 inhibitor within a biological compartment of a patient and disposing a lymphotoxin beta receptor (LTBR) agonist within the biological compartment of the patient. In some instances, the TLS-resistant tumor is a pancreatic ductal adenocarcinoma tumor. In some instances, the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient overcomes resistance to TLS generation by the LTBR agonist.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1A are representative bioluminescence images representing PK5 (PdxlCre/LSL- KRas^G12D/LSL-p53^R172H) and PK5L (PdxlCre/LSL- KRas^G12D/LSL- p53^R172H/LSL-Luciferase- SIYRYYGL) cell lines orthotopic PK5 or PK5L mouse PDAC models. Seven days following implant, PK5L or PK5 tumors were exposed to bioluminescent imaging.

[0013] FIG. IB is a graphical representation of PK5 (PdxlCre/LSL- KRas^G12D/LSL- p53^R172H) and PK5L (PdxlCre/LSL- KRas^G12D/LSL- p53^R172H/LSL-Luciferase-SIYRYYGL) cell lines orthotopic PK5 or PK5L mouse PDAC models. Representative flow cytometry plots of SIY antigen-specific CD8+ T cell population in PK5 and PK5L tumors. Cells are gated on viable CD8+ T cells.

[0014] FIG. 1C is a graphical representation of PK52014 and PK51975 cells stimulated for 24 hrs. by either 5 ng/ml TNFa, IFNP, or IFNg or 2 pg/ml aLTBR to monitor increases in MHC- I (H2-Kb) expression. MFI = median fluorescence intensity. All graphs represent mean + SEM. [0015] FIG. ID is a graphical representation of PK52014 and PK51975 cells stimulated for 24 hrs. by either 5 ng/ml TNFa, IFN|3, or IFNg or 2 pg/ml aLTBR to monitor increases in PD-L1 expression. MFI = median fluorescence intensity. All graphs represent mean + SEM.

[0016] FIG. IE is a graphical representation of PK52014 and PK51975 cells stimulated for 24 hrs. by either 5 ng/ml TNFa, IFN , or IFNg or 2 pg/ml aLTBR to monitor increases in LTBR expression. MFI = median fluorescence intensity. All graphs represent mean + SEM. [0017] FIG. IF is a graphical representation of composition of major tumor-resident cell types in PK5 tumors including leukocytes, tumor cells, endothelial cells, and fibroblasts (n = 8 mice per group). All graphs represent mean + SEM.

[0018] FIG. 1G is graphical representation of orthotopic PK5 PDAC mouse models that have heterogeneous response to LTBR agonism. CXCL13⁺ cells determined by IHC (left panel) and T cell density determined by flow cytometry (right panel) in control and a LTBR treated PK5 tumors. Quantification of CXCL13⁺ cell density and abundance of T cell infiltration in PK5 tumors + LTBR agonism (n = 3-5 mice per group as indicated). All graphs represent mean + SEM. Groups were compared by an unpaired student’s t test. * p < 0.05.

[0019] FIG. 2 A is a graphical representation of murine PK5 cell lines inducing heterogenous PDAC tumors and responding to LTBR agonism. Tumor kinetics (mm²) for KPC-derived PK5/PK5L PDAC cell lines implanted orthotopically in C57BL/6 mice (n=3 mice per cell line). [0020] FIG. 2B is a profile representation of murine PK5 cell lines inducing heterogenous PDAC tumors and responding to LTBR agonism. Cytokine/chemokine profiling of supernatants from PK5/PK5L cell lines by 48-plex Luminex assay normalized to the cell line with the lowest values in each analyte. Analytes quantified below detection limit are not shown.

[0021] FIG. 2C is a graphical representation of murine PK5 cell lines inducing heterogenous PDAC tumors and responding to LTBR agonism. Analysis of surface LTBR expression on tumor-infiltrating cell subsets as indicated in a PK51975 orthotopic model (n=5 mice).

[0022] FIG. 2D is a graphical representation of murine PK5 cell lines inducing heterogenous PDAC tumors and responding to LTBR agonism. Tumor growth curves for PK51975 tumors following orthotopic or subcutaneous tumor implant and treatment with either IgG2a isotype control or aLTBR antibodies. Dotted lines indicate aLTBR treatment.

[0023] FIG. 2E is an image representation of murine PK5 cell lines inducing heterogenous PDAC tumors and responding to LTBR agonism. Micrographs of aLTBR-treated PK51975 tumor H&E-stained sections (left) with corresponding IHC of PNAd+ high endothelial venules and CXCL13+ cells (right). T = tumor and arrows demarcate TLS aggregates and HEV structures.

[0024] FIG. 3A is a graphical representation of distinct CAF phenotypes associating with sensitivity to TLS formation in PDAC tumors. Tumor measurements of TLS-permissive (PK51975) and TLS-resistant (PK5L1948) tumors implanted orthotopically into C57BL/6 mice. Mice were treated with either aLTBR or an IgG control antibody at indicated days (dotted lines) and followed for tumor area by ultrasound. All graphs depict mean + SEM. Statistical comparisons between two groups were performed by an unpaired student’s t test. * p < 0.05; *** p < 0.001; **** p < 0.0001.

[0025] FIG. 3B is a graphical representation of distinct CAF phenotypes associating with sensitivity to TLS formation in PDAC tumors. Flow cytometry analysis of PDPN⁺ CAF composition in TLS-permissive and TLS-resistant tumors including myCAF (Ly6c'MHC-IT), iCAF (Ly6c⁺MHC-IT), apCAF (Ly6c MHC-II⁺CD74⁺), and other CAF (Ly6c MHC-ITCD74 ). [0026] FIG. 3C is a graphical representation of distinct CAF phenotypes associating with sensitivity to TLS formation in PDAC tumors. Uniform manifold approximation projection (UMAP) cluster analysis of total CAFs determined from flow cytometry data. The heat map below shows the mean percent of each subpopulation cluster pooled from 4-5 tumors.

[0027] FIG. 3D is a graphical representation of distinct CAF phenotypes associating with sensitivity to TLS formation in PDAC tumors. Quantification of VCAM-1 TCAM-1 ¹ CAF gated on the indicated CAF subsets in tumors from FIG. 3B is shown. All graphs depict mean + SEM. Statistical comparisons between two groups were performed by an unpaired student’s t test. * p < 0.05; *** p < 0.001; **** p < 0.0001.

[0028] FIG. 3E is a graphical and image representation of distinct CAF phenotypes associating with sensitivity to TLS formation in PDAC tumors. Analysis of aSMA-positive tumor area by IHC and representative images from untreated TLS-permissive and TLS-resistant tumors. All graphs depict mean + SEM. Statistical comparisons between two groups were performed by an unpaired student’s / test. * p < 0.05; *** p < 0.001; **** p < 0.0001.

[0029] FIG. 3F is a graphical and image representation of distinct CAF phenotypes associating with sensitivity to TLS formation in PDAC tumors. Analysis of phospho-SMAD2 positive area in tumors by IHC and representative images from untreated TLS-permissive and TLS-resistant tumors. All graphs depict mean + SEM. Statistical comparisons between two groups were performed by an unpaired student’s / test. * p < 0.05; *** p < 0.001; **** p < 0.0001.

[0030] FIG. 3G is a graphical representation of distinct CAF phenotypes associating with sensitivity to TLS formation in PDAC tumors. Characterization of CD4+ and CD8+ T cell phenotypes in TLS-permissive orthotopic tumors +/- aLTBR treatment. Relative frequency of Tfh (CD4⁺ PD-1 ⁺ CXCR5⁺ Foxp3 ), Tfr (CD4⁺ PD-1 ⁺ CXCR5⁺ Foxp3⁺), Treg (CD4⁺ PD-1 ⁺ CXCR5’ Foxp3⁺), stem-like (CD8⁺ PD-1⁺ TCF1⁺), and exhausted (CD8⁺ PD-1⁺ TCF1 ) T cells are shown for TLS-permissive tumors, n = 3-5 mice per group as indicated. All graphs depict mean + SEM. Statistical comparisons between two groups were performed by an unpaired student’s t test. * p < 0.05; *** p < 0.001; **** p < 0.0001. All tumor experiments were repeated in at least 2 independent replicates.

[0031] FIG. 3H is a graphical representation of distinct CAF phenotypes associating with sensitivity to TLS formation in PDAC tumors. Characterization of CD4+ and CD8+ T cell phenotypes in TLS-resistant orthotopic tumors +/- aLTBR treatment. Relative frequency of Tfh (CD4⁺ PD-1⁺ CXCR5⁺ Foxp3 ), Tfr (CD4⁺ PD-1⁺ CXCR5⁺ Foxp3⁺), Treg (CD4⁺ PD-1⁺ CXCR5’ Foxp3⁺), stem-like (CD8⁺ PD-1⁺ TCF1⁺), and exhausted (CD8⁺ PD-1⁺ TCF1 ) T cells are shown for TLS-resistant tumors, n = 3-5 mice per group as indicated. All graphs depict mean + SEM. Statistical comparisons between two groups were performed by an unpaired student’s t test. * p < 0.05; *** p < 0.001; **** p < 0.0001. All tumor experiments were repeated in at least 2 independent replicates.

[0032] FIG. 4A is a graphical representation of CAF phenotype and expression of TLS modulating cytokines in TLS-permissive and resistant PK5 models. Flow cytometry characterization of VCAM-1⁺ICAM-1⁺ CAF phenotype in untreated TLS-permissive and TLS- resistant PDAC tumors.

[0033] FIG. 4B is a representative plot of CAF phenotype and expression of TLS modulating cytokines in TLS-permissive and resistant PK5 models. UMAP plots of individual surface marker expression on CAFs from FIG. 3C.

[0034] FIG. 4C is a graphical representation of CAF phenotype and expression of TLS modulating cytokines in TLS-permissive and resistant PK5 models. Bulk transcriptome analysis of endogenous TNFR and LTBR receptors/ligands in TLS-permissive (PK51975) and TLS- resistant (PK5L1948) tumors (fold change normalized to TLS-resistant tumors) (n = 4 mice). All graphs represent mean + SEM. Groups were compared by an unpaired student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

[0035] FIG. 4D is a graphical representation of CAF phenotype and expression of TLS modulating cytokines in TLS-permissive and resistant PK5 models. Bulk transcriptome analysis of endogenous TNFR and LTBR receptors/ligands in TLS’ and TLS⁺ human PDAC tumors (fold change normalized to TLS-tumors) (n=19 TLS' and n=20 TLS⁺ patients). All graphs represent mean + SEM.

[0036] FIG. 4E is a graphical representation of CAF phenotype and expression of TLS modulating cytokines in TLS-permissive and resistant PK5 models. Latent TGFp/LAP expression in tumor-resident cell types in untreated (i) TLS-permissive and (ii) TLS-resistant tumors. All graphs represent mean + SEM. Groups were compared by an unpaired student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

[0037] FIG. 5A is a graphical representation of how TGFp antagonizes TNFa and LTBR induction of reticular programming in fibroblasts. Flow cytometric analyses of surface marker expression on treated fibroblasts in vitro. Reticular fibroblast marker expression on primary mouse ear fibroblasts treated with 5 ng/ml TNFa, 1 pg/ml aLTBR, or a combination for 24 hrs. MFI = median fluorescence intensity, denoted on the y axis of each panel. All graphs depict mean + SEM. All groups compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001. Experiments were repeated 3 times independently with 3 culture wells/treatment group.

[0038] FIG. 5B is a graphical representation of how TGFp antagonizes TNFa and LTBR induction of reticular programming in fibroblasts. Flow cytometric analyses of surface marker expression on treated fibroblasts in vitro. Surface protein expression as determined by flow cytometry. Primary fibroblasts were treated with either 1 pg/ml aLTBR or 5 ng/ml TGFp for 24 hours or six-hour pretreatment with TGFP followed by aLTBR for 24 hrs. MFI = median fluorescence intensity, denoted on the y axis of each panel. All graphs depict mean + SEM. All groups compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001. Experiments were repeated 3 times independently with 3 culture wells/treatment group.

[0039] FIG. 5C is a graphical representation of how TGFP antagonizes TNFa and LTBR induction of reticular programming in fibroblasts. RT-PCR quantification for chemokines (Cell 9, Cxcll3) and myCAF markers (Acta2, Ctgf) in primary mouse fibroblasts. Fibroblasts were treated with either TGFP or TNFa/aLTBR combination for 24 hrs. or 6 hr. pre-treatment with TGFp followed by 24 hrs. of TNFa/aLTBR stimulation. All graphs depict mean + SEM. All groups compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01 ; **** p < 0.0001 . Experiments were repeated 3 times independently with 3 culture well s/treatm ent group.

[0040] FIG. 6A is a graphical representation of TNFa, aLTBR, and TGFP-mediated changes in fibroblast phenotype and chemokine expression that are interferon-independent. VCAM-1 expression on primary mouse fibroblasts treated with either TGF0, aLTBR, or conditioned media from TLS-permissive or TLS-resistant tumor cells. Graphs depict mean + SEM and all groups were compared by one-way ANOVA with Tukey’s multiple comparisons test, ns = not significant. *p < 0.05; ** p < 0.01; **** p < 0.0001. All experiments were repeated 3 times independently.

[0041] FIG. 6B is a graphical representation of TNFa, aLTBR, and TGFP-mediated changes in fibroblast phenotype and chemokine expression that are interferon-independent. Analysis of T cell chemokines from in vitro activated fibroblasts: i) secretion of CXCL9 by ELISA and ii) qRT-PCR of CCL21 transcripts was assessed. Graphs depict mean + SEM and all groups were compared by one-way ANOVA with Tukey’s multiple comparisons test, ns = not significant. *p

< 0.05; ** p < 0.01; **** p < 0.0001. All experiments were repeated 3 times independently.

[0042] FIG. 6C is a graphical representation of TNFa, aLTBR, and TGFP-mediated changes in fibroblast phenotype and chemokine expression that are interferon-independent. Mouse chemokine array was performed on activated fibroblasts treated as indicated and the relative levels of secreted chemokines determined by integrated density measurements in ImageJ. Hsp60 was used as a loading control to normalize all chemokine values.

[0043] FIG. 6D is a graphical representation of TNFa, aLTBR, and TGFP-mediated changes in fibroblast phenotype and chemokine expression that are interferon-independent. RT-PCR for CXCL13 in primary fibroblasts treated for 24 hrs. with TNFa/aLTBR or TNFa/LIGHT combinations or 6 hr. TGFb pre-treatment. Graphs depict mean + SEM and all groups were compared by one-way ANOVA with Tukey’s multiple comparisons test, ns = not significant. *p

< 0.05; ** p < 0.01; **** p < 0.0001. All experiments were repeated 3 times independently.

[0044] FIG. 6E is a graphical representation of TNFa, aLTBR, and TGFP-mediated changes in fibroblast phenotype and chemokine expression that are interferon-independent. Analysis of fibroblast phenotype in response to IL-1 a, IFNP, or IFNg treatment alone or in combination with aLTBR: i) RT-PCR for CXCL13 transcripts or ii-iii) flow cytometry expression of reticular fibroblast markers VCAM-1 and PDGFRa. MFI = median fluorescence intensity. Graphs depict mean + SEM and all groups were compared by one-way ANOVA with Tukey’s multiple comparisons test, ns = not significant. *p < 0.05; ** p < 0.01; **** p < 0.0001. All experiments were repeated 3 times independently.

[0045] FIG. 6F is a plot representation of TNFa, aLTBR, and TGFP-mediated changes in fibroblast phenotype and chemokine expression that are interferon-independent. Surface marker expression on primary mouse fibroblasts treated with TGFP and/or TNFa/aLTBR as indicated and described previously.

[0046] FIG. 7A is a graphical representation of lymphocyte migration to rCAF suppressed by TGFp. ELISA for CXCL13 in the supernatant of primary fibroblasts stimulated with TNFa, aLTBR, or combination for 24 hrs. TCM = tumor-conditioned media from PK52003 tumor cells. All graphs represent the mean + SEM. All experimental groups (n=3) were compared by oneway ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001; **** p < 0.0001. All experiments were conducted 3 times independently.

[0047] FIG. 7B is a graphical representation of lymphocyte migration to rCAF suppressed by TGFp. ELISA for CCL19 in primary mouse fibroblasts. Cells were treated with either 1 pg/ml aLTBR or 5 ng/ml TGFp for 24 hours or six-hour pretreatment with TGFp followed by aLTBR for 24 hrs. All graphs represent the mean + SEM. All experimental groups (n=3) were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001; **** p

< 0.0001. All experiments were conducted 3 times independently.

[0048] FIG. 7C is a graphical representation of lymphocyte migration to rCAF suppressed by TGFp. ELISA for CXCL13 in primary mouse fibroblasts. Cells were treated with either 1 pg/ml aLTBR or 5 ng/ml TGFP for 24 hours or six-hour pretreatment with TGFP followed by aLTBR for 24 hrs. All graphs represent the mean + SEM. All experimental groups (n=3) were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001; **** p

< 0.0001. All experiments were conducted 3 times independently.

[0049] FIG. 7D is a schematic representation of lymphocyte migration to rCAF suppressed by TGFp. Schematic for transwell migration assays of lymphocyte populations to activated fibroblasts. Fibroblasts cultured in a monolayer were exposed to the indicated cytokines for 16- 18 hours followed by addition of lymphocytes into the top chamber of 3-micron transwells for 24 hours. [0050] FIG. 7E is a graphical representation of lymphocyte migration to rCAF suppressed by TGFp. Migration analysis of naive B cells to treated fibroblasts. # of cells/field was calculated as described in methods. Spontaneous migration without fibroblasts was subtracted to determine final values. All graphs represent the mean + SEM. All experimental groups (n=3) were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001; **** p < 0.0001. All experiments were conducted 3 times independently.

[0051] FIG. 7F is a graphical representation of lymphocyte migration to rCAF suppressed by TGFp. Migration analysis of naive CD8⁺ cells to treated fibroblasts. # of cells/field was calculated as described in methods. Spontaneous migration without fibroblasts was subtracted to determine final values. All graphs represent the mean + SEM. All experimental groups (n=3) were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001; **** p < 0.0001. All experiments were conducted 3 times independently.

[0052] FIG. 7G is a graphical representation of lymphocyte migration to rCAF suppressed by TGFp. Migration analysis of CD4¹ THI cells to treated fibroblasts. # of cells/field was calculated as described in methods. Spontaneous migration without fibroblasts was subtracted to determine final values. All graphs represent the mean + SEM. All experimental groups (n=3) were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001; **** p < 0.0001. All experiments were conducted 3 times independently.

[0053] FIG 8A is a plot representation of chemokine receptor profiles of T and B cell subsets isolated from naive splenocytes. Flow cytometry purity assessment of B cells (CD90.2' CD19⁺) and CD4⁺ T cells (CD90.2⁺ CD4⁺ CD8 ) purified from bulk mouse spleenocytes by magnetic column separation.

[0054] FIG. 8B is a plot representation of chemokine receptor profiles of T and B cell subsets isolated from naive splenocytes. Flow cytometry plots indicating Tbet⁺ IFNy Thl cell phenotype following three days of in vitro culture as described.

[0055] FIG. 8C is a graphical representation of chemokine receptor profiles of T and B cell subsets isolated from naive splenocytes. Flow cytometry histograms of CXCR5 expression on differentiated THI cells.

[0056] FIG. 8D is a graphical representation of chemokine receptor profiles of T and B cell subsets isolated from naive splenocytes. Flow cytometry histograms of CCR7 expression on differentiated THI cells. [0057] FIG. 8E is a graphical representation of chemokine receptor profiles of T and B cell subsets isolated from naive splenocytes. Chemokine receptor cell surface expression on naive B cells or 24/48 hr. IgM-activated B cells.

[0058] FIG. 8F is a graphical representation of chemokine receptor profiles of T and B cell subsets isolated from naive splenocytes. Transwell migration assay of naive B cells or 48 hr. IgM-activated B cells to treated primary mouse fibroblasts. Number of cells/field was calculated as described in methods and normalized to spontaneous migration without fibroblasts or stimulating factors. All errors bars represent mean + SEM and all groups were compared by a one-way ANOVA with Tukey’s multiple comparisons test. ** p < 0.01; **** p < 0.0001.

[0059] FIG. 9A is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. qRT-PCR for CXCL13 in primary mouse fibroblasts treated as described previously with the addition of either 10 pM TGFpRli (LY3200882) for 24 hrs. or TGFpRli treatment for 1 hr. followed by TGFP for six hours and then TNFa/aLTBR stimulation for 24 hrs. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.001; *** p < 0.001; **** p < 0.0001. Every tumor study was repeated with 2 independent replicates.

[0060] FIG. 9B is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. qRT-PCR for CCL19 in primary mouse fibroblasts treated as described previously with the addition of either 10 pM TGFpRli (LY3200882) for 24 hrs. or TGFpRli treatment for 1 hr. followed by TGFP for six hours and then TNFa/aLTBR stimulation for 24 hrs. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.001; *** p < 0.001; **** p < 0.0001. Every tumor study was repeated with 2 independent replicates.

[0061] FIG. 9C is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. Transwell migration assays with naive B cells. TGFpRl inhibition was added one hour prior to TGFpi treatment as in FIG. 9A and neutralizing antibodies to CXCL13 (5 g/ml) were added at the time of B cell addition. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.001 ; *** p < 0.001 ; **** p < 0.0001. Every tumor study was repeated with 2 independent replicates.

[0062] FIG. 9D is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. Transwell migration assays with THI polarized CD4⁺ T cells. CCR7 neutralizing antibodies were pre-incubated with Thl cells for 30 min. prior to adding these cells in the transwell. Number of cells migrated/field in the bottom chamber was calculated as described in methods. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.001; *** p < 0.001; 0.0001. Every tumor study was repeated with 2 independent replicates.

[0063] FIG. 9E is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. Flow cytometric analysis of CAF composition in TLS-resistant PK5L1948 tumors treated with IgG isotype antibody, aLTBR (day 10 and 14), TGFpRli (SM16) formulated in chow (day 10-17), or a combination of both treatments.

[0064] FIG. 9F is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. Frequency of VCAM-1⁺ICAM-1⁺ rCAF among total CAFs from FIG. 9E. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.001; *** p < 0.001; **** p < 0.0001. Every tumor study was repeated with 2 independent replicates.

[0065] FIG. 9G is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. TLS-resistant PK5L1948 orthotopic tumor area was measured after treatment from FIG. 9E. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.001; *** p < 0.001; **** p < 0.0001. Every tumor study was repeated with 2 independent replicates.

[0066] FIG. 9H is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. TLS-resistant PK5L1948 orthotopic tumors were treated with Gem/Nab- PTX chemotherapy at day 12, 16, and 20 (dotted lines), aLTBR at day 10, 14, and 18 (dashed lines), or TGFpRli (SM16) in chow form for the period indicated by the blue shaded region. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.001; *** p < 0.001; **** p < 0.0001. Every tumor study was repeated with 2 independent replicates.

[0067] FIG. 91 is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. IHC analysis of aSMA in treated PK5L1948 tumors defined as % SMA⁺ area of the tumor. All graphs represent the mean + SEM. All experimental groups were compared by oneway ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.001; *** p < 0.001; **** p < 0.0001. Every tumor study was repeated with 2 independent replicates.

[0068] FIG. 9J is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. Frequency of VCAM-1⁺ICAM-1⁺ cells among total CAFs in PK5L1948 tumors following the indicated treatments. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.001; *** p < 0.001; **** p < 0.0001. Every tumor study was repeated with 2 independent replicates.

[0069] FIG. 9K is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. UMAP visualization of CAF clustering from flow cytometry of PK5L1948 tumors gated on PDPN⁺ CAF.

[0070] FIG. 9L is a graphical representation showing blockade of TGFpRl signaling reverses suppression of the rCAF phenotype and enhances treatment efficacy against TLS resistant PDAC tumors. Average percentage of CAFs from each treatment group (n=6) in individual clusters as defined in FIG. 9K.

[0071] FIG. 10A is a graphical representation of TGFpRli reversing suppression of rCAF phenotypes and promoting chemotherapeutic efficacy. RT-PCR for myCAF markers Acta2 and Ctgf in primary fibroblasts treated with indicated combinations of TGFpRli, TGFp, TNFa, or aLTBR. All errors bars represent mean + SEM and all groups were compared by a oneway ANOVA with Tukey’s multiple comparisons test, ns = not significant. *p < 0.05; ** p < 0.01 ; ***p < 0.001; **** p < 0.0001.

[0072] FIG. 10B is a graphical representation of TGFpRli reversing suppression of rCAF phenotypes and promoting chemotherapeutic efficacy. Transwell migration assay to assess effects of CCR7 neutralization on fibroblast-mediated naive B cell migration. All errors bars represent mean + SEM and all groups were compared by a oneway ANOVA with Tukey’s multiple comparisons test, ns = not significant. *p < 0.05; ** p < 0.01; ***p < 0.001; **** p < 0.0001.

[0073] FIG. 10C is a graphical representation of TGFpRli reversing suppression of rCAF phenotypes and promoting chemotherapeutic efficacy. VCAM-1 expression on primary fibroblasts stimulated with TGFp prior to TGFpRli (LY3200882) +/- aLTBR. All errors bars represent mean + SEM and all groups were compared by a oneway ANOVA with Tukey’s multiple comparisons test, ns = not significant. *p < 0.05; ** p < 0.01; ***p < 0.001; **** p < 0.0001.

[0074] FIG. 10D is a schematic representation of TGFpRli reversing suppression of rCAF phenotypes and promoting chemotherapeutic efficacy. Schematic for orthotopic transplantation of PK51975 cells and aLTBR and/or TGFpRli (LY2157299) treatments in tumor-bearing mice. [0075] FIG. 10E is a graphical representation of TGFpRli reversing suppression of rCAF phenotypes and promoting chemotherapeutic efficacy. Orthotopic tumor growth curves for treatments described in FIG. 10D. All errors bars represent mean + SEM and all groups were compared by a oneway ANOVA with Tukey’s multiple comparisons test, ns = not significant. *p < 0.05; ** p < 0.01; ***p < 0.001; **** p < 0.0001.

[0076] FIG. 10F is a graphical representation of TGFpRli reversing suppression of rCAF phenotypes and promoting chemotherapeutic efficacy. Orthotopic tumor growth curves for TLS- resistant tumors treated with Gem/Nab-PTX chemotherapy (dashed lines) alone or in combination with either aLTBR (dotted lines) or TGFpRli (SM16) (shaded region). All errors bars represent mean + SEM and all groups were compared by a oneway ANOVA with Tukey’s multiple comparisons test, ns = not significant. *p < 0.05; ** p < 0.01; ***p < 0.001; **** p < 0.0001. [0077] FIG. lOG is a graphical representation of TGFpRli reversing suppression of rCAF phenotypes and promoting chemotherapeutic efficacy. Survival of TLS resistant PK5L1948 tumor bearing mice receiving the indicated treatments.

[0078] FIG. 10H is a plot representation of TGFpRli reversing suppression of rCAF phenotypes and promoting chemotherapeutic efficacy. UMAP plots of individual CAF surface marker expression on CAFs from FIG. 9K.

[0079] FIG. 11 A is a schematic representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. Experimental procedure for purifying PDPN+ CAF from PK5L1948 tumors.

[0080] FIG. 1 IB is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. qRT-PCR for (i) CCL19, (ii) CCL21, and (iii) CXCL13 in CAFs sorted from PDAC tumors. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001. Shown is 1 representative experiment reflective of 2 independent replicates.

[0081] FIG. 11C is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. Density of CD 19+ B cells in TLS-resistant tumors by flow cytometry analysis following the indicated treatments of tumor bearing mice. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001. Shown is 1 representative experiment reflective of 2 independent replicates.

[0082] FIG. 1 ID is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. Density of CD4+ T cells in TLS-resistant tumors by flow cytometry analysis following the indicated treatments of tumor bearing mice. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001. Shown is 1 representative experiment reflective of 2 independent replicates. [0083] FIG. 1 IE is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. Density of CD8+ T cells in TLS-resistant tumors by flow cytometry analysis following the indicated treatments of tumor bearing mice. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001. Shown is 1 representative experiment reflective of 2 independent replicates.

[0084] FIG. 1 IF is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. Percent of CD62L-CD44+ effector memory cells (i) and PD1+TIM3+ exhausted cells (ii) of CD8+ T cells. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < o.OOl. Shown is 1 representative experiment reflective of 2 independent replicates.

[0085] FIG. 11G is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. T cell immune infiltrate in TLS-resistant tumors as determined by flow cytometry following chemotherapy and the indicated combination treatments. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001. Shown is 1 representative experiment reflective of 2 independent replicates.

[0086] FIG. 11H is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. Frequency of CD8+ T-stem cells (PD1+ TCF1+), and CD8+ exhausted T cells (PD1+ TIM3+) of total CD8+ T cells in tumors following the indicated treatments. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001. Shown is 1 representative experiment reflective of 2 independent replicates.

[0087] FIG. 1 II is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. Fraction of SIY-pentamer+ tumor-reactive CD8+ T cells in TLS- resistant tumors following the indicated treatments. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001. Shown is 1 representative experiment reflective of 2 independent replicates.

[0088] FIG. 11 J is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. Percent CD19+ B cells and CD90.2+ T cells of total viable cells as determined by flow cytometry on end-stage tumors of mice receiving either anti-CXCL13 antibody, aLTBR/TGFbRli, or both. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001. Shown is 1 representative experiment reflective of 2 independent replicates.

[0089] FIG. 1 IK is a graphical representation of myCAF to rCAF reprogramming inducing T and B cell chemokine gene expression leading to increased lymphocyte infiltration in TLS- resistant PDAC tumors. Tumor area of end-stage tumors (day 21) following the indicated treatments. All graphs represent the mean + SEM. All experimental groups were compared by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05; *** p < 0.001. Shown is 1 representative experiment reflective of 2 independent replicates.

[0090] FIG. 12A is a graphical representation of decreased myCAF differentiation following TGFbRl inhibition associating with an increase in tumor reactive stem-like CD8+ T cells. qRT- PCR for myCAF markers Acta2 and Ctgf on CAFs sorted from PK5L1948 tumors as described in FIG. 11 A and treated as described in FIG. 9E. All errors bars represent mean + SEM and all groups were compared by a one-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001.

[0091] FIG. 12B is a graphical representation of decreased myCAF differentiation following TGFbRl inhibition associating with an increase in tumor reactive stem-like CD8+ T cells. Amount of CD8+ T cells in the respective treatment groups. All errors bars represent mean + SEM and all groups were compared by a one-way ANOVA with Tukey’s multiple comparisons test. **** _p < 0.0001.

[0092] FIG. 12C is a graphical representation of decreased myCAF differentiation following TGFbRl inhibition associating with an increase in tumor reactive stem-like CD8+ T cells. Frequency of antigen-specific (SIY+) of tumor-infiltrating CD8+ T cells in the respective treatment groups. All errors bars represent mean + SEM and all groups were compared by a oneway ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001.

[0093] FIG. 12D is a graphical representation of decreased myCAF differentiation following TGFbRl inhibition associating with an increase in tumor reactive stem-like CD8+ T cells. Stemlike (PD1+TCF1+) phenotype of SIY-reactive and polyclonal CD8+ T cells as assessed by flow cytometry from PDAC tumors treated as indicated. All errors bars represent mean + SEM and all groups were compared by a one-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001.

[0094] FIG. 12E is a graphical representation of decreased myCAF differentiation following TGFbRl inhibition associating with an increase in tumor reactive stem-like CD8+ T cells. Quantification of the frequency of TNFa+ cells by flow cytometry in indicated tumor-resident cell types in TLS-resistant tumors treated with combinations of Gem/Nab-PTX, aLTBR, and TGFpRli (SMI 6). All errors bars represent mean + SEM and all groups were compared by a one-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001.

[0095] FIG. 13 is a graphical representation of the effects of disclosed treatments on TLS- resistant PK5L 1941 tumors.

[0096] Further implementations, features, and aspects of the disclosed technology, and the advantages offered thereby, are described in greater detail hereinafter, and can be understood with reference to the following detailed description, accompanying drawings, and claims.

DETAILED DESCRIPTION

[0097] Embodiments described herein can be understood more readily by reference to the following detailed description and examples. Elements, apparatus and methods described herein, however, are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present disclosure. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the disclosure. [0098] All publications, patents and patent applications mentioned in this specification are incorporated herein in their entirety by reference, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

[0099] In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9. All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10,” “from 5 to 10,” or “5-10” should generally be considered to include the end points 5 and 10.

[0100] Further, when the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity. For example, a material present in an amount “up to” a specified amount can be present from a detectable amount and up to and including the specified amount.

[0101] Additionally, in any disclosed embodiment, the terms “substantially,” “approximately,” and “about” may be substituted with “within [a percentage] of’ what is specified, where the percentage includes 0.1, 1, 5, and 10 percent.

[0102] It is also to be understood that the article “a” or “an” refers to “at least one,” unless the context of a particular use requires otherwise.

[0103] [0027] The terms “treatment”, “treat” and “treating” as used herein refers a course of action initiated after the onset of a clinical manifestation of a disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition. Such treating need not be absolute to be useful.

[0104] The terms such as “administering” or “administration” include acts such as prescribing, dispensing, giving, or taking a substance such that what is prescribed, dispensed, given, or taken is actually contacts the patient’s body externally or internally (or both). It is specifically contemplated that instructions or a prescription by a medical professional to a subject or patient to take or otherwise self-administer a substance is an act of administration.

[0105] The term “in need of treatment” as used herein refers to a judgment made by a medical professional that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a medical professional’s expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a condition that is treatable by a method or composition of the present disclosure.

[0106] The term “therapeutically effective amount” as used herein refers to an amount of a compound, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state or condition. Such effect need not be absolute to be beneficial.

[0107] The term “individual”, “subject” or “patient” as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female.

[0108] Tertiary lymphoid structures (TLS) are aggregations of lymphocytes and antigen presenting cells that form in chronically inflamed tissues, like solid tumors. Many of the functions of TLS resemble that of what is provided to the immune system by a tissue draining lymph node or secondary lymphoid organ. However, only some cancer patients elicit TLS formation in their tumors, and the presence of TLS is prognostic for survival and predictive of response to certain immunotherapies.

[0109] In mouse models of pancreatic cancer, TLS formation can be transiently promoted by treating mice with an agonist antibody to lymphotoxin beta receptor (anti-LTBR). One of the primary cellular targets for anti-LTBR is cancer associated fibroblasts (CAF). In vitro, primary mouse fibroblasts can be activated by treatment with anti-LTBR, which is enhanced by treatment with the cytokine TNF-alpha. Fibroblasts treated with anti-LTBR/TNFa upregulate VCAM1 and the critical lymphocyte chemokines CXCL13 and CCL19. However, most CAF in pancreatic cancer exhibit a phenotype called myofibroblasts, induced by the cytokine transforming growth factor beta (TGFb).

[0110] It was found that when cultured fibroblasts were pretreated with TGFb prior to anti- LTBR/TNFa treatment, the expression of CXCL13, CCL19 and VC AMI were all completely repressed. Correspondingly, B and T cell migration is enhanced towards aLTBR/TNFa activated fibroblasts, dependent on CXCL13, and reduced by TGFb pre-treatment of fibroblasts. Treating cultured fibroblasts and PDAC tumor-bearing mice with a TGFb-receptor 1 inhibitor prior to LTBR agonism reversed the effects of TGFb leading to increased B and T cell migration and greater tumor control. [0111] Not intending to be bound by theory, it is believed that the foregoing results implicate a mechanism whereby lymphocyte recruitment and LTBR-induced TLS formation is suppressed by TGFb-regulated myofibroblast phenotypes in pancreatic cancer. Blockade of TGFbRl can reverse this suppression by relieving the repression of CXCL13 and CCL19 expression in CAF leading to enhanced B and T cell migration to tumors and ultimately greater tumor control and survival in PDAC mouse models.

[0112] The invention described herein, in some embodiments, is directed to the therapeutic induction of TLS formation. Not intending to be bound by theory, it is believed that the treatment with TGFb inhibition and LTBR agonism promotes TLS formation and tumor immunity in cancer patients.

[0113] In one aspect, methods of treating a patient in need thereof are described herein. In some embodiments, the method comprises disposing a TGFb-receptor 1 inhibitor within a biological compartment of the patient, and disposing a lymphotoxin beta receptor (LTBR) agonist within the biological compartment of the patient. Moreover, in some cases, the step of disposing a TGFb-receptor 1 inhibitor within the biological compartment of the patient is carried out before the step of disposing the LTBR agonist within the biological compartment of the patient (e.g., as a pre-treatment step).

A. Compositions and Medicaments

[0114] The present disclosure provides for compositions for treatment of cancer patients in need of such treatments. The compositions herein include one or more active agents, including an LTBR agonist and/or a TGFb-receptor 1 inhibitor. Without intending to be bound by theory, it is believed that the LTBR agonist upregulates VCAM1 and the critical lymphocyte chemokines CXCL13 and CCL19 in fibroblasts and thus promotes TLS formation where the tumor microenvironment is permissive for TLS formation. Without intending to be bound by theory, it is believed that inhibiting TGF0 receptor (TGF0R1) signaling with a TGFb-receptor 1 inhibitor reverses TGF0 suppression of the recruitment of lymphocytes to reticular fibroblasts and promotion of a TLS permissive reticular CAF phenotype, rCAF. Without intending to be bound by theory, it is believed that a composition including both an LTBR agonist and a TGFb-receptor 1 inhibitor may promote rCAF differentiation, increase lymphocyte recruitment, and improve tumor control. [0115] The present disclosure contemplates the use of active agent derivatives in the methods of treatment disclosed herein. An active agent derivative is one that includes one or more fragments, insertions, deletions, or substitutions. The active agent derivative may have an activity that is comparable to or increased relative the active agent wild-type activity; alternatively, the active agent derivative may have an activity that is decreased relative the active agent wild-type activity.

[0116] Derivatives have some degree of identity with native active agents. For example, many derivative will have from 90% to 100% identity with the native active agent. There is also a likelihood that the derivative has functionality retention at a different range of activity relative the native active agent, such as the following range of identity: 75-100%, 80-100%, 85-100%, 90-100%, and 95%-100%.

[0117] Useful compositions of the present disclosure may comprise one or more active agents as described above. In one embodiment, such compounds are in the form of compositions, such as but not limited to, pharmaceutical compositions and medicaments. The compositions disclosed may comprise one or more of such compounds, in combination with a pharmaceutically acceptable carrier. To form a pharmaceutically acceptable composition suitable for administration, such compositions will contain a therapeutically effective amount of a compound(s).

[0118] The compositions of the disclosure may be used in the treatment methods of the present disclosure. Such compositions are administered to a subject in amounts sufficient to deliver a therapeutically effective amount of the compound(s) so as to be effective in the treatment methods disclosed herein. The therapeutically effective amount may vary according to a variety of factors such as, but not limited to, the subject’s condition, weight, sex and age.

Other factors include the mode and site of administration. The compositions may be provided to the subject in any method known in the art. Exemplary routes of administration include, but are not limited to, subcutaneous, intravenous, topical, epicutaneous, oral, intraosseous, intramuscular, intranasal and pulmonary.

[0119] In some instances, the TGFb-receptor 1 inhibitor of the present disclosure is administered at a range of about 200 mg to about 400 mg daily. In preferred embodiments, the TGFb-receptor 1 inhibitor is administered at a dose of about 300 mg daily. In some cases, the TGFb-receptor 1 inhibitor is administered orally at about 150 mg twice daily. The dose may be administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some cases, the TGFb-receptor 1 inhibitor is administered orally at about 150 mg twice daily for 14 days in two separate courses.

[0120] In some instances, the LTBR agonist is administered at a dose ranging from about 1 mg/kg to about 5 mg/kg.

[0121] The compositions and active agents of the present disclosure may be administered only one time to the subject or more than one time to the subject. Furthermore, when the compositions are administered to the subject more than once, a variety of regimens may be used, such as, but not limited to, one per day, once per week, once per month or once per year. The compositions may also be administered to the subject more than one time per day. The therapeutically effective amount of the active agents and appropriate dosing regimens may be identified by routine testing in order to obtain optimal activity, while minimizing any potential side effects. In addition, co-administration or sequential administration of other agents may be desirable.

[0122] The compositions of the present disclosure may be administered systemically, such as by intravenous administration, or locally such as by subcutaneous injection or by application of a paste or cream.

[0123] The compositions of the present disclosure may further comprise agents which improve the solubility, half-life, absorption, etc. of the compound(s). Furthermore, the compositions of the present disclosure may further comprise agents that attenuate undesirable side effects and/or or decrease the toxicity of the compounds(s).

[0124] The compositions of the present disclosure can be administered in a wide variety of dosage forms for administration. For example, the compositions can be administered in forms, such as, but not limited to, tablets, capsules, sachets, lozenges, troches, pills, powders, granules, elixirs, tinctures, solutions, suspensions, elixirs, syrups, ointments, creams, pastes, emulsions, or solutions for intravenous administration or injection. Other dosage forms include administration transdermally, via patch mechanism or ointment. Further dosage forms include formulations suitable for delivery by nebulizers or metered dose inhalers. Any of the foregoing may be modified to provide for timed release and/or sustained release formulations. [0125] In the present disclosure, the pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspending agents, emulsifying agents, inert fillers, diluents, excipients, wetting agents, binders, lubricants, buffering agents, disintegrating agents and carriers, as well as accessory agents, such as, but not limited to, coloring agents and flavoring agents (collectively referred to herein as a carrier). Typically, the pharmaceutically acceptable carrier is chemically inert to the active compounds and has no detrimental side effects or toxicity under the conditions of use. The nature of the pharmaceutically acceptable carrier may differ depending on the particular dosage form employed and other characteristics of the composition.

[0126] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the patient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound(s) may be administered in a physiologically acceptable diluent, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, glycols, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as, but not limited to, a soap, an oil or a detergent, suspending agent, such as, but not limited to, pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

B. Methods of Treatment

[0127] The teachings of the present disclosure provide for the treatment of cancer and autoimmune disease in a subject in need of such treatment. Cancers include pancreatic cancer, such as pancreatic ductal ademocarcinoma. Cancers include cancer types associated with immunosuppression, such as cancers with tumor microenvironments exhibiting a myCAF phenotype.

[0128] The method of treatment comprises administering to the subject any of the active compounds or compositions containing active compounds disclosed herein. The method may further comprise identifying a subject in need of treatment. [0129] Treatment may be accomplished by disposing an agonist antibody to lymphotoxin beta receptor (anti-LTBR) within a biological compartment of the subject. Without intending to be bound by theory, it is believed that the LTBR agonist upregulates VCAM1 and the critical lymphocyte chemokines CXCL13 and CCL19 in fibroblasts and thus promotes TLS formation where the tumor is TLS permissive.

[0130] Treatment may be accomplished by disposing a TGFb-receptor 1 inhibitor within a biological compartment of a subject, and by disposing an LTBR agonist within the biological compartment of the subject, where a tumor of the subject is TLS resistant. Treatment may involve the admixing of the TGFb-receptor 1 inhibitor and LTBR agonist and disposing the admixed compounds within the subject. Treatment may involve first disposing the TGFb- receptor 1 within the subject before disposing the LTBR agonist within the subject.

[0131] Without intending to be bound by theory, it is believed that inhibiting TGFp receptor (TGFpRl) signaling with the TGFb-receptor 1 inhibitor reverses TGFp suppression of the recruitment of lymphocytes to reticular fibroblasts.

[0132] The following non-limiting Examples describe the foregoing and other embodiments in further detail.

EXAMPLE 1

Tertiary lymphoid structures in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a cancer with low survival rates and currently no immunotherapy options. However, many tumors from PDAC patients can be heavily infiltrated with T and B cells associated with favorable survival outcomes indicating anti-tumor immunity is functional in certain patients. In some of these lymphocyte-inflamed PDAC tumors, spontaneous organization of tertiary lymphoid structures (TLS) is evident upon surgical resection and these patients have a clear survival advantage. TLS are predictive of immune checkpoint blockade response in some cancer types, yet PDAC patients, with or without TLS, are still insensitive to these strategies.

To address this, surgically resected PDAC tumors were evaluated from previously untreated patients, and it was found that that tumors with TLS (TLS+) recruited T and B cells with specific anti-tumor and memory phenotypes suggesting immunosurveillance of disease was enhanced. Furthermore, a subset of TLS+ patients contained germinal centers and improved humoral immune function corresponding to an increased neoantigen burden and long-term survival.

[0133] A method for inducing TLS formation in implantable mouse PDAC tumors in the orthotopic setting utilizing an antibody that agonizes the lymphotoxin beta receptor (LTBR) in mice bearing established PDAC tumors. These TLS are complexed with T cells, B cells, and CXCL13 cells, and are associated with PNAd+ HEV and reduced tumor growth, the presence of lymphocyte-recruiting cancer associated fibroblasts and increases in anti-tumor T and B cell phenotypes. Interestingly, some mouse PDAC cell lines are resistant to TLS formation by LTBR agonism, while others are susceptible, offering a model to study cancer patient heterogeneity. A TLS+ PDAC mouse model is used to address how TLS may directly improve antigen-specific T cell and B cell immunity and elucidate the pathways that regulate TLS formation. These data offer new strategies to overcome immunotherapy resistance in cancer patients.

EXAMPLE 2

Myofibroblast programming blocks differentiation of TLS-organizing fibroblastic reticular cells in pancreatic cancer

[0134] Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and highly lethal malignancy (13% 5-year overall survival) due to the lack of early detection and therapeutic insensitivity. Nearly half of PDAC patients are diagnosed with metastatic disease, while also having a less than 5% objective response rate to current immunotherapies. Thus, there is an need to develop improved therapies and novel biomarkers that will improve survival. Despite the lack of clinical benefit from immunotherapies, significant T and B cell infiltration in PDAC tumors in a subset of patients has been reported that is equivalent or even superior to other cancer types, many of which do respond to immunotherapy. Furthermore, these lymphocyte-inflamed signatures associate with longer overall survival. Thus, despite the belief that pancreatic cancer is a poorly immunogenic tumor type, many PDAC patients mount nascent immune responses that conceivably can be harnessed to control tumors. One unique lymphocyte inflamed phenotype, a tertiary lymphoid structure (TLS), is prognostic for survival in all solid tumor types, including PDAC, and predictive of response to immune checkpoint blockade therapies in melanoma and sarcoma. TLS are a lymph node-like follicle comprised of heterogeneous aggregations of T cells, B cells, and antigen-presenting cells observed in chronically inflamed peripheral tissues, such as solid tumors. One study of treatment-naive PDAC patients showed that 44% of surgically resected tumors displayed histological presence of TLS or TLS-like lymphocytic aggregates associated with improved survival and downregulated transforming growth factor beta (TGFb) signaling. In other mouse tumor models depletion or suppression of Tregs, a major source of TGFb in the tumor microenvironment, elicits TLS formation. Without intending to be bound by theory, this may indicate that in addition to expressing the requisite pro-inflammatory mediators of TLS formation, some tumors lack or have overcome local immunosuppression to form TLS. [0135] Others have demonstrated the presence of CAF subsets critical for promoting adaptive immunity in tumors. Reflecting the fibroblastic reticular cell with lymphoid tissue organizing (LTO) properties during lymph node organogenesis, parallel functions have been described for TLS-organizing rCAF subsets. Signaling activation from tumor necrosis factor receptor (TNFR) and the lymphotoxin beta receptor (LTBR) converge to activate these TLS- promoting CAF phenotypes in complimentary ways. While TNFR1/2 is required for the initial aggregation of lymphocytes and the reticular network, LTBR expands and organizes the TLS. These signaling pathways are chiefly responsible for upregulation of CCL19, CCL21, and CXCL13 in CAF while other groups have also reported on the pro-inflammatory effects of type 1 and type 2 interferons in stromal-mediated T cell recruitment. However, most cancer patients do not present with TLS in their tumors and these CAF phenotypes are rare in cancers like PDAC where the trademark desmoplastic stroma is governed overwhelming by TGFb- programmed myofibroblasts (myCAF). Ergo, while the positive signals that reinforce TLS formation have been studied, little is understood about the negative regulators that restrict this biology and how to overcome them therapeutically. Here, heterogenous mouse models of PDAC are utilized to investigate the mechanisms that dampen CAF-mediated lymphocyte recruitment and develop treatment strategies aimed at repolarizing CAF phenotypes to promote TLS formation. It was discovered that LTBR agonism can trigger lymphocyte aggregates in some PDAC models, but not others, strongly associated with the baseline CAF phenotype. Treating fibroblasts with TGFbl to induce myCAF programming abrogated the effects of LTBR/TNFR activation primarily by silencing expression of T and B cell chemoattractants. Inhibiting TGFbRl signaling in TLS-resistant PDAC models reversed these effects and combined with LTBR agonism promoted rCAF differentiation, increased lymphocyte recruitment, and improved tumor control. These data indicate that in TLS-resistant tumor microenvironments, immunosuppressive factors counteract the effects of TLS-promoting inflammatory mediators by silencing reticular CAF transcriptional programs.

Mouse PK5 cell lines exhibit PDAC tumor heterogeneity and respond to LTBR agonism [0136] To understand the heterogeneity of the stromal immune response and its relationship to PDAC progression, the phenotype of nine PDAC cell lines derived from KPC30 (Pdxl- Cre/LSL-KRasG12D/+/LSL-p53R172H/+) transgenic mouse tumors were sought to be characterized. Five of these cell lines were termed PK5 (Pdxl-Cre/LSL-KRas^G12D/p53^{R172H wt}) and four were termed PK5L (Pdxl-Cre/LSL-KRas^G12D/LSL-p53^{R172H w,t}/LSL-Luciferase- SIYRYYGL) which includes a fourth transgene encoding for a luciferase fusion protein and the model H2-Kb antigen, SIY, for tracking of tumor-reactive CD8+ T cells (FIG. 1A and FIG. IB). These cell lines were implanted orthotopically into C57BL/6 mice and tumor growth kinetics monitored until tumors reached 100 mm² or greater. Despite their common genetic origin, it was observed that there were substantial differences in orthotopic tumor growth kinetics upon implantation cells into the pancreas of naive animals (FIG. 2A). Cytokine/chemokine profiling of PK5/PK5L cell lines in vitro revealed disparities in the inflammatory phenotype including secretion of influential myeloid factors such as GMCSF, IL-6, CCL2, CXCL1, CXCL2, and CXCL5 across both PK5 and PK5L lines (FIG. 2B). All tumor cell lines expressed low basal levels of MHC-I and PD-L1 expression but treatment with type I (IFNP) and type II (IFNy) interferon induced upregulation of MHC-I and PD-L1 expression indicating their responsiveness to T cell mediated inflammation (FIG. 1C and FIG. ID). Lymphotoxin beta receptor (LTBR) is a necessary gene for secondary lymphoid organogenesis. Others have shown LTBR agonism can elicit TLS formation in mouse tumor models. In the PK5 tumor models, surface LTBR expression was present on numerous tumor-resident cell types including myeloid cells, tumor cells, endothelial cells, and fibroblasts (FIG. 2C). However, stromal fibroblasts and endothelial cells consistently expressed the highest levels of surface LTBR across all PK5 models at multiple timepoints tested FIG. 2C) and proinflammatoiy cytokines did not increase tumor cell LTBR expression (FIG. IE). Podoplanin expressing (PDPhT) CAF were numerically higher than CD31⁺ endothelial cells suggesting CAF are a primary target of LTBR ligands in PK5 tumors (FIG. IF).

[0137] To assess the capacity of PDAC tumors to allow TLS formation in vivo, the response of the PK51975 cell line to an LTBR monoclonal antibody with agonist activity (oLTBR) was tested in an orthotopic and subcutaneous setting. Only the orthotopic tumors displayed a significant reduction in tumor growth rate with aLTBR treatment compared to isotype control groups (FIG. 2D). Histological evaluation of the orthotopic tumor sections showed evidence of lymphoid aggregates on the periphery of the tumors and extensive PNAd⁺ high endothelial venules (HEV) and CXCL13⁺ cells present within the aggregates whereas this was never observed in isotype treated controls (FIG. 2E). Even though PK5 tumor cells maintained robust expression of LTBR, these cells did not upregulate PDL1 or MHCI in response to aLTBR treatment (FIG. 1C - FIG. IE) indicating the pro-inflammatory response to this agonist in vivo is tumor cell extrinsic. In line with detection of HEV and lymphocyte recruitment in these tumors, the density of CXCL13⁺ cells and density of total tumor-infiltrating T cells were enriched in aLTBR-treated PK51975 tumors (FIG. 1G). These data demonstrate the extensive heterogeneity that persists in these PK5 PDAC tumor models. Furthermore, by agonizing LTBR there exists a reproducible method to induce lymphocyte recruitment and TLS formation, allowing assessment of cellular and molecular mechanisms that influence TLS development and function.

CAF phenotypes dictate TLS permissiveness of PDAC tumors in vivo

[0138] The response of other PK5 cell lines to aLTBR treatment was then queried. A significant reduction in tumor growth was observed in the PK51975 “TLS-permissive” tumors treated with aLTBR but this was not detected in the PK5L1948 “TLS-resistanf ’ lines where no visible lymphoid aggregates were observed in the tumor following treatment (FIG. 3A). Others have demonstrated that specific CAF phenotypes can dictate lymphocyte recruitment and organization of the reticular network in other mouse tumor models. CAF that lack expression of fibroblast-activating protein (FAP) and alpha smooth muscle actin (aSMA) while co-expressing the cell adhesion molecules, ICAM-1 and VCAM-1, mark immunofibroblasts that initiate TLS formation in vivo.

[0139] Flow cytometry assessment of fibroblasts (PDPN⁺CD3 FCD45") in our TLS- permissive and TLS-resistant PK5 tumors revealed a substantial contrast in CAF phenotype. TLS-permissive tumors were dominated by ICAM-1⁺VCAM-1⁺ rCAFs whereas TLS-resistant tumors had a predominant ICAM-1⁺VCAM-L myCAF phenotype (FIG. 3B and FIG. 4A). Deeper profiling of these CAF populations in untreated tumor-bearing mice confirmed these findings showing considerable differences in CAF phenotypic marker expression at baseline between models including ICAM1 and VCAM1 surface expression (FIG. 3C, FIG. 3D, and FIG. 4B). The percent of cells expressing aSMA or exhibiting activated SMAD2 signaling was higher in TLS-resistant tumors, reflecting an increase in myCAF and TGF0R signaling in these PDAC tumors (FIG. 3E and FIG. 3F). These differences in CAF phenotype associated with significant increases in intratumoral PD1⁺TCF1⁺ stem-like CD8⁺ T cells, PD1⁺TCFF exhausted CD8⁺ T cells and PD1⁺CXCR5⁺Foxp3-CD4⁺ T-follicular helper cells in aLTBR-treated TLS-permissive PK51975 tumors (FIG. 3G). Conversely, none of these increases were observed in the TLS- resistant PK5L1948 model (FIG. 3H). This finding is in line with previous reports of the importance of ICAM-1⁺VCAM-1⁺ CAF populations in lymphocyte recruitment and organization and the recruitment of stem-like CD8⁺ T cells in LTBR-agonized mouse tumor models. Bulk transcriptome analysis of PK5 tumors showed that TLS-permissive PK51975 tumors expressed higher levels of TNFR and LTBR ligands than TLS-resistant PK5L1948 tumors, but this was not consistent across other TLS-resistant cell lines such as PK5L1941 (FIG. 4C). Additionally, there were no significant differences in these genes between TLS' and TLS⁺ human PDAC tumors (FIG. 4D). Finally, expression of pro-TGFpi/LAP between tumor models was broader among cells in the tumor in TLS-resistant lines with macrophages expressing the highest in both models and B cell and tumor cells expressing the lowest (FIG. 4E). Taken together these data show that individual mouse PDAC models have variable responses to TLS-induction therapies, such as LTBR agonism. This variation associates with CAF phenotypic heterogeneity that cannot readily be explained by intrinsic differences in cancer cell lines or enhanced presence of endogenous TNFR and LTBR signaling elements.

TGFp antagonizes TNFa and LTBR induction of reticular programming in fibroblasts [0140] Previously, it has been shown tumor necrosis factor receptor (TNFR1/2) and LTBR signaling cooperate to promote CAF as a lymphoid tissue organizing (LTO) cells in tumors. To understand how these signaling pathways regulate fibroblast phenotypes that enhance these functions, primary fibroblasts were isolated from naive mice and exposed to various treatments in vitro. Fibroblasts were treated with either TNFa, aLTBR, or in combination and analyzed by flow cytometry for reticular fibroblast markers. TNFa and aLTBR treatment alone upregulated expression of VCAM-1, which was enhanced by co-treatment (FIG. 5A, top left panel). Expression of PDGFRa was also upregulated in treated fibroblasts with a concomitant downregulation of PDGFRp (FIG. 5A, top right and bottom left panels) while TNFa treatment alone slightly reduced LTBR expression on fibroblasts (FIG. 5A, bottom right panel). These observations combined with the variable response to aLTBR agonism in vivo associated with CAF hetereogeneity (FIG. 3A-C) prompted identification of negative regulators of TNFR/LTBR signaling in fibroblasts.

[0141] PDGFRp has been implicated as a direct target of TGFp signaling, and a marker for TGFp-induced myCAF differentiation. Furthermore, previous work showed TLS⁺ PDAC patients have downregulated TGFP signaling and myCAF markers such as LRRC15. To that end, and without intending to be bound by theory, it was hypothesized that TGFp induction of myCAF programming modulates the response to LTBR agonism. In contrast to aLTBR treatment, TGFP suppressed VCAM-1, PDGFRa, and LTBR expression but upregulated PDGFRp expression (FIG. 5B). Moreover, TGFp pre-treatment of fibroblasts prevented aLTBR- mediated increases in VCAM-1 and PDGFRa cell surface expression (FIG. 5B, top left and right panels). Co-culture of naive fibroblasts with tumor conditioned media from TLS-permissive and resistant cell lines did not reproduce these effects indicating paracrine tumor cell-fibroblast crosstalk is not responsible for CAF differentiation in these contexts (FIG. 6A). Interestingly, upregulation and secretion of the lymphocyte chemoattractants, CXCL13 and CCL19, required co-treatment with both TNFa and aLTBR (FIG. 5C, top left and right panels; FIG. 7A). Again, this was completely repressed by pre-treatment with TGFp at the transcriptional (FIG. 5C, top left and right panels) and protein level (FIG. 7B-C). This corresponded to upregulation of canonical myCAF markers Acta2 and Ctgf in TGFpi-treated groups (FIG. 5C, bottom left and right panels). Other T cell chemoattractants, such as CXCL9 and CCL21 were not significantly induced by TNFa/aLTBR treatment (FIG. 6B, left and right panels) but numerous other chemokines were and could be inhibited by TGFP pre-treatment (FIG. 6C). By comparison, cotreatment of primary fibroblasts with TNFa and LIGHT resulted in a 3-fold increase in CXCL13 mRNA expression, but this was markedly less than with TNFa/aLTBR (FIG. 6D) indicating the agonist antibody delivers a more potent signal to fibroblasts than endogenous LTBR ligands. Furthermore, induction of the iCAF phenotype by IL- la treatment or exposure to interferons alone or in combination with aLTBR had no impact on rCAF differentiation (FIG. 6E). Flow cytometry analysis of in vitro activated fibroblasts showed global changes in CD 105, CD34, Ly6A, CD74, CD21/CD35, TNFR and HVEM between rCAF and myCAF differentiation conditions (FIG. 6F). Taken together, these data show that TNFR/LTBR-mediated reticular cell programming in fibroblasts is abrogated by TGFp induction of the myCAF phenotype. Lymphocyte migration to reticular fibroblasts is suppressed by TGFp

[0142] The induction of rCAF phenotypes by TNFR/LTBR and lack of CXCL13 secretion from our PK5 cancer cell lines (FIG. 7A) suggest lymphocyte recruitment to tumors is primarily regulated by CAF similar to the functions of fibroblastic reticular cells during lymph node organogenesis. In support of this, TNFa/aLTBR treatment of cultured fibroblast resulted in secretion of CXCL13 and CCL19 protein that was abrogated by TGF 1 pre-conditioning (FIG. 7B-C). Transwell migration assays were then conducted by culturing naive fibroblasts in the bottom well followed by addition of purified B or T cells (FIG. 8A-E) to the upper transwell (FIG. 7D). Naive B cells were first isolated from mouse splenocytes and assessed their migratory potential to activated fibroblasts. Migration of naive B cells to TNFa/aLTBR-activated fibroblasts was significantly increased compared to untreated fibroblasts (FIG. 7E). Both TGFp treatment alone and TGF pretreatment prior to TNFa/aLTBR stimulation suppressed this migration (FIG. 7E). Naive CD8⁺ T cells and THI (Tbet⁺ IFNY⁺) polarized CD4⁺ T cells (FIG. 8A-B) also displayed enhanced migration to TNFR/LTBR-activated fibroblasts and this migration was again repressed when fibroblasts underwent myCAF differentiation (FIG. 7F-G). B cell receptor activated B cells exhibited similar results albeit at reduced migration numbers as compared to naive B cells (FIG. 8F). Both T and B cell subsets expressed varying levels of the cognate CXCL13 and CCL19 receptors, CXCR5 and CCR7 (FIG. 8C-E), but these expression patterns changed upon BCR-stimulation of B cells (FIG. 8E). These results demonstrate that myCAF programming can suppress migration of lymphocytes towards TNFR/LTBR differentiated rCAF.

Blockade of TGFpRl signaling reverses suppression of the reticular fibroblast phenotype and enhances chemotherapeutic efficacy against PDAC tumors

[0143] Given that TGFp suppressed the recruitment of lymphocytes to reticular fibroblasts, and not intended to be bound by theory, it was hypothesized that inhibiting TGFP receptor (TGFpRl) signaling would reverse this effect. Naive fibroblasts treated with a small molecule antagonist to TGFpRl prior to TGFpi relieved the repression of CXCL13 and CCL19 in TNFa/aLTBR stimulated fibroblasts (FIG. 9A-B) and blocked the myCAF phenotype (FIG. 10A, left and right panels). Importantly, treatment of fibroblasts alone with TGFpRli did not upregulate CXCL13 indicating a requirement for TNFR and LTBR signaling engagement to induce chemokine expression (FIG. 9A-B). Correspondingly, inhibiting TGFpRl signaling in TNFa/aLTBR stimulated fibroblasts alleviated TGFP-mediated suppression of naive B cell migration (FIG. 9C). Antibody neutralization of CXCL13 prevented B cell migration to activated reticular fibroblasts (FIG. 9C) but not CCR7 blockade (FIG. 10B) as has been shown in other inflammatory conditions and tumor models. Conversely, Tul cell migration towards rCAF was partially dependent on CCR7 (FIG. 9D). Importantly, TGFpRl inhibition following TGFpi treatment also restored aLTBR mediated upregulation of VCAM1 indicating myCAF programming was not an irreversible phenotype (FIG. IOC). Not to be bound by theory, it was then hypothesized that blocking TGFpR signaling would overcome resistance to LTBR agonist therapy and TLS formation in the PK5 tumor models. Beginning at day 10 post-implant, the TLS-permissive PK51975 tumor-bearing mice were treated orally with TGFpRli once per day for 7 days, followed by a second oral course at day 23 as monotherapy or in combination with aLTBR (FIG. 10D). As before, tumor growth to end stage was attenuated in the aLTBR treated group but the addition of TGFpRli added no additional benefit (FIG. 10E) supporting the findings that the stromal biology in these tumors is not regulated by myCAF (FIG. 3B-C). Conversely, LTBR agonism and TGFpRl -inhibition in the PK5L1948 TLS-resistant model reprogrammed CAF away from myCAF (FIG. 9E) and towards rCAF phenotypes (FIG. 9F) associated with improved tumor control (FIG. 9G). Additionally, combining gemcitabine and Nab-paclitaxel chemotherapy with aLTBR and TGFpRl inhibition was explored to increase treatment responses in the TLS-resistant PDAC models. Indeed, intervention with double and triple combination treatments in TLS-resistant PK5L1948 and PK5L1941 tumor bearing mice decreased tumor growth over chemotherapy alone (FIG. 10F-H). Histologically, decreased aSMA⁺ was observed in TGFpRl -inhibited tumors indicative of attenuation of myCAF controlled stroma (FIG. 91). These treatment responses also associated with a repolarization of the CAF compartment away from PDGFRp^hlVCAMLLTBR^loCD105⁺ myCAFs and towards VCAM1⁺ICAM1⁺PDGFRP¹⁰CD105- rCAF (FIG. 9J-L and FIG. 10H). These data show that inhibiting TGFpRl signaling in mouse PDAC tumors blocks myCAF differentiation allowing TNFR/LTBR-mediated induction of fibroblastic reticular cells associated with improved tumor control. myCAF to rCAF reprogramming increases T and B cell infiltration and anti-tumor immune phenotype [0144] The tumor immune response associated with reprogramming to rCAF was then investigated. Supporting the increased rCAF differentiation in tumors of aLTBR/TGFpRli treated groups (FIG. 9F and 9J-L), significant upregulation of lymphocytes chemokines was observed in CAF sorted from tumors (FIG. 11 A). While LTBR agonism alone induced expression of Ccll9, Ccl21, and Cxcll3 from CAF, blocking TGFpRl had the most significant impact on Ccll9 and Cxcll3 upregulation (FIG. 1 IB). Furthermore, TGFpRli reduced myCAF markers Acta2 and Ctgf, while aLTBR and TGFpRli further suppressed myCAF gene expression in treated tumors (FIG. 12A, left and right panels). Correspondingly, tumors from aLTBR/TGFpRli treated animals demonstrated an increase in total B cell, CD4⁺ T cell, and CD8⁺ T cell infiltration (FIG. 11C-E). CD8⁺ T cells also demonstrated a change in phenotype with enrichment in effector memory and exhausted subsets significantly increased in treated tumors (FIG. 1 IF). However, when combined with chemotherapy it was observed that LTBR agonism largely drove CD4⁺ T cell infiltration while TGFpRl inhibition increased CD8⁺ T cell infiltration into tumors (FIG. 11G and FIG. 12B). There was also a significant enrichment in CD8⁺PD1⁺TCF1⁺ stem-like T cells in the chemo/aLTBR/TGFpRli treated animals concordant with an increase in PD1⁺TIM3⁺TCF1‘ exhausted T cells (FIG. 11H, left and right panels). There were no significant differences in the frequency of tumor-reactive CD8⁺ T cells with or without chemotherapy (FIG. I ll and FIG. 12C) but stem -like phenotypes did increase in the tumor antigen-reactive population in the absence of cytotoxic agents (FIG. 12D) indicative of an enhanced chronic immune response. Despite the critical role of TNFa in driving chemokine expression in CAF (FIG. 7A), an increase in TNFa expression was not detected on a per cell basis due to treatment (FIG. 12E). Because CAF from treated tumors upregulated lymphocyte chemokines (FIG. 1 IB) it was determined whether this was the primary mechanism of increased T and B cell infiltration. Neutralization of CXCL13 in vivo ameliorated the increase in B cell but not T cell infiltration in tumors of treated groups confirming that TGFP and LTBR signaling in CAF are the primary regulators of this B cell chemotaxis pathway (FIG. 11 J). However, CXCL13 blockade had no impact on aLTBR/TGFpRli treatment efficacy suggesting intratumoral B cells did not mediate tumor control in this context (FIG. 1 IK). Taken together, these data show that CAF phenotype can be therapeutically modulated to support T and B cell infiltration in PDAC tumors to overcome the TLS-resistant tumor microenvironment. TLS-promoting therapeutic regimen is T cell dependent and can be in observed in TLS⁺ human PDAC patients.

[0145] Because of the chronic exposure to antigen, CD8+ T cell responses in tumors frequently become exhausted and upregulate inhibitory receptors such as PD1. The observation that LTBR agonism plus TGF0R1 inhibition increased both PD1⁺ stem like T cells and PD1⁺TIM3⁺ exhausted T cells, without intending to be bound by theory, it was hypothesized that T cells may be supporting a treatment response. Indeed, depletion of CD4⁺ and CD8⁺ T cells reversed efficacy of aLTBR/TGFpRli treatment demonstrating that the therapeutic efficacy was dependent on T cells (FIG. 13).

Discussion

[0146] Cancer associated fibroblasts comprise a significant functional component of the tumor microenvironment and can regulate tumor progression in multiple ways. Considering the outsized presence of fibrosis and collagen deposition in PDAC tumors, understanding the phenotypic plasticity and how to therapeutically modulate CAF function in pancreatic cancer has been a priority but also paradoxical. Depletion of CAF via FAP or aSMA targeting in PDAC have demonstrated contradictory effects on tumor progression. Whereas FAP+ CAF secrete CXCL12 and sequester T cell trafficking within tumors, genetic ablation of aSMA-expressing cells or blocking sonic hedgehog signaling cells reduced collagen-mediated containment of cancer cell metastasis. However, more recent efforts to characterize CAF with single cell approaches have revealed significant phenotypic heterogeneity in cancer patients. These studies have shown that TGF -educated myCAF subsets comprise the largest fraction of CAF within the PDAC microenvironment, significantly restrict T cell immunity in tumors and eventually contribute to immunosuppressive senescent associated secretory activity. Yet in lymph nodes and other inflammatory conditions, fibroblasts readily acquire reticular phenotypes with immune stimulating functions including rare populations that promote lymphocyte recruitment, organization, and activation reflective of the rCAF subset described here. In tumors, rCAF phenotypes are distinct in gene expression and function from iCAF and apCAF. Specifically, IFNy synergized with TNFa to promote expression of CXCR3 ligands from CAF in mouse PDAC tumors. In immunogenic B 16 melanoma models, TNFR and LTBR activation of CAF were necessary for B cell recruitment and TLS formation when implanted in the intraperitoneal cavity. Metastatic lesions in colorectal patients and mouse models contained a population of CCL19-expressing CAF that augmented TLS formation while CCL19/CCL21- expressing rCAF could be detected in tumors from breast cancer patients located proximal to TLS. This work demonstrates that not only is LTBR agonism coupled with TNFR1/2 activation required for optimal rCAF differentiation and function, imprinting of the myCAF state by TGFp dramatically silences this transcriptional program. Given that the majority of patient tumors are devoid of TLS and myCAF are the dominant CAF phenotype across cancer types associated with immunosuppression, these data help to mechanistically explain these observations. Increasing the proportion of cancer patients with TLS could provide a survival benefit and a tool for patient stratification for immunotherapy treatment.

[0147] Despite the common genomic origin of the disclosed PK5 cell models, these tumors exhibited a nascent transcriptional and cellular heterogeneity reflective of PDAC patients (FIG. 1 and FIG. 2). This corresponded to a variable response to LTBR-mediated TLS formation in these tumors that strongly associated with baseline CAF phenotype (FIG. 3). Relieving myCAF transcriptional programming by TGFPR1 inhibition in TLS-resistant tumors allowed agonism of LTBR to upregulate critical LTO-genes such as VCAM1, ICAM1, CXCL13 and CCL19 and enhance tumor control dependent on T cell recruitment (FIG. 12 and FIG. 13). This supports other studies that repolarize myCAF differentiation using TGFP-blocking agents often alleviating the restriction of interferon-transcriptional signatures. In contrast, CD105 was not observed as strictly demarcated pro-tumor from anti-tumor CAF subsets, as has been proposed in other PDAC models. This work is distinct in that by concomitantly providing a strong LTBR agonist during myCAF blockade, CAF are skewed to actively support T and B cell immune responses, independent of interferon, in tumor microenvironments strongly influenced by TGFp. This therapy may circumvent the need to stimulate endogenous lymphotoxin signaling for TLS formation, as can be provided when boosting ILC2 activation in other PDAC models.

[0148] Understanding how to modify or repolarize CAF phenotypes into those more favorable to antitumor immunity is an important barrier to promoting immunotherapy response. It was previously demonstrated that TGFpRl inhibition was effective in the context of cytotoxic therapy in colorectal cancer mouse models and rectal cancer patients by directly suppressing CD8+ T cell trafficking. Stromal TGFp signatures and the myCAF phenotype associate with a reduced T cell infiltrate and poor response to immune checkpoint blockade such as anti- PDl/anti-CTLA4. Indeed, TGFp education of CAFs was one of the strongest predictors of immunotherapy failure across all cancer types indicating there are conserved mechanisms of CAF to immune cell crosstalk. As TGFp blockade in the clinic has not yet significantly improved responses for metastatic PDAC patients, the present disclosure offers an alternative therapeutic strategy with associated histological and molecular biomarkers of response suitable for clinical translation.

Methods

Tumor Models

[0149] PK5/PK5L PDAC cell lines used for orthotopic tumor implantation were derived from KPC (Pdxl-Cre/LSL-KRas^{G12D +}/LSL-p53^R172H/+) transgenic mouse tumors as previously described. Five of these cell lines were termed PK5 (Pdxl-Cre/LSL-KRas^G12D/p53^R172H/wt) including PK51972, PK51975, PK52003, PK52005, and PK52014 while four were termed PK5L (Pdxl-Cre/LSLKRas^G12D/ LSL-p53^{R172H W}7LSL-Luciferase-SIYRYYGL) including PK5L1932, PK5L1940, PK5L1941, and PK5L1948. All cells were cultured at 37°C with 5% CO₂.

PK5/PK5L tumor cells were implanted orthotopically into C57BL/6 mice (Jackson Labs). Prior to implant, tumor cells were cultured in RPMI complete medium until 70-90% confluency. On the day of implant, cells were harvested, washed, and resuspended in a 50:50 RPMI serum-free + 5 mg/ml Cultrex (R&D Systems) and implanted at 5 x 10^A3 cells/mouse in 50 pl. For subcutaneous implant, tumor cells were washed of culture media and resuspended in lx PBS and implanted under the skin of the hind limb with 2x10^A5 cells/100 pl injections. Tumors were randomized at day 7 by either a Vevo2100 ultrasound (Visual sonics) for PK5 tumors or by bioluminescence utilizing the IVIS system for PK5L tumors. For IVIS, mice were injected with 150 mg/kg D-luciferin (GoldBio) 10 min. prior to exposure to determine tumor luciferase fluorescence. All tumors were subsequently monitored by ultrasound following randomization.

Treatment of tumor-bearing mice

[0150] Tumor-bearing mice were not treated until mice were randomized by tumor volume and tumors reached ~25 mm² which occurred around day 7-10 unless otherwise indicated. In vivo treatments were performed as follows: anti-LTBR monoclonal antibody (Dr. Carl Ware) i.p. 3mg/kg, TGFpRli - LY2157299 (SelleckChem) via oral gavage at 150 mg/kg, TGF0Rli - SM16 (MedChemExpress) in mouse chow at 0.3 g/kg (Tocris Biosciences), gemcitabine (i.v. 15 mg/kg) and nab-paclitaxel (i.p. 33 mg/kg) dual chemotherapy, anti-PDl (BioXCell) i.p. 200 pg/mouse, and rat IgG2a isotype control antibody (BioXCell) i.p. 50 pg/mouse. Flow Cytometry

[0151] Orthotopic tumors were removed and half of the tumor with spleen attached was saved for histology (see Immunohistochemistry). Remaining tumor portion was weighed and collected into 1 ml of DMEM serum-free media and placed on ice. Tumors were minced into small pieces, transferred to stir bottles containing 15 ml digestion media (1 mg/ml collagenase IV (Worthington) + 50 U/ml DNase I (Worthington)), and digested on a stirring platform for 35 min. at 400 rpm at 37°C. Following digestion, cells and remaining tissue chunks were gently grinded and filtered through a 100 pm cell strainer. Cells were washed by centrifugation for 5 min. at 1300 rpm and resuspended in FACS buffer prior to downstream flow cytometry staining or freezing. Flow cytometry data was analyzed by FlowJo software (vlO.10.0) and advanced clustering analysis was performed using OMIQ.

Flow Cytometry Antibodies

Table 1 - Antibodies for Flow Cytometry

Generation, culture, and treatment of primary mouse fibroblasts

[0152] Orthotopic tumors were removed and half of the tumor with spleen attached was saved for histology (see Immunohistochemistry). Remaining tumor portion was weighed and collected into 1 ml of DMEM serum-free media and placed on ice. Tumors were minced into small pieces, transferred to stir bottles containing 15 ml digestion media (1 mg/ml collagenase IV (Worthington) + 50 U/ml DNase I (Worthington)), and digested on a stirring platform for 35 min. at 400 rpm at 37°C. Following digestion, cells and remaining tissue chunks were gently grinded and fdtered through a 100 pm cell strainer. Cells were washed by centrifugation for 5 min. at 1300 rpm and resuspended in FACS buffer prior to downstream flow cytometry staining or freezing. Flow cytometry data was analyzed by FlowJo software (vlO.10.0) and advanced clustering analysis was performed using OMIQ. Primary mouse fibroblasts were cultured in RPMI complete medium as described above for all in vitro experiments. Cytokines, antibodies, and inhibitors used for treatments as indicated included: recombinant murine LIGHT (25 ng/ml - Peprotech), recombinant murine TNFot (5 ng/ml - Peprotech), recombinant human TGF01 (5 ng/ml - Peprotech), mouse monoclonal anti-CXCL13 (15 pg/ml - Invitrogen), mouse monoclonal anti- CCR7 (5 pg/ml - R&D Systems), TGF[3Rli - LY3200882 (10 pM - SelleckChem), and mouse monoclonal anti-LTBR (1 pg/ml - Dr. Carl Ware).

CAF sorting from PDAC tumors

[0153] For sorting of CAFs from PK5 or PK5L tumors, single-cell tumor suspensions were enriched for PDPN⁺ CAF by dead cell removal (EasySep mouse dead cell removal kit, StemCell) and CD45⁺ cell depletion (EasySep mouse CD45 positive selection kit, StemCell). Cells were then stained for 30 minutes with anti-mouse CD45-BV510 (30-F11; BD), CD31-BUV496 (MEC13.3; BD), and PDPN-PerCP/EF710 (8.1.1; ThermoFisher). Immediately prior to sorting, 5 pl of propidium iodide (Invitrogen) was added to the sample, mixed well, and incubated for 5 min. prior to acquisition of PT CD45' CD3 F PDPN⁺ CAF on an Aurora CS (Cytek).

RT-PCR

[0154] RNA lysates were collected from primary fibroblast cultures after 24 hr. treatments by adding Buffer RLT (Qiagen) to lyse the cells. Prior to RNA isolation, lysates were homogenized using QIAshredder columns (Qiagen) and RNA isolation was carried out using the Qiagen RNeasy Micro RNA isolation kit (Qiagen) according to manufacturer’s instructions. Purified RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) and cDNA synthesis carried out in a T100 thermal cycler (Bio-Rad). 50 ng of RNA was added to each qPCR reaction and prepared in TaqMan Fast Advanced master mix (Applied Biosystems). qRT-PCR reaction was carried out in a QuantStudio 3 (Applied Biosystems) and RT-PCR data analyzed by QuantStudio Design and Analysis software version 2.7.0. Sequences of PrimeTime qPCR assays (IDT) for target genes are outlined. All qRT-PCR data is represented as fold change normalized to cellular Tbp levels. Table 2 - PCR Sequences

ELISA and Luminex assays

[0155] Supernatants for ELISA were collected from primary fibroblast cultures and frozen at -80°C prior to protein detection. Mouse CXCL13/BLC (Biolegend), CCL19/MIP-3 beta (Fisher Scientific), and CXCL9 (Fisher Scientific) ELISA kits were used according to manufacturer’s instructions to quantify protein expression. Luminex assays were performed using the ProcartaPlex Mouse immune monitoring panel, 48-plex (ThermoFisher Scientific) as per the manufacturer’s instructions. Supernatants from PK5/PK5L cells were collected after each cell line reached 70-90% confluency in a T-75 culture flask (2-3 days culturing). Luminex plates were read and analyzed on a MAGPIX instrument (ThermoFisher) with absolute protein concentrations (pg/ml) determined via a standard curve.

In vitro T cell differentiation

[0156] Naive CD4⁺ T cells were isolated from the spleens of 6-8-week-old BL/6 female mice and purified using the EasySep mouse naive CD4⁺ T cell isolation kit (StemCell Technologies) according to manufacturer’s instructions. Purity was confirmed by flow cytometry to be >94% by CD45⁺ CD4⁺ CD8' staining. One day prior to T cell differentiation, plates were coated with anti-CD3e (145-2C11, Biolegend) and anti-CD28 (37.51, Biolegend): Thl (5 pg/ml of each). Naive CD4⁺ T cells were then polarized for 3 days according to the following conditions: Thl (cytokines - IL-12 (5 ng/ml - Peprotech), IL-2 (250 U/ml - Millipore Sigma) and neutralizing antibodies - anti-IL-4 (10 pg/ml - 11B11, Invitrogen)). Differentiated Thl cells were then harvested for downstream analysis of activation by flow cytometry or in vitro migration assays.

In vitro migration assays

[0157] Primary mouse fibroblasts were cultured and treated in a monolayer as previously described. B cells were isolated from the spleens of 6-8-week-old BL/6 female mice and purified using the EasySep mouse B cell isolation kit (StemCell Technologies) according to manufacturer’s instructions. Purity was confirmed by flow cytometry to be >95% by CD90.2- CD19+ staining. 1 x 10⁵ B cells, naive CD8 T cells, or in vitro differentiated Thl cells were added to 3.0 pm Transwell filters (Corning) and allowed to migrate to the lower chamber containing a fibroblast monolayer for 24 hrs. Following migration, five independent and representative images of each well were taken using an inverted Rebel microscope (VWR). The number of migrated cells in each image was quantified in ImageJ (NIH - version 1.54d) using thresholding to eliminate background debris and size (pixels² - B cells (20-80) and Thl cells (20-120) to account for increase in size during activation) and circularity (0.4-1.0 for all cells) parameters to count cells of interest.

Immunohistochemistry

[0158] Standard staining procedures were utilized for IHC staining on 5-micron FFPE sections. Tissue was fixed overnight in 10% NBF prior to paraffin embedding. Primary antibodies for IHC included anti-mouse aSMA (ab 150301; Abeam), pSMAD2 (ab 188334; Abeam), and CD8 (14-0808-82; Invitrogen). Sequential IHC for all primary antibodies was performed with DAB using ImmPACT DAB substrate kit (#SK-4105; Vector Laboratories). Hematoxylin was used as a nuclear counterstain. Hematoxylin and eosin staining of mouse tissue sections was performed according to standard procedures. Whole slide scans of stained tissue sections were acquired with an Olympus VS200 digital scanner and images were analyzed in QuPath version 0.5.1. aSMA and staining was quantified as the percent area of highly stained pixels relative to total tumor area. CD8 staining was quantified as the number of positively stained cells relative to total cells detected in the tumor tissue area.

Bulk RNA sequencing of mouse and human PDAC tumors

[0159] RNA isolation and sequencing human PDAC tumors was performed as previously described. Mouse PK5 or PK5L tumors used for RNA-sequencing were implanted subcutaneously and harvested at day 21 post implant and snap frozen in liquid nitrogen. mRNA was isolated using a RNeasy plus mini kit (Qiagen) according to manufacturer’s instructions. RNA quality was determined on a nanodrop (ND- 1000) and the quantity of RNA was determined on Qubit 4 flourometer. To prepare for sequencing, samples were processed using an Illumnia TruSeqR Stranded mRNA Library Prep kit with Illumina TruSeq RNA Single Indexes Set A and Set B barcoding kits. An Illumnia NovaSeq 6000 SI Reagent Kit vl.5 was used to make RNA libraries along with a NovaSeq XP 2-Lane Kit vl.5. RNA libraries were sequenced on NovaSeq 6000. Demultiplexed fastq fdes for all samples were first processed with FastQC for general quality control. All the samples in the sequencing run passed read level QC with at least 17 million reads per sample. Average read depth across all samples were 51 million reads. Raw illumina BCL data was demultiplexed using Illumina bcl2fastq2 v2.20. Gene expression counts were quantified using salmon-v.1.1.0 76 for all samples sequenced. Differential gene expression analysis was performed using the R software package edgeR77.

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Claims

1. A method of treating a condition of a patient in need thereof, the method comprising: disposing a TGFb-receptor 1 inhibitor within a biological compartment of the patient; and disposing a lymphotoxin beta receptor (LTBR) agonist within the biological compartment of the patient.

2. The method of claim 1, wherein the step of disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient is carried out before the step of disposing the LTBR agonist within the biological compartment of the patient.

3. The method of claim 1 or claim 2, wherein the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient alleviates TGFb-mediated suppression of lymphocyte recruitment to reticular fibroblasts.

4. The method of any one of claims 1-3, wherein the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient overcomes resistance to a therapeutic effect of the LTBR agonist.

5. The method of any one of claims 1-4, wherein the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient overcomes resistance to generation of tertiary lymphoid structures in tumors of the patient.

6. The method of any one of claims 1-5, wherein the condition is a cancer.

7. The method of claim 6, wherein the cancer is pancreatic cancer.

8. The method of claim 7, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

9. The method of any one of claims 1-5, wherein the patient has a myofibroblastic cancer associated fibroblast (myCAF) phenotype.

10. The method of claim 9, wherein the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient reprograms the mCAF phenotype to a reticular cancer associated fibroblast (rCAF) phenotype.

11. The method of any one of claims 1-5, wherein the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient reverses suppression of an rCAF phenotype.

12. The method of any one of claims 1-5, wherein the disposing of the TGFb-receptor 1 inhibitor is disposed within the biological compartment of the patient at about 200 mg to about 400 mg daily.

13. The method of any one of claims 1-5, wherein the disposing of the LTBR agonist is disposed within the biological compartment of the patient at about 1 mg/kg to about 5 mg/kg.

14. The method of any one of claims 1-5, further comprising the step of disposing at least one chemotherapeutic compound within the biological compartment of the patient.

15. The method of claim 14, wherein the at least one chemotherapeutic compound is a combination of gemcitabine and nab-paclitaxel.

16. A composition for treating a condition of a patient in need thereof comprising: a lymphotoxin beta receptor (LTBR) agonist; and a TGFb-receptor 1 inhibitor.

17. The composition of claim 16, wherein the LTBR agonist is an anti-LTBR monoclonal antibody.

18. The composition of claim 16 or claim 17, wherein the composition is formulated for oral delivery or intravenous delivery.

19. The composition of any one of claims 16-18, wherein the LTBR agonist and the TGFb- receptor 1 inhibitor are admixed for simultaneous administration.

20. A method of inducing lymphocyte recruitment to a tumor, the method comprising: disposing a TGFb-receptor 1 inhibitor within a biological compartment of a patient; and disposing a lymphotoxin beta receptor (LTBR) agonist within the biological compartment of the patient.

21. A method of generating tertiary lymphoid structures (TLS) in a TLS-resistant tumor, the method comprising: disposing a TGFb-receptor 1 inhibitor within a biological compartment of a patient; and disposing a lymphotoxin beta receptor (LTBR) agonist within the biological compartment of the patient.

22. The method of claim 21, wherein the TLS-resistant tumor is a pancreatic ductal adenocarcinoma tumor.

23. The method of claim 21 or claim 22, wherein the disposing the TGFb-receptor 1 inhibitor within the biological compartment of the patient overcomes resistance to TLS generation by the LTBR agonist.