Methods of use of agents targeting mesenchymal stem cell- derived tumors and tumor associated cells

Abstract

The present disclosure provides various methods of using a radiotheranostic agent targeting LRRC15 expressed on cells derived from or associated with mesenchymal stem cells. The LRRC15 targeting agent improves treatment of a broad range of diseases and malignancies. In one embodiment, the methods using the LRRC15 targeting agent use a cytotoxic agent conjugated to DUNP19, a humanized monoclonal antibody that has high binding affinity to LRRC15 and is internalized by cells expressing LRRC15. In one embodiment, the antibody targeting LRRC15 is administered in combination with an immune checkpoint inhibitor.

Description

METHODS OF USE OF AGENTS TARGETING MESENCHYMAL STEM CELL-DERIVED TUMORS AND TUMOR ASSOCIATED CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application 63 / 624,083, filed January 23, 2024, which is incorporated here by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing conforming the rules of WIPO Standard ST.26 which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on January 15, 2025, is named “P-634894-PC_SQL_15JAN25.xml” and is 6,640 bytes in size.FIELD

[0003] The present disclosure relates generally to the field of disease therapy. In one embodiment, the present disclosure provides radiotheranostic agents that target LRRC15 expressed on cells derived from mesenchymal stem cells.BACKGROUND

[0004] Recent breakthroughs in immunobiotechnology have led to exciting therapeutic technologies and platforms using antibodies in the form of single-chain variable fragments, such as bispecific T-cell engagers (BiTE) and chimeric antigen receptors (CAR). However, most of these are only applicable to treat a limited number of solid tumors. The main factor contributing to therapeutic inefficacy and resistance is the tumor stromal compartment’ s ability to decrease infiltration of activated effector cells to the tumor bed. In addition, stromal cells actively orchestrate resistance through a myriad of other processes such as immune cell regulation, metabolic reprogramming, and hypoxia. The tumor stroma is comprised of a large array of epithelial, fibroblast, endothelial and inflammatory cell populations that coordinate together to regulate tumor growth and progression. Of these, cancer-associated fibroblasts (CAFs) are a unique cellular subset within the stroma due to their complex interaction with cancer cells. CAFs are a heterogeneous cell population with high degrees of cellular plasticity thought to arise from numerous cell types including resident fibroblasts, endothelial cells, cells undergoing epithelial-to-mesenchymal transition (EMT), and mesenchymal stem cells. This is a rapidly evolving area of tumor biology and many outstanding questions still remain regarding the origin, prevalence, and biological function of CAF populations across tumor types.Development of pharmaceuticals abating tumor specific immunosuppression have been focused on identifying targets that are specifically associated with cancer stroma, and the most coveted are plasma membrane proteins specific to CAFs. An absolute majority of these efforts have been targeting fibroblast activated protein a (FAP), a membrane bound serine protease overexpressed by CAFs. However, FAP is also expressed by normal tissues and healthy activated fibroblasts during wound healing, such as the activation of myofibroblasts post myocardial infarction. FAP-targeted therapeutic approaches (including antibody-drug conjugates, C AR-T cells and enzyme-inhibitors) have failed resulting in adverse effects or lack of efficacy due to low tumor specificity.

[0005] An alternative approach to pharmacologically regulate the immunosuppressive effects of the tumor stroma has been to target specific cytokines or regulatory pathway receptors. One of the leading endeavors is to attenuate the TGF-0 pathway, which supports the evasion of cancer cells from immune surveillance and contributes to the subversion of the immune system from acting as an extrinsic tumor suppressor to a promoter of malignant growth and invasiveness. Along with numerous effects on neoplastic cells and tumor stroma, multipronged effects of TGF-0 on immune cells shape the cancer microenvironment. For example, TGF-0 activity dictates several aspects of T-cell fate decisions, NK cell functions and y5 Tregs. In addition, crosstalk between the canonical TGF-0 signaling (SMADs) and several non-canonical pathways including MAPK, PI3K, WNT, HH, and NOTCH has been described. TGF-0 inhibitors primarily exert their antitumor activity by affecting fibroblastic and endothelial cell growth as well as T-cell activation in the tumor microenvironment. Of the TGF-0 pathway targeting drugs, small molecule TGF-0 receptor inhibitors such as galunisertib (LY2157299) have shown the most promising results. Unfortunately, these inhibitors have been impeded by a narrow therapeutic window and toxic effects. Therefore, non-invasive biomarkers for patient selection and dose titration are currently needed in order to fully utilize TGF-0 receptor inhibitors.

[0006] LRRC15 is a 581 amino acid type I membrane protein with an N-terminal transmembrane domain, 15 Leucine-rich repeats (LRRs), and a short C-terminal cytoplasmic tail. In general, proteins containing LRRs can have a wide variety of functions, including innate immunity, inflammation and nervous system development. The specific physiological function of LRRC15, a highly conserved molecule, is largely unknown and understudied but is likely impacting cell-matrix adhesion and cell migration. LRRC15 is governed by TGF-0, which has a leading role in tissue healing and immune regulation. The normal tissues that express LRRC15are sites where TGF-P is reported to be present and where mesenchymal stem cells (MSCs) are known to reside, such as hair follicles, tonsil and sites of wound healing. Bone marrow and adipose derived MSCs, and to some extent umbilical cord derived MSCs, express LRRC15 under TGF-P stimulation. This feature of TGF-P governance is unique to LRRC15 compared to other mesenchymal associated markers such as FAP. Studies have shown that LRRC15 impacts osteogenesis through MSC differentiation and is a highly upregulated gene in focal erosions of rheumatic arthritis. Interestingly, LRRC15 expression also impedes adenovirus infection, supporting evidence for the protein’s role as an important immune regulator.

[0007] LRRC15 is highly expressed on CAFs within the tumor stroma of a wide range of malignancies, as well as directly on cancer cells from a subset of mesenchymal tumors (e.g., sarcomas, glioblastoma multiforme). LRRC15+CAFs emerge from a LRRC15+fibroblast population as a result of TGF-P activity. Compared to FAP, LRRC15 has a significantly lower baseline expression and a higher differential RNA expression between cancer and adjacent normal tissues. In pancreatic adenocarcinoma (PDAC), LRRC15+CAFs specifically surround tumor islets, while absent from normal pancreatic tissue. Preclinical and clinical studies evaluating genes associated with metastatic progression have identified LRRC15 to be among the top genes expressed in metastasis. LRRC15 has been shown to promote metastatic spread to bone and bowel in breast and ovarian cancer patients, respectively, while knockdown by siRNA significantly inhibited progression in preclinical models. Overexpression has also been associated with aggressive behavior in metastatic lesions of androgen independent metastatic prostate cancer. Importantly, analysis of tissues obtained from clinical trials show that high avidity of LRRC 15+CAFs is associated with resistance to immune checkpoint blockade.

[0008] There is an unmet need for non-invasive tools for identifying and treating molecular events and cellular phenotypes associated with immunotherapy resistance mechanisms. Specific eradication of the tumor stroma and pharmacological manipulation of the TGF-P pathway have both been exploited as monotherapies or in combination with immunotherapies to pharmacologically alleviate resistance. However, previous compounds designed to eliminate the stroma has been unsuccessful due to off-target expression, while compounds decreasing TGF- activity are rarely applicable due to narrow therapeutic windows. Radiotheranostic compounds for molecularly precise eradication of tumor associated stroma and non-invasive imaging of downstream TGF-P activity could be particularly useful for curbing resistance mechanisms and ameliorating the therapeutic potential of modem immunotherapeutic compounds.SUMMARY

[0009] In one aspect, a method is provided for treating a tumor in a patient comprising administering to the patient a combination of: a. a binding moiety comprising SEQ ID NO:1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent; and b. an immune checkpoint inhibitor.

[0010] In some embodiments, the tumor and / or stroma thereof expresses LRRC15. In some embodiments, the tumor and / or stroma thereof produces TGFp. In some embodiments, the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma. In some embodiments, the prostate cancer is androgen receptor negative or androgen independent. In some embodiments, the tumor is metastatic. In some embodiments, the tumor comprises stroma which comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

[0011] In some embodiments, the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR). In some embodiments, the binding moiety has picomolar affinity for LRRC15. In some embodiments, the binding moiety is an antibody. In some embodiments, the antibody binds to a mammalian LRRC15. In some embodiments, the antibody is a humanized monoclonal antibody. In some embodiments, the monoclonal antibody is an afucosylated antibody or a low-fucose variant thereof.

[0012] In some embodiments, the cytotoxic agent is a therapeutic radionuclide. In some embodiments, the radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. In some embodiments, the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium. In some embodiments, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. In some embodiments, the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine. In some embodiments, the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium. In some embodiments, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, ataxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

[0013] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD- Ll, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and various B-7 family ligands. In some embodiments, the inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NEG 919, INCB024360, PF-05082566, urelumab or MEDI6469. In some embodiments the immune checkpoint inhibitor is a combination of two or more immune checkpoint inhibitors, such as a CTLA-4 inhibitor and a PD-1 inhibitor.

[0014] In some embodiments, the tumor or stroma thereof exhibits an immunotherapy resistant signature. In some embodiments, the signature comprises one or more of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and / or Prfl. In some embodiments, the signature In some embodiments, LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl. In some embodiments, the signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl.

[0015] In some embodiments, the combination is synergistic in treating the tumor.

[0016] In one aspect, a method is provided for increasing susceptibility of an immunotherapy resistant tumor in a patient to immunotherapy comprising exposing the tumor or tumor stroma to a binding moiety comprising SEQ ID NO:1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent. In one embodiment, the tumor or stroma thereof exhibits an immunotherapy resistant signature. In one embodiment, the signature comprises one or more of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and / or Prfl. In one embodiment, the signature comprises LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl. In one embodiment, the signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl.

[0017] In one embodiment, the resistance to immunotherapy is resistance to immune checkpoint blockade. In one embodiment, the immune checkpoint is CTLA-4, PD-L1 , PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and various B-7 family ligands.

[0018] In one embodiment, the tumor and / or stroma thereof expresses LRRC15. In one embodiment, the tumor and / or stroma thereof produces TGFp. In one embodiment, the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma. In one embodiment, the prostate cancer is androgen receptor negative or androgen independent. In one embodiment, the tumor is metastatic. In one embodiment, the tumor comprises stroma which comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

[0019] In one embodiment, the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR). In one embodiment, the binding moiety has picomolar affinity for LRRC15. In one embodiment, the binding moiety is an antibody. In one embodiment, the antibody binds to a mammalian LRRC15. In one embodiment, the antibody is a humanized monoclonal antibody. In one embodiment, the monoclonal antibody is an afucosylated antibody or a low-fucose variant thereof.

[0020] In one embodiment, the cytotoxic agent is a therapeutic radionuclide. In one embodiment, radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. In one embodiment, the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium. In one embodiment, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. In one embodiment, the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine. In one embodiment, the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium. In one embodiment, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

[0021] In one embodiment, the patient is subsequently treated with an immunotherapeutic agent.

[0022] In one embodiments, the exposing further comprises exposing the tumor or stroma to an immunotherapeutic agent. In one embodiments, the exposing to the binding moiety and theimmunotherapeutic agent is synergistic in increasing susceptibility.

[0023] In one embodiment, the immunotherapeutic agent is at least one immune checkpoint inhibitor, T cell therapy, cytokine therapy, chemokine therapy, immunomodulatory drug therapy, monoclonal antibody therapy, or any combination thereof. In one embodiment, the at least one immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-L1 , PD-L2, PD1 , B7- H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN- 15049, CHK 1 and CHK2 kinases, A2aR, or a B-7 family ligand. In one embodiment, the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF-05082566, urelumab, MEDI6469 or any combination thereof.

[0024] In one embodiment, the patient is subsequently treated with an immunotherapeutic agent. In one embodiment, the immunotherapeutic agent is at least one immune checkpoint inhibitor, T cell therapy, cytokine therapy, chemokine therapy, immunomodulatory drug therapy, monoclonal antibody therapy, or any combination thereof. In one embodiment, the at least one immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-L1, PD-L2, PD1, B7- H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN- 15049, CHK I and CHK2 kinases, A2aR, or various B-7 family ligands. In one embodiment, the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF-05082566, urelumab, MEDI6469 or any combination thereof.

[0025] In one aspect, a method is provided for depleting TFGP expressing cells in a tumor or tumor microenvironment comprising exposing the tumor or tumor microenvironment to a binding moiety comprising SEQ ID NO:1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent. In one embodiment, the TFGP expressing cells are tumor cells. In one embodiment, the TGFp expressing cells are tumor stoma cells. In one embodiment, the tumor and / or stroma thereof expresses LRRC15. In one embodiment, the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, ormelanoma. In one embodiment, the prostate cancer is androgen receptor negative or androgen independent. In one embodiment, the tumor is metastatic. In one embodiment, the stroma comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

[0026] In one embodiment, the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR). In one embodiment, the binding moiety has picomolar affinity for LRRC15. In one embodiment, the binding moiety is an antibody. In one embodiment, the antibody binds to a mammalian LRRC15. In one embodiment, the antibody is a humanized monoclonal antibody. In one embodiment, the monoclonal antibody is an afucosylated antibody or a low-fucose variant thereof.

[0027] In one embodiment, the cytotoxic agent is a therapeutic radionuclide. In one embodiment, the radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. In one embodiment, the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium. In one embodiment, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. In one embodiment, the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine. In one embodiment, the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium.

[0028] In one embodiment, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

[0029] In one embodiment, the exposing further comprises exposing the tumor or stroma to an immunotherapeutic agent. In one embodiment, the combination is synergistic in depleting TFGP expressing cells. In one embodiment, the immunotherapeutic agent is at least one immune checkpoint inhibitor, T cell therapy, cytokine therapy, chemokine therapy, immunomodulatory drug therapy, monoclonal antibody therapy, or any combination thereof. In one embodiment, the at least one immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, or various B-7 family ligands. In one embodiment, the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559,atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF- 05082566, urelumab, MEDI6469 or any combination thereof.

[0030] In one aspect, a method is provided for treating a tumor in a patient comprising administering to the patient a combination of: a. a bispecific or trispecific targeting molecule that binds to LRRC15 and TGFp, said targeting molecule comprising at least one occurrence of amino acid sequences SEQ ID NO:1 and SEQ ID NO:2, and at least one occurrence of a targeting sequence to TGFP; and b. an immune checkpoint inhibitor.

[0031] In one embodiment, the targeting molecule comprises amino acid sequences SEQ ID NO:1 and SEQ ID NO:2, and amino acid sequences SEQ ID NO:3 and SEQ ID NO:4. In one embodiment, the targeting molecule comprises one occurrence of amino acid sequences SEQ ID NO:1 and SEQ ID NO:2, and two occurrences of amino acid sequences SEQ ID NO:3 and SEQ ID NO: 4. In one embodiment, the targeting molecule comprises two occurrences of amino acid sequences SEQ ID NO: 1 and SEQ ID NO:2, and one occurrence of amino acid sequences SEQ ID NO:3 and SEQ ID NO:4. In one embodiment, the targeting molecule is humanized. In one embodiment, the targeting molecule is afucosylated or a low-fucose variant thereof.

[0032] In one embodiment, the bispecific or trispecific targeting molecule is associated with a cytotoxic agent. In one embodiment, the cytotoxic agent is a therapeutic radionuclide. In one embodiment, the therapeutic radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. In one embodiment, the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium. In one embodiment, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. In one embodiment, the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine. In one embodiment, the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium. In one embodiment, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

[0033] In one embodiment, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-LI, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and / or a B-7 family ligand. In one embodiment, the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF-05082566, urelumab or MEDI6469. In one embodiment, the combination is synergistic in treating the tumor.

[0034] In one aspect, a method is provided for inducing dormancy in a LRRC15-expressing tumor in a patient comprising exposing the tumor or stroma thereof to a binding moiety comprising SEQ ID NO:1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent. In one embodiment, the tumor and / or stroma thereof produces TGFp. In one embodiment, the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma. In one embodiment, the prostate cancer is androgen receptor negative or androgen independent. In one embodiment, the tumor is metastatic. In one embodiment, the tumor comprises stroma which comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

[0035] In one embodiment, the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR). In one embodiment, the binding moiety has picomolar affinity for LRRC15. In one embodiment, the binding moiety is an antibody. In one embodiment, the antibody binds to a mammalian LRRC15. In one embodiment, the antibody is a humanized monoclonal antibody. In one embodiment, the monoclonal antibody is an afucosylated antibody or a low-fucose variant thereof.

[0036] In one embodiment, the cytotoxic agent is a therapeutic radionuclide. In one embodiment, the therapeutic radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. In one embodiment, the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium. In one embodiment, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. In one embodiment, the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine. In one embodiment, the gamma-ray emitter is selected fromthe group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium. In one embodiment, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

[0037] In one embodiment, the exposing is in combination with an immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD- Ll, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and / or a B-7 family ligand. In one embodiment, the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF-05082566, urelumab or MEDI6469. In one embodiment, the combination is synergistic in inducing dormancy of the tumor.

[0038] In one embodiment, the tumor or stroma thereof exhibits an immunotherapy resistant signature. In one embodiment, the signature comprises one or more of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and / or Prfl. In one embodiment, the signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl.

[0039] These and other aspects of the disclosure will be appreciated from the ensuing descriptions of the Figures and Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figures 1A-1F depict in vitro evaluation of DUNP19’s binding profile to LRRC15. Figure 1A. Flow cytometry demonstrates that DUNP19 binds with picomolar affinity to LRRC15 molecules on various cell lines, exhibiting distinct expression levels and tissue origins. Detection of LRRC15 was based on the assessment of antigen- antibody equilibrium. Figure IB. Analysis across a diverse range of cell lines unveils correlations between LRRC15 mRNA expression and the abundance of LRRC15 molecules bound by DUNP19. Figure 1C. Confocal microscopy of various cell lines incubated with AlexaFluor647-labeled DUNP19 at room temperature, followed by staining for plasma membrane-associated calcium ATPase (PCMA) and DNA (DAPI). Images reveal that DUNP19 binding corresponds with LRRC15 expression levels and co-localizes with LRRC15. Figure ID. Internalization rates of AlexaFluor647-labeled DUNP19 at 37°C were examined in LRRC 15 -expressing HuO9 andSA0S2 cells using live confocal microscopy. The endocytic process of DUNP19 is accelerated in cells with higher LRRC15 abundance. Figure IE. Confocal microscopy of SAOS2 cells incubated with AlexaFluor647-labeled DUNP19 at 4°C or 37°C, co-stained for lysosomes (LAMP1) and DNA (DAPI). DUNP19 is exclusively found in the plasma membrane at the lower temperature demonstrating that the rapid endocytosis after binding to LRRC15 is an active, energy -requiring process. Figure IF. LigandTracer sensorgram of [177Lu]-DUNP19 binding to LRRC 15 -expressing Hu09 cells, measured at 1 nM and 3 nM concentrations. Cellbound activity, presented as CPS, was used to determine association, dissociation rates, and equilibrium dissociation constants (KD). The table shows KD values for various LRRC15- expressing cell lines.

[0041] Figures 2A-2G depict the specificity and biodistribution of DUNP19 in multiple mouse models with LRRC 15 expressing tumors. Figure 2A. Representative PET images of s.c. SAOS2 osteosarcoma xenografts obtained at different time points post i.v. administration of [^CuJ-DUNP , highlighting significant tumor-specific uptake with minimal accumulation in normal tissues. In contrast, PET with the clinical bone scanning agent [18F]-NaF showed low activity in tumor tissue, with the majority of the tracer dose observed in bone (Bn) and bladder (Bl). Figure 2B. In vivo assessment of LRRC15 targeting specificity by [177Lu]-DUNP19. At 48 h post i.v. injection, [177Lu]-DUNP19 displayed significantly higher uptake (p < 0.001) in LRRC 15+ U118MG (blue bar) and HuO9 (red bar) tumors compared to LRRC 15-negative LNCaP tumors (light gray bar). The accumulation of non-specific [177Lu]-IgGl in LRRC15+ U 118MG tumors (dark grey bar) was significantly lower than that of [177Lu]-DUNP19. Figure 2C. |177LU]-DUNP19 tumor uptake in multiple s.c. tumor models at 72 h p.i. , correlating with the LRRC15 expression level in the respective model. Figures 2D, 2E. Kinetics of [177Lu]- DUNP19 in healthy organs and LRRC15+ SAOS2 and HuO9 osteosarcoma lesions. Ex vivo tissue biodistributions of [177Lu]-DUNP19 obtained at multiple time points after i.v. injection showed a continuous decline in activity levels in healthy organs, but sustained uptake by malignant lesions. Figures 2F, 2G. Microanatomy of tumor tissues obtained from animals treated with fluorescently-labeled DUNP19. Confocal images of s.c. SAOS2 (LRRC15+ cancer cells / LRRC15+ CAF) and HCC1954 (LRRC15- cancer cells / LRRC15+ CAF) tumors harvested at 72 h post- i.v. injection of AF594-DUNP19 (yellow). Tumor sections were costained for Actin (red), DNA (DAPI, blue) and LAMP1 (lysosomal marker, green). Images show that DUNP19 accumulates in the cellular cytoplasm and co-localized with LAMP1 indicating intracellular trafficking of the mAb to the lysosomal compartments (arrow) afterbinding membranous LRRC15.

[0042] Figures 3Ai-3Cv depict the evaluation of [177Lu]-DUNP19 therapy and monitoring in HuO9 osteosarcoma tumors. Figure 3AI-II. Tumor volumes in mice bearing s.c. HuO9 xenografts were randomized for a single i.v. administration of 30 MBq [177Lu]-DUNP19 when tumors reached 203+64 mm3 (blue line; day 21) or 504+152 mm3 (red line; day 39), or received no treatment (black line). The results demonstrated a significant delay in disease progression, with treatment efficacy being influenced by tumor volume (Figure 3Ai). Kaplan- Meier survival analysis revealed that [177Lu]-DUNP19 extended survival, with the impact varying based on the timing of intervention (Figure 3An). Figures 3Bi-n. Representative coronal SPECT / CT images showing orthotopic HuO9 osteosarcoma tumors (indicated by arrow) after initial (left) and follow-up (right) i.v. administrations of 20 MBq [177Lu]-DUNP19. In all treated mice (right), no tumor associated uptake was observed at 163 days after treatment (Figure 3Bi). Kaplan-Meier survival analysis revealed a significant increase in survival for the [177LU]-DUNP19 treated group during the observed 190-days period (Figure 3BII). Figures 3Ci-v. Mice with s.c. HuO9 osteosarcoma xenografts were randomized for three treatment cycles (red: 10+20+10 MBq; blue: 20+10+20 MBq) of i.v. [177Lu]-DUNP19 (at day 0, 32, and 75), resulting in a total administered activity of 50 MBq, or no treatment (black). Assessment of tumor volumes demonstrated that repeated cycles of LRRC15-RIT effectively inhibit tumor growth (Figure 3Ci). Kaplan-Meier survival analysis confirmed significantly improved survival for mice randomized for [177Lu]-DUNP19 over no treatment (Figure 3Cn). Four tumors from the treatment and control (non-treatment) arm, harvested 72 hours after administration of an imaging dose of |177Lu|-DUNP19 (3 MBq), were imaged ex vivo by SPECT and CT (Figure 3Cm). Tissue activity levels (%IA / g), assessed by gamma counter and normalized to tissue weight, revealed significantly lower uptake of the antibody in treated vs. non-treated tumors (p < 0.001), reflecting reduction in total LRRC15-expressing cells posttreatment (Figure 3Crv). Quantification of radiopacity in CT images showed significantly higher ossification levels in treated vs. non-treated tumor tissues (p < 0.001) (Figure 3Cv). Together, these findings illustrate that repeated cycles of [177Lu]-DUNP19 effectively reduce tissue viability and calcification.

[0043] Figures 4A-4D depict the therapeutic efficacy of [177Lu]-DUNP19 in LRRC15- expressing human xenograft models. Figures 4A, 4B. [177Lu]-DUNP19 demonstrates antitumor activity in other cancer indications. BALB / c nude mice bearing s.c. U118MG glioblastoma xenografts were treated with two fractions of [177Lu]-DUNP19 at days 0 and 34for a cumulative activity of 20 MBq (10+10 MBq, red) or 30 MBq (20+10 MBq, blue). Despite lower LRRC15 expression by U118MG tumors, treatment with [177Lu]-DUNP19 significantly controlled tumor growth and prolonged survival in both [177Lu]-DUNP19 doses (median survival; untreated = 74 days, 20 MBq = not reached, 30 MBq = not reached, p < 0.001). Figures 4C, 4D. [177Lu]-DUNP19 is effective in HCC1954 breast cancer (LRRC15-null cancer cells, LRRC15+ stroma). Results demonstrate delayed s.c. HCC1954 growth in female mice intravenously administered a single dose of [177Lu]-DUNP19 (20 MBq; day 7). Figure 4D. Median survival was not reached for the treated group by the end of the observation period (day 62), while median survival of treated mice was 30.5 days (p < 0.005).

[0044] Figures 5A-5F depict [177Lu]-DUNP19 induced radio-immunotherapy signatures in LRRC15+ cancer cells. Figure 5A. Schematic of transcriptomic analysis of Hu09, U118MG, and HCC1954 tumors after [177Lu]-DUNP19 treatment. Treated or untreated tumor samples were harvested for RNA isolation, before sequencing and alignment to either murine or human genomes. Overlapping or ambiguous reads were discarded. Figure 5B. Volcano plot of the top up- (red) and downregulated (blue) DEGs after treatment with [177Lu]-DUNP19 in U118MG (left) and Hu09 (right) cancer cells (FDR < 0.05). DEGs were ranked by fold-change. The top and bottom genes were labeled. Figures 5C, 5D. Gene ontology (GO) biological pathway enrichment analysis of DEGs in treated (Figure 5C) U118MG and (Figure 5D) Hu09 cancer cells (adjusted p-value < 0.05). Enriched biological pathways with more than 10 overlapping terms (genes) were plotted by Padj value to indicate processes most significantly enriched after [177Lu]-DUNP19 RIT. Figures 5E, 5F. Overlapping differentially expressed genes (40 genes, FDR < 0.05, FC > 1 ) in (Figure 5E) Ul i 8MG and (Figure 5F) HuO9 cancer cells after 1177Lu |- DUNP19 treatment were plotted for visualization of [177Lu]-DUNP19-induced changes. Relative expression per gene was plotted to indicate up- (in red) or downregulated (blue) genes by Z-score normalization.

[0045] Figures 6A-6C depict [177Lu]-DUNP19 therapy eradicates a LRRC15+ TGFf) signature associated with immunotherapy resistance. DEGs overlapped in [177Lu]-DUNP19- treated stroma from U1 18MG (Figure 6A, 26 genes), Hu09 (Figure 6B, 23 genes), and HCC1954 (Figure 6C, 26 genes) tumors. Relative expression (Z-score normalization) was plotted to indicate upregulated (red) or downregulated (blue) genes.

[0046] Figures 7A-7F show that DUNP19 binds to human and murine LRRC15 protein. Figure 7A. DUNP19 binding kinetics to human (left) and murine (right) LRRC 15 recombinant protein using the Bio-Layer Interferometry Octet system. Association of lOOug / mL DUNP19(pink trace) or lOOug / mL IgGl isotype control (green trace) are shown in real time over 850 s. Association and dissociation of DUNP19 to hLRRC15 or mLRRC15 is measured by binding rate (nm). Figure 7B. The 1 1 unique proteins present after an immunoprecipitation-mass spectrometry (IP-MS) analysis of crude protein lysates from Ul i 8MG cells incubated with Protein G magnetic bead-conjugated DUNP19. Pulldown-MS composition was compared to protein composition present in non-specific IgGl with U118MG lysates. LRRC15 is among the top proteins in complex with DUNP19. Figures 7C. Box-and-whisker plots representing relative transcript expression of LRRC15 (top) and TGFB1 (bottom), comparing untreated tumors to tumors after [177Lu]-DUNP19 therapy. Hu09 transcripts are plotted in red (left), U118MG in blue (middle), and HCC 1954 in black (right). Samples were separated by transcript signature based on PC A plots and hierarchical clustering into 2 (HCC 1954) or 3 (U118MG, Hu09) clusters. Expression of LRRC15 and TGFB1 in treated samples from cluster 3 are significantly (p<0.005) decreased in U118MG and Hu09, while no changes are observed in the LRRC15- HCC1954 cancer cells. Figure 7D. Consistently, in two mice with HCC1954 tumors which progressed despite [177Lu]-DUNP19 RIT, neither Lrrcl5 nor the LRRC15+ CAF gene-signature decreased. Figures 7E and 7F. Transcript data from clustered cancer cells (6E) or tumor stroma (6F) show decreased expression of the LRRC15+ TGF|3 signature. E. Untreated U118MG (top) and Hu09 (middle) cancer cells lose expression of the LRRC15+ TGFP signature after [177Lu]-DUNP19 treatment (red = high, blue = low expression). F. Loss of the LRRC15+ TGF[3 signature is observed across all tumor stroma after [177Lu]-DUNP19 RIT (green = high, orange = low expression) G. HCC 1954 tumors that were resistant to [177Lu]- DUNP19 treatment (defined as reaching 1000m3 endpoint before conclusion of study) had no significant reduction of the 11 -gene LRRC15+ TGFP signature within tumor stroma.

[0047] Figures 8A-8B depict live confocal microscopy-based assays to determine internalization kinetics of DUNP19 after chelator conjugation and in various cell types. Figure 8A. AF647-DUNP19 or AF647-DUNP19-DOTA conjugate internalize into U118MG cells at similar rates. To determine internalization rate, images were taken every hour across 12 h at 37C. Time to 50% internalization (Tl / 2) of AF647-DUNP19 (measured by ratio of cytosolic intensity of AF647-DUNP19 compared to membrane integrated intensity) was 5.16 + 0.83 h. The AF647-DUNP19-DOTA conjugate internalized at a similar rate, with a Tl / 2 of 5.75 ± 0.93 h. Figure 8B. Internalization of AF647-DUNP19 in fibroblast-derived Hs819.T cells compared to osteosarcoma SAOS2 cancer cells. To determine Internalization rate was determined by an 8 h microscopy-based assay with images were taken every 10m at 37C. Time to 50%internalization (Tl / 2) of AF647-DUNP19 in SAOS2 cells was 1.31 ± 0.14 h. Tl / 2 internalization of AF647-DUNP19 into Hs819.T cells was significantly slower with a Tl / 2 of 1.66 + 0.09 h.

[0048] Figure 9 depicts expanded tumor biodistribution of [177Lu]-DUNP19 in U118MG, SAOS2, Hu09, K7M2LRRC15+ and HCC1954 xenograft models across 6 timepoints; 6h, 24h, 48h, 72h, 168h, and 336h. Tumor accumulation of [177Lu]-DUNP19 peaks between 48-72 h for all models and is represented as %IA / g (percent injected activity per gram tissue). At 72h, tumoral accumulation is as follows; U118MG: 14.31 ± 2.01 %IA / g, SAOS2: 23.07 ± 2.90 %IA / g, Hu09: 43.93 + 7.89 %IA / g, K7M2LRRC15+: 13.60 +1.49 %IA / g, HCC1954: 11.83 + 2.50 %IA / g.

[0049] Figures 10A-10B show IHC and IF analysis of ex vivo LRRC15+ tumor models. Figure 10A. Ex vivo immunohistochemistry analysis of HuO9, K7M2LRRC15+, and K7M2 wildtype tumor sections harvested from untreated animals. Images are stained for LRRC15 and counterstained with hematoxylin, shown at 4x and 20x. Figure 10B. Confocal microscopy of tumor tissues obtained from animals treated with fluorescently labeled DUNP19. Confocal images of s.c. HuO9 (LRRC15+ cancer cells / LRRC15+ CAF) tumors harvested at 72 h post- i.v. injection of AF594-DUNP19 (yellow). Tumor sections were co-stained for Actin (red), DNA (DAPI, blue) and LAMP1 (lysosomal marker, green). Images show that DUNP19 accumulates in the cellular cytoplasm and co- localized with LAMP! indicating intracellular trafficking of the mAb to the lysosomal compartments (arrow) after binding to LRRC15.

[0050] Figures 11A-11C depict animal weights across tumor models (11A: HuO9, 11B: U118MG, 11C: HCC1954) corresponding to therapy studies in Figure 3 (HuO9), and Figure 4A-4B (U118MG), and 4C-4D, HCC1954). Gray arrows denote administration of [177Lu]- DUNP19 or PBS. Overall, weight was stable throughout [177Lu]-DUNP19 therapy studies across tumor models.

[0051] Figures 12A-12C depicts toxicity studies examining blood cell counts during treatment course across tumor models (12A: Hu09, 12B: U118MG, 12C: HCC1954). HuO9, U118MG and HCC1954 exhibited transient reductions in lymphocytes and monocytes after administration of [177Lu]-DUNP19, which recovered to baseline levels within 3 weeks, recapitulating toxicity profiles observed in clinical cases.

[0052] Figure 13 depicts principal component analysis (PCA) plots of whole transcriptome cancer cell reads (left) and stromal cell reads (right). PCI and PC2 show separation of treatedsamples in distinct clusters that were determined to correlate with LRRC15 transcript responses to [177LU]-DUNP19 RIT. In Hu09 and HCC1954 tumor models, treated cancer cells and stromal cells from the same sample clustered in similar patterns. U 118MG and Hu09 treated tumors were grouped into three distinct clusters, whereas HCC1954 treated tumors formed two clusters that were differentiated from untreated mice.

[0053] Figure 14 shows relative cell characterization of individual tumor samples using Syllogist. Overall, 43 cell types were analyzed and relative expression was averaged into seven summary cell types as shown (Germ / ESC, Neural / Glial, Mesenchymal, Endothelial, Epithelial, HSPC, Myeloid, Lymphoid). Normalized relative expression of cell types is presented on a scale of 0 (underrepresented cell type / not present in sample) to 1 (overrepresented cell type / present in sample). U118MG and Hu09 cancer cells were characterized as mesenchymal phenotypes, whereas HCC1954 cancer cells were majority epithelial (left plots), in line with relative LRRC15 cancer cell expression between the three models. In stromal cells, no significant cell phenotype was observed in any of the three models.

[0054] Figure 15A-15B depict stromal cell expression of LRRC15 and TGFpi. Figure 15A. Transcript data from clustered (Fig.13) tumor stroma of Lrrcl5 (top) and TGFpi (bottom) relative expression, represented by box-and-whisker plots. Hu09 transcripts are plotted in green (left), U118MG in orange (middle), and HCC1954 in grey (right). In HuO9 and U118MG stromal cells, expression of TGFpi is significantly reduced in cluster 3 (p<0.05). Across tumor models, changes in Lrrcl 5 transcript expression were not significant between treated and untreated samples. Figure 15B. Comparison of relative baseline transcript expression of Lrrc 15 in tumor stroma, comparing HCC1954 (right), Hu09 (middle) and U118MG (left). In HCC1954 tumors, median stromal Lrrcl5 expression is high (8.91) compared to moderate expression in Hu09 (3.02) and U118MG (3.48).

[0055] Figure 16 depicts growth curves (mean tumor volume, mm3) of s.c. 4T 1 tumors in mice receiving control (PBS) [triangles], non-labelled mDUNP19 [diamonds], one dose i.v. 177Lu- DUNP19 [circles], i.p. ICI treatments (anti-CTLA4 + anti-PD-1) [squares], and i.v. 15MBq 177Lu-DUNP19 combined with i.p. ICI treatments (anti-CTLA4 + anti-PD-1) [inverted triangles].DETAILED DESCRIPTION

[0056] The tumor stroma plays a critical role in tumor initiation, progression and resistance to therapeutic compounds as well as inherent immunological defense mechanisms. Leucine-richrepeat-containing protein 15 (LRRC15) is a TGF- P governed protein that is expressed by cancer-associated fibroblasts (CAFs) and by a subset of cancer cells of mesenchymal origin. Importantly, expression is coupled to poor response to checkpoint blockade in several tumor types. The present disclosure presents radiotheranostic applications based on a recently developed monoclonal anti-LRRC15 antibody (mAb DUNP19; described in PCT / US2021 / 025054 and published as WO2021 / 202642) that is specifically internalized by cells expressing LRRC15. LRRC15-targeted radiotheranostics (such as DUNP19 antibody) could significantly improve treatment of a broad range of malignancies. Previously, the efficacy of LRRC15-RIT had been demonstrated using tumor models expressing LRRC15 in both cancer cells and tumor stroma (typically found in glioblastomas, sarcomas, and malignant melanomas). In one embodiment, treatment effects are further demonstrated in a tumor model expressing LRRC15 exclusively in the tumor stroma, which is a more prevalent scenario. This significantly broadens the potential treatment applications, particularly in, by way of nonlimiting examples, aggressive breast, ovarian, lung, bladder, head and neck, and pancreatic cancers.

[0057] Thus, the present disclosure addresses a fundamental unmet need in clinical management of several late-stage and disseminated cancers by employing a unique strategy targeting LRRC15, a TGF-P governed biomarker specifically overexpressed in mesenchymal stem cell-derived cancer cells and tumor stroma, in one embodiment in combination with immune checkpoint inhibition. Preclinical results obtained in vitro and in rodent models demonstrate intracellular delivery of this LRRC 15 -directed platform. In one embodiment, the present disclosure describes therapeutic utilities of |89Zr]DUNP19, |225Ac|DUNP19 and [177Lu]DUNP19. In one embodiment, combination therapy with [177Lu]DUNP19 and immune checkpoint inhibitors shows greater efficacy against a tumor than either therapy separately. Such combination may enhance or synergize efficacy. In other embodiments, factors that govern the magnitude and distribution of the a- and P-particle emitting constructs can be evaluated. These studies will significantly advance radio-theranostic approaches toward clinical application for sustained treatment and monitoring of several late stage malignant diseases.

[0058] For decades, an absolute majority of cancer treatments have focused on targeting malignant cells without consideration of the tumor microenvironment (TME) and its unique role in tumor maintenance and progression. Aggressive malignancies such as pancreatic adenocarcinoma, glioblastoma multiforme (GBM) and osteosarcoma rely on the TME for tumor growth and maintenance, presenting a unique opportunity to target this criticalinteraction for therapeutic benefit. An integral component of the TME is the tumor stroma, a collection of fibroblasts, mesenchymal and epithelial cells vital to tumor progression. The present disclosure identifies new uses for a binding moiety targeting the tumor- specific membrane protein leucine-rich repeat containing 15 (LRRC 15) as a highly upregulated protein across a wide range of malignancies of mesenchymal origin and in cancer-associated fibroblasts (CAFs). LRRC 15 is thought to play a role in cell migration, cell adhesion and is highly associated with immunological response and resistance to immunotherapy. Additional studies identify its expression on tumor stroma, and in some embodiments, only or predominantly by the tumor stroma.

[0059] The present disclosure describes new uses for the anti-LRRC15 antibody DUNP19, a humanized monoclonal antibody (mAb) that is rapidly internalized into target cells by binding to LRRC15 with high specificity and picomolar affinity (3.29e -10M). The DUNP19 mAb, or its derivatives, can be used as a radiotherapeutic. Targeting tumor and stromal cells expressing LRRC 15 via DUNP19 provides novel radio-theranostic strategies for currently untreatable malignancies, and as described herein, in combination with one or more immune checkpoint inhibitors increases effectiveness. Targeting tumor stromal cells expressing LRRC 15 via DUNP19 in combination with immune checkpoint inhibition provides novel radio-theranostic strategies for currently untreatable malignancies.

[0060] In another embodiment, the tumor or stroma thereof exhibits an immunotherapy resistant signature that is depleted by LRRCL5-targeted therapy such as with DUNP19 having binding moieties comprising SEQ ID NO:1 and SEQ ID NO:2. Such therapy may be combined with immune checkpoint blockade to enhance or synergize effectiveness. Such signature may be determined, for example, by biopsy of the tumor and assessing transcriptomic signatures using any method, such as but not limited to those described herein. In one embodiment, the gene signature comprises one or more of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and / or Prfl. Such signature may comprise any two, three, four, five, six, seven, eight, nine or ten of the foregoing genes. In one embodiment, the gene signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl. Depletion of this signature, along with other genes related to immunotherapy resistance, is achieved through LRRC 15 -targeted RIT. Increased susceptibility to immunotherapy is provided in some embodiments with concurrent or subsequent therapy with one or more immune checkpoint inhibitors such as but not limited to those described herein.

[0061] Disclosed herein is a highly innovative and unique non-invasive immuno-theranosticplatform applicable for molecularly specific treatment of a wide range of primary, late-stage and disseminated malignancies. In one embodiment, the central component of the new methods is a highly specific mAb (DUNP19) targeting LRRC15, which is applicable for targeting TGF- P driven CAFs within the tumor stroma of many cancer indications, as well as direct targeting of cancer cells of mesenchymal tumors (e.g., sarcomas, glioblastoma multiforme). A second component of such platform in some embodiments comprises one of more immune checkpoint inhibitors, that enhance or synergize efficacy. While the potential of stromal targeting is widely recognized, innovative approaches have been difficult to implement, largely because of a scarcity of optimal targets with high tumor specificity. Unlike other CAF proteins, LRRC15 expression is very low in non-cancerous tissues and absent in lymph node stroma and normal pancreas. The cancer specific expression profile, correlation to immunotherapy resistance and inducibility by TGF- differentiate LRRC15 from other commonly used mesenchymal markers.

[0062] DUNP19 has several advantages over other anti-LRRC15 antibodies. Tn addition to having significantly higher affinity, DUNP19 binds to a developmentally advantageous and phylogenetically conserved epitope. This feature enables evaluations in wild-type (wt) animal models, avoiding the need to genetically engineer human LRRC15 expression into the host genome. Importantly, DUNP19 is rapidly and effectively internalized by target cells, which is critical for targeted radiopharmaceuticals emitting short-range particles (e.g., high-LET alpha or Auger emitters). The internalization also renders other possibilities such as exploiting DUNP19 for targeting intracellular proteins with molecules that do not naturally cross the cell membrane. As shown herein, in combination with immune checkpoint inhibitor therapy, use of DUNP19 is particularly effective.

[0063] Thus, LRRC15 is an optimal targetable biomarker that can be leveraged for therapy, in particular by a binding moiety such as an antibody that is internalized by LRRC15-expressing cells. The present disclosure further advances a mAb, DUNP19, which demonstrates specific targeting and effective internalization by LRRC15-expressing cells. Other LRRC15-targeted antibodies that are internalized by LRRC15-expressing cells are also embraced herein. In one embodiment, DUNP19 can be applied for the following clinical applications. In any such applications, combination therapy with immune checkpoint inhibition increases efficacy.

[0064] Moreover, targeting to LRRC15-expressing, disease-associated tissues other than tumors is another application useful, for example, in lung disease and in autoimmune diseases such as but not limited to rheumatoid arthritis. Combination therapy with immune checkpointinhibition enhances effectiveness.

[0065] In one embodiment, it is expected that pharmacological strategies aimed at eliminating tumor-associated stroma or impeding immune-suppressive pathways will have impactful antitumor effects either as monotherapies, or in some embodiments in combination with compounds directing the innate immune system.

[0066] In one embodiment, it is anticipated that the anti-neoplastic effects of anti-LRRC15 radioimmunotherapy (RIT) would be contingent on the applied linear energy transfer (LET) and the cellular localization of target expression. In brief, it is believed that intracellular dose deposition of short-range high-LET in LRRC15-expressing CAFs will result in specific eradication of the tumor stoma, causing anti-neoplastic effects through acute microenvironmental changes, exhaustion of paracrine support, improved drug infiltration and increased susceptibility to immunotherapies and components of the intrinsic immune system. Although utilization of low-LET LRRC 15 -radioimmunotherapy (RIT) may result in less effective obliteration of the targeted CAFs, crossfire effects from the long track path-length will translate into damage of the near-by tumor cells.

[0067] The present disclosure harnesses the unique characteristics of a humanized IgGi antibody, DUNP19, designed to specifically bind to a phylogenetically stable epitope of LRRC 15. Such harnessing includes treatment of cancer using DUNP19 or binding moieties with its variable heavy chain and variable light chain regions, in some embodiments in combination with immune checkpoint inhibitor therapy. The proficient cellular internalization exhibited by the target-expressing cells provides an optimal foundation for leveraging DUNP19 as an effective cellular delivery vehicle. As described in the examples herein, the versatile functionality of DUNP19 has been substantiated by demonstrating its capacity for functionalization with therapeutic radioisotopes. This modality facilitates the identification of patients harboring LRRC 15+ lesions, allowing for precise therapeutic interventions through the implementation of PET and SPECT with anatomical imaging. Subsequently, patients with LRRC 15+ tissues can be selected for personalized therapeutic dosing, delivering tumorspecific ionizing radiation with minimal off-target effects. Examples include the effective application of radioactive iodine in thyroid cancer therapy and the use of radioligands binding to specific membrane antigens, such as [177LU]-PSMA for prostate-specific membrane antigen and [177LU]-DOTATATE for somatostatin receptors. Patients exhibiting high tumoral uptake of the diagnostic radioligand on PET / CT imaging, reflecting elevated target expression and successful drug delivery, are deemed eligible for treatment with these therapeutic radioligands.Combination with immune checkpoint inhibitor therapy enhances or even synergizes with the efficacy of DUNP19 based RIT.

[0068] The LRRC15-RIT approach using DUNP19 disclosed herein exhibited effective targeting across various models, each representing distinct tumor biology and LRRC15 expression patterns. Notably, the specific depletion of LRRC15+ cancer and stromal cells through a singular systemic administration of [177Lu]-DUNP19 significantly slowed tumor progression and conferred a survival benefit in all models. Including immune checkpoint inhibition therapy further enhanced anti-tumor effectiveness. The findings disclosed herein additionally establish the potential of DUNP19 as a carrier for177Lu, known for inducing DNA breaks across multiple cell diameters. Notably, crossfire effects by177Lu radiation can significantly improve responses in tumors with low or heterogeneous target expression. The strategic application of a177Lu antibody conjugate addresses the challenge of intra-tumoral heterogeneity in LRRC15 expression within tumor tissues, thereby enhancing the therapeutic efficacy of the treatment. Such approach surpasses the efficacy of an LRRC15-targeted antibody-drug-conjugate or a potential small molecule, and in combination with immune checkpoint targeted therapy, further enhances its efficacy.

[0069] Given DUNP19’s binding to an epitope shared by human and murine LRRC15, it is anticipated that no vastly different biodistribution upon translation to patients. Moreover, in vivo cellular internalization, as observed for the DUNP 19-based radioconjugate, has been reported to enhance radioisotope retention and reduce extracellular shedding of the radioisotope. In agreement with other beta-emitting RIT, a decrease was observed in white and red blood cells after administration of [177Lu]-DUNP19. However, the bone marrow recovered within an expected time frame after treatment injection, allowing for serial dosing.

[0070] Given LRRC15’s known association with TGF-P, immunosuppression, and the observed potential immunomodulatory effects of RIT, in one embodiment the anti-tumor activity induced by [177Lu]-DUNP19 was shown to reverse the signaling profile associated with immunosuppression and resistance to immunotherapy. Development of pharmaceuticals abating tumor-specific immunosuppression have been focused on identifying targets that are specifically associated with cancer stroma, with the most coveted being plasma membrane proteins specific to CAFs. An absolute majority of these efforts target fibroblast activation protein-a (FAP), a membrane bound serine protease overexpressed by CAFs. However, normal tissues and healthy activated fibroblasts also express FAP during wound healing, such as the activation of myofibroblasts post-myocardial infarction. The feature of TGFp governance isunique to LRRC 15 compared to other mesenchymal associated markers such as FAP. Although TGFp induces upregulation of many targets, including FAP, both directly and through crosstalk, in one embodiment, a target can only be considered TGF -driven if its expression is also downregulated after TGFP blockade or withdrawal, which has not been demonstrated for FAP.

[0071] LRRC 15 -targeted RIT using DUNP19 was shown to lead to differential expression of genes in stromal cells related to immune cell function, including genes indicative of activation and proliferation of T-cells (Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes) and natural killer cells (Cxcrd, Eomes, Prfl). In the stroma of U118MG tumors, the gene encoding PD-1, a negative regulator of T-cell activity was downregulated, suggesting that [177Lu]-DUNP19 may contribute to the relief of immune cell suppression and T-cell exclusion. In light of these findings, immune-based adjuvant therapies such as but not limited to immune checkpoint blockade are complementary to LRRC15-RIT, and the combination providing enhanced efficacy. The transcriptomic remodeling of immune-related signaling pathways and expression of immune activating cytokines after RIT may increase immune cell activation and infiltration in previously immunologically “cold” tumors. Additionally, ablation of radio-resistant stroma may further increase immune cell invasion and help amplify the anti-tumor immune response.

[0072] As disclosed herein, a novel techniques for treatment of a wide range of aggressive tumors is provided. Critically, [177Lu]-DUNP19 therapy was demonstrated to be effective despite heterogenous LRRC15 expression and targeted radiation by [177Lu]-DUNP19 treatment is not limited to LRRC 15+ tumor cells, taking advantage of the crossfire effects of betaradioimmunotherapy. Crossfire refers to the radiation damage inflicted on neighboring cells by emitted particles that travel beyond the targeted cell. In tumors, the targeted biomarker is often heterogeneously expressed; some cancer cells may have a lower expression or lack the target antigen. This poses a challenge for conventional targeted therapies. Beta particles travel short to moderate distances in tissues. Even if a cancer cell does not express the target antigen, in some embodiments it can still be affected by radiation from neighboring cells that do. By exploiting crossfire effects, in some embodiments, radioimmunotherapy can potentially increase the therapeutic efficacy of treatment across the entire tumor mass. It allows for a more comprehensive approach to targeting cancer cells within a tumor with heterogeneous LRRC 15 expression (non-limiting examples include commonly used beta-emitting radionuclides in the context of RIT: Iodine-131 (1311), Yttrium-90 (90Y), Lutetium-177 (177Lu), Samarium-153 (153Sm), Holmium- 166 (166Ho)). Furthermore, combination therapy with immune checkpoint inhibition therapy further enhances or even synergizes with the efficacy of RIT.

[0073] In addition, LRRC15’s regulation by TGFP allows for successful targeting of pro- tumorigenic TGFP signaling mechanisms that have been shown to contribute to immunotherapy resistance and poor prognosis. As disclosed herein, at a transcriptomic level, these TGFP-LRRC15 signatures are largely erased in [177Lu]-DUNP19-treated tumors and expression of other anti-tumor immune pathways increases. The eradication of this TGFP- LRRC15 signature in tumor cells would be synergistic with existing immunotherapies and allow for immune cell infiltration. Thus, targeting LRRC15+ cells with [177Lu]-DUNP19 as a novel theranostic strategy provides sustained tumor control across models of LRRC15+ disease, improves survival, and reprograms the transcriptomic landscape of pro-tumorigenic and immunosuppressive mechanisms within the TME, all with minimal side effects.METHODS OF USE

[0074] In one embodiment, disclosed herein is method of using a binding moiety targeting LRRC15 disclosed herein associated with a cytotoxic agent, in combination with at least one immunotherapeutic agent, to treat a disease having disease-associated tissue that is derived from or associated with mesenchymal stem cells. In one embodiment, the disease-associated tissue may be tumor tissue. In one embodiment, the disease associated tissue may be tumor stroma tissue. In one embodiment, the disease-associated tissue may be tumor tissue and tumor stroma tissue. In one embodiment, the disease-associated tissue may be tumor tissue and not tumor stroma tissue. In one embodiment, the disease-associated tissue may be tumor stroma tissue and not tumor tissue.

[0075] In one embodiment, disclosed herein is method of using the binding moiety targeting LRRC15 disclosed herein associated with a cytotoxic agent, in combination with immune checkpoint inhibitor therapy, to treat a disease having disease-associated tissue that is derived from or associated with mesenchymal stem cells. In one embodiment, the disease-associated tissue may be tumor tissue. In one embodiment, the disease associated tissue may be tumor stroma tissue. In one embodiment, the disease-associated tissue may be tumor tissue and tumor stroma tissue. In one embodiment, the disease-associated tissue may be tumor tissue and not tumor stroma tissue. In one embodiment, the disease-associated tissue may be tumor stroma tissue and not tumor tissue.

[0076] In another embodiment, the binding moiety targeting LRRC15 disclosed herein is provided for a use in treating a disease having disease-associated tissue that is derived from or associated with mesenchymal stem cells. The method or use comprises the step ofadministering to a patient a combination of at least one immune checkpoint inhibitor and a binding moiety targeting LRRC15 expressed on cells in the disease-associated tissue, wherein the binding moiety is associated with a cytotoxic agent and the binding moiety is internalized by cells in the disease-associated tissue, thereby delivering the cytotoxic agent to the disease- associated tissue. In one embodiment, the disease is an autoimmune disease (e.g., rheumatoid arthritis). In another embodiment, the disease is lung injury. In another embodiment, the disease-associated tissue comprises tumor or tumor stromal cells. Representative examples of tumors include, but are not limited to, pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma. The prostate cancer can be androgen receptor negative or androgen independent. In another embodiment, the tumor is metastatic. In another embodiment, the tumor stromal cells comprise fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

[0077] In one embodiment, the binding moiety can be a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR). The binding moiety can be part of a bispecific antibody or a trispecific antibody. In one embodiment, the binding moiety has picomolar affinity for LRRC15. In one embodiment, the binding moiety is an antibody. In one embodiment, the antibody binds to a phylogenetically conserved epitope on LRRC15 (e.g. to an epitope on mammalian LRRC15). In one embodiment, the antibody is a humanized monoclonal antibody, for example, a monoclonal antibody comprising a variable heavy chain having the sequence of SEQ ID NO:1 and a variable light chain having the sequence of SEQ ID NO: 2. In another embodiment, the monoclonal antibody is an afucosylated antibody or a low-fucose variant thereof.

[0078] In one embodiment, the cytotoxic agent associated with the binding moiety is a therapeutic radionuclide. The therapeutic radionuclide can be an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. Examples of alpha particle emitters include, but are not limited to, uranium, radium, thorium, and actinium. Examples of beta particle emitters include, but are not limited to, uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. Examples of Auger electron emitters include, but are not limited to, technetium, indium, and iodine. Examples of gamma-ray emitters include, but are not limited to, uranium, thorium, actinium,cobalt, cesium, and technetium. In another embodiment, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof. In one embodiment, the cytotoxic agent is covalently bound to the binding moiety. In one embodiment the cytotoxic agent is non-covalently bound to the binding moiety. In one embodiment the binding moiety is radiolabeled with the cytotoxic agent. In one embodiment cytotoxic agent is conjugated to the antibody by a chelating agent. In some embodiments the conjugation is direct, i.e., the cytotoxic agent is directly conjugated to the binding moiety. In some embodiments, the conjugation is indirect, i.e., the binding moiety and the cytotoxic agent are conjugated in vivo, also known as a pretargeting method. Such conjugation methods are known in the art, for example in the making of antibody drug conjugates (ADC), and will be readily prepared for the particular agent being conjugated.

[0079] In some embodiments, methods are provided for treating a disease having disease- associated tissue that is derived from or associated with mesenchymal stem cells as described herein, by administering to a subject in need an effective amount of a combination of a bispecific or trispecific targeting molecule is provided targeting both LRRC15 and TGFp, and an immunotherapeutic agent such as an immune checkpoint inhibitor. In some embodiments, the bispecific or trispecific targeting molecule comprises at least one occurrence of SEQ ID NO:1 and SEQ ID NO:2, and at least one occurrence of SEQ ID NO:3 and SEQ ID NO:4. In some embodiments, a bispecific or trispecific targeting molecule is provided targeting both LRRC15 and TGFp, comprising at least one occurrence of SEQ ID NO: 1 and SEQ ID NO:2, and at least one sequence that binds to TFGp. In some embodiments, the bispecific or trispecific molecule is associated with a cytotoxic agent. The treatable diseases, such as cancers and autoimmune diseases, and selections of cytotoxic agents and immune checkpoint inhibitors are as set forth elsewhere herein.

[0080] In some embodiments, methods are provided for treating a disease having disease- associated tissue that is derived from or associated with mesenchymal stem cells as described herein, by administering to a subject in need an effective amount of a combination of a bispecific or trispecific targeting molecule is provided targeting both LRRC15 and at least one binding specificity for another ligand, in combination with an immune checkpoint inhibitor. In one embodiment, the other ligand is TFG-p. In one embodiment, a bispecific or trispecific antibody that binds LRRC15 and TFG-P may be used to reduce TGF-P levels or inhibit activity at the site of cells or tissues expressing LRRC15. In one embodiment, becauseTGF-P receptor activity governs LRRC15 expression, use of the bi- or trispecific antibody will decrease the effect of the TGF- . In one embodiment, decreasing the effect of TGF-P will reduce the expression of LRRC15. In one embodiment, such modality is generated by incorporating the LRRC15 specific-binding site of DUNP19 as one, or two, of a bi-specific or tri-specific antibody’s binding site / sites, combined with two, or one, binding sites specific for TGF-p. Examples of anti-TGF-P antibodies include, but are not limited to, fresolimumab. In one embodiment, fresolimumab comprises a variable heavy chain having the sequence of SEQ ID NO:3 and a variable light chain having the sequence of SEQ ID NO:4.

[0081] In the foregoing embodiment, the bi or trispecific antibody may be associated with a cytotoxic agent, such as any of those described herein. The immune checkpoint inhibitor may be any of those described herein.

[0082] In one embodiment, a method for treating a lung injury in a patient is provided by administering to the patient a combination of an immune checkpoint inhibitor and a binding moiety targeting LRRC15 wherein the binding moiety is associated with a cytotoxic agent and the binding moiety is internalized by cells in the lungs.

[0083] In any of the foregoing embodiments, the LRRC 15 binding moiety comprises a variable heavy chain having the sequence of SEQ ID NO:1 and a variable light chain having the sequence of SEQ ID NO:2. In one embodiment, the binding moiety is DUNP19. In any of the foregoing embodiments, a LRRC 15 binding moiety may comprise the complementarity determining regions of the aforementioned sequences, or of an antibody to LRRC 15 that is internalized by its target cells.

[0084] Thus, in one embodiment, provided is LRRC 15 targeting on a bispecific T Cell Engagers (BiTEs), e.g., an antibody, designed to engage CD19 on B cells and CD3 on T cells. In another embodiment, provided are trifunctional antibodies, such as antibodies engaging LRRC15 on the tumor cells, CD3 on T cells, and Fey receptors on accessory immune cells such as macrophages. In another embodiment, provided are dual-affinity re-targeting (DART) molecules; which are bispecific antibodies that can engage two different antigens. In one nonlimiting example, a DART molecule might simultaneously target CD3 on T cells and LRRC 15 on the tumor cells. In one embodiment, LRRC15-targeting is provided on Fc-enhanced bispecific antibodies. In one embodiment, these antibodies include an Fc region that can interact with Fc receptors on immune cells, providing additional effector functions. Such designs enhance antibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC). Moreover, such BiTEs and DARTs and Fc-enhanced bispecific antibodies are useful for any or all of the methods described herein, such as but not limited to the treatment of cancer, treatment of autoimmune diseases, treatment of lung diseases, increasing susceptibility of a cancer to immunotherapy, inducing dormancy in a tumor, and depleting TGFP expressing cells in a tumor or tumor microenvironment. Any such methods may further be administered in combination with at least one immune checkpoint inhibitor.COMBINATION WITH AN IMMUNE CHECKPOINT INHIBITOR

[0085] In some embodiments, DUNP19, or any antigen binding fragment (Fab), F(ab’)2 fragment, single chain variable fragment (scFv), bispecific T cell engager, a chimeric antigen receptor (CAR), monoclonal antibody, bispecific or trispecific antibody comprising at least one occurrence of SEQ ID NO: 1 and SEQ ID NO:2 (referred to herein generally as DUNP19 therapy), optionally associated with a cytotoxic agent such as a radionuclide or another cytotoxic agent as described herein, is administered with at least one immune checkpoint inhibitor (ICI). In some embodiments, ICI treatment comprises two or more immune checkpoint inhibitors, for example, targeting the same or different checkpoint proteins. In some embodiments, immune checkpoints regulate T cell function in the immune system. Checkpoint proteins interact with specific ligands which send a signal into the T cell and essentially switch off or inhibit T cell function. Cancer cells take advantage of this system by driving high levels of expression of checkpoint proteins on their surface which results in control of the T cells expressing checkpoint proteins on the surface of T cells that enter the tumor microenvironment, thus suppressing the anticancer immune response. As such, in combination with DUNP19 therapy, inhibition of checkpoint proteins would result in restoration of T cell function and an immune response to the cancer cells. An immune checkpoint inhibitor (or checkpoint inhibitor) is a compound or agent that blocks or inhibits immune checkpoint proteins (i.e., that blocks or inhibits checkpoint receptors or checkpoint receptor ligands). As noted herein, the combination treatment is not necessarily both the binding moiety to LRRC15 and the one or more immune checkpoint inhibitors in the same formulation, but each administered separately but the administrations of each concurrent or overlapping, such that the benefit of both components is achieved to provide or enhance efficacy.

[0086] In some embodiments, the combination of LRRC15-targeted RIT, such as DUNP19 RIT, and immune checkpoint inhibition (ICI) therapy is synergistic. In some embodiments, the efficacy of the combination of LRRC 15 -targeted RIT, e.g., DUNP19 RIT, and ICI therapy is greater than the sum of the efficacy of individual treatments. Such synergy may bedemonstrated by, for example, comparing each treatment individual to the combination of both.

[0087] Examples of checkpoint proteins for targeted immune checkpoint therapy include, but are not limited to, CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK cells, and memory CD8+T cells), CD 160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and any of various B-7 family ligands. Programmed Death- 1 (PD-1) is a member of the immunoglobulin superfamily (IGSF) of molecules involved in regulation of T cell activation. The structure of PD- 1 is composed of one IGSF domain, a transmembrane domain, and an intracellular domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosinebased switch motif (ITSM). PD-1 has two binding partners: PD-L1 (B7-H1, CD274) and PD- L2 (B7-DC, CD273). PD-L1 is expressed broadly on both hematopoietic and non- hematopoietic lineages. It is found on T cell, B cells, macrophages, NK cells, DCs, and mast cells as well as in peripheral tissues. PD-1 engagement represents one means by which tumors evade immunosurveillance and clearance. It is noted that such proteins may be referred to as checkpoint proteins or immune checkpoint proteins, and their inhibitors as checkpoint inhibitors or immune checkpoint inhibitors, or other syntactic variations of each, which are intended to be synonymous. Furthermore, the terms immune checkpoint blockade and checkpoint blockade, and variants thereof, are similarly used synonymously referring to the use of (immune) checkpoint inhibitors.

[0088] Non-limiting examples of checkpoint inhibitors include small molecules, peptides, and antibodies. Non-limiting examples of antibodies include nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011), MEDI0680 (AMP-514), AMP-224, AUNP-12, BMS 936559, atezolizumab (MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS935559 (MDX-1105), rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab (BMS-986015), IPH2101 (1-7F9), Indoximod (NLG 9189), NLG 919, INCB024360, PF-05082566, Urelumab (BMS-663513), and MEDI6469.

[0089] Nivolumab (OPDIVO) is a fully human IgG4 monoclonal antibody targeted against PD-1 receptor on activated T and B lymphocytes. Pembrolizumab (KEYTRUDA) is another non-limiting example of an antibody that targets PD- 1. Other compounds and agents that block, inhibit or target checkpoint proteins include compounds undergoing testing and not yet available on the market. The disclosure is not limited by the specific checkpoint inhibitor. Nonlimiting examples of checkpoint inhibitors that may be used are listed below.

[0090] In one embodiment, a combination of two or more checkpoint inhibitors is administered to the subject with DUNP19 or any LRRC15 binding moiety comprising SEQ ID NO:1 andSEQ ID N0:2, associated with a cytotoxic agent. In one embodiment, the combination of checkpoint inhibitors is selected from among those disclosed herein, such as targeting CTLA- 4 and targeting PD-1 (e.g., nivolumab or pembrolizumab, and ipilumumab). The two or more checkpoint inhibitors can be administered simultaneously or consecutively with respect to one another and with respect to the herein-described LRRC15-targeted, e.g., DUNP19, therapy. In a further embodiment, the combination of two or more checkpoint inhibitors target two different checkpoint proteins, such as PD-1 (e.g., nivolumab or other PD-1 inhibitor) and CTLA-4 (e.g., ipilimumab or other CTLA-4 inhibitor), are administered to the subject simultaneously or consecutively with respect to one another and with respect to DUNP19 therapy. In one embodiment, the combination of two or more checkpoint inhibitors target two or more different checkpoint proteins from among: CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN- 15049, CHK 1 kinase, CHK2 kinase, A2aR, and B-7 family ligands. In one embodiment, the combination of two or more checkpoint inhibitors targeting two or more different checkpoint proteins is selected from among those disclosed herein.

[0091] The dose level, frequency of dosing, duration of dosing and other aspects of administration of the checkpoint inhibitor may be optimized in accordance with the patient’s clinical presentation, duration or course of the disease, comorbidities, and other aspects of clinical care. The disclosure is not so limiting with regard to the particular aspects of the checkpoint inhibitor component of the methods embodied herein. As noted herein, the combination therapy of the LRRC15-targeted antibody associated with a cytotoxic agent and the immune checkpoint inhibitor for any of the methods or embodiments disclosed herein, may be in different formulations and / or on different administration schedules, provided the combination therapy or activity of each component is concurrent or overlapping or at least shows benefits from both components. Such activity may derive from the pharmacokinetics of the component, such as its half-life, such that one therapy may be administered before the other begins, but the activity provided by the therapy of one continues during the course of the therapy of the other. By way of example, antibody infusions may be given weekly, biweekly or monthly, or even more infrequently. Thus, combination treatment embraces that the effectiveness of each component is concurrently active.

[0092] In some embodiments, other forms of immunotherapy in addition to or in place of immune checkpoint blockade is described. Non- limiting examples of such immunotherapy that may be use in any of the methods disclosed herein include T cell therapy, such as TIL or CAR-T therapies; cytokine therapy, such as interleukins (e.g., IL-2, IL-7, IL-12), cytokines (e.g., interferons, G-CSF), chemokines (e.g., CCL3, CCL26, CXCL7); immunomodulatory drugs (e.g., thalidomide); monoclonal antibody therapy (e.g. rituximab, BiTEs), among many others. The disclosure is not limited with regard to the immunotherapy administered with DUNP19 therapy.IMMUNOTHERAPY RESISTANT SIGNATURE

[0093] In one embodiment, the tumor or stroma thereof exhibits an immunotherapy resistant signature. Such signature may be determined, for example, by biopsy of the tumor and assessing transcriptomic signatures using any method, such as but not limited to those described herein. Such signature comprises two or more genes among LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and / or Prfl. Such signature may comprise any two, three, four, five, six, seven, eight, nine or ten of the foregoing genes. In one embodiment, the gene signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl .Detection of the depletion of this signature, optionally along with other genes related to immunotherapy resistance, is achieved through LRRC15-targeted RIT. In one embodiment, the effect of LRRC15 treatment may be monitored by determining the signature on a treated tumor or tumor stroma, wherein reduction of the expression of one or more signature genes indicates effectiveness. In one embodiment, a tumor may be evaluated for susceptibility to any of the methods disclosed herein by identifying the aforementioned signature in the tumor or stroma, wherein expression of any one or more genes in the signature indicates susceptibility. In one embodiment, the susceptibility of a tumor to a method described herein comprising treatment targeting LRRC15, e.g., DUNP19, associated with a cytotoxic agent, optionally in combination with immune checkpoint blockade may be evaluated by identifying the signature genes in a tumor or tumor stroma. Furthermore, once the susceptibility to immunotherapy is increased by such treatment (e.g., targeting LRRC15 optionally in combination with at least one immune checkpoint inhibitor), subsequently the patient may be administered or the tumor exposed to immune checkpoint blockade.DEPLETING TGFp EXPRESSING CELLS

[0094] In one embodiment, a method is provided for depleting TGFP expressing cells in a tumor or in a tumor microenvironment by exposing the tumor or tumor microenvironment to a cytotoxic agent such as a radionuclide conjugated to a binding moiety comprising SEQ ID NO: 1 and SEQ ID NO:2. In some embodiments, such method also includes combinationtherapy with an immunotherapeutic agent such as but not limited to an immune checkpoint inhibitor. Such combination therapy may be synergistic. The various binding moieties comprising SEQ ID NO: 1 and SEQ ID NO:2, cytotoxic agents, immune checkpoint inhibitors and other forms of immunotherapy are as described herein.INDUCING DORMANCY

[0095] In one embodiment, tumor dormancy is a phase in cancer progression where dispersed tumor cells stay concealed and inactive for an extended period. This dormancy may occur at the onset of tumor development or following treatment. Various mechanisms, including cellular quiescence, angiogenic inhibition, or immune surveillance, can shift aggressive tumor growth to dormancy after treatment. These mechanisms either hinder the proliferation of tumor cells or trigger their apoptosis. Thus, in one embodiment, the methods provided herein result in dormancy of a tumor. In one embodiment, the methods provided herein reduce aggressivity of a tumor. Thus, in some embodiments, dormancy is induced by treatment of a tumor with an LRRC 15 -targeting binding moiety or antibody (e.g., DUNP19) associated with a cytotoxic agent. In some embodiments, dormancy is induced by treatment of a tumor with a combination of an LRRC 15 -targeting binding moiety or antibody (e.g., DUNP19) associated with a cytotoxic agent, and at least one immune checkpoint inhibitor, wherein such combination enhances efficacy or is synergistic.

[0096] Various selections of these components in the combination for such use are as set forth elsewhere herein, such as selections of the LRRC 15 -targeting binding moiety, radionuclides and other cytotoxic agents, etc. Non-limiting examples of immune checkpoint inhibitors are as described herein.

[0097] Thus, the following embodiments are among those embraced herein.

[0098] In one embodiment, a method is provided for treating a tumor in a patient comprising administering to the patient a combination of: a. a binding moiety comprising SEQ ID NO: 1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent; and b. an immune checkpoint inhibitor.

[0099] In some embodiments, the tumor and / or stroma thereof expresses LRRC15. In some embodiments, the tumor and / or stroma thereof produces TGFp. In some embodiments, the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breasttumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma. In some embodiments, the prostate cancer is androgen receptor negative or androgen independent. In some embodiments, the tumor is metastatic. In some embodiments, the tumor comprises stroma which comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

[0100] In some embodiments, the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR). In some embodiments, the binding moiety has picomolar affinity for LRRC15. In some embodiments, the binding moiety is an antibody. In some embodiments, the antibody binds to a mammalian LRRC15. In some embodiments, the antibody is a humanized monoclonal antibody. In some embodiments, the monoclonal antibody is an afucosylated antibody or a low-fucose variant thereof.

[0101] In some embodiments, the cytotoxic agent is a therapeutic radionuclide. In some embodiments, the radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. In some embodiments, the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium. In some embodiments, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. In some embodiments, the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine. In some embodiments, the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium. In some embodiments, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

[0102] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD- Ll, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and various B-7 family ligands. In some embodiments, the inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF-05082566, urelumab or MEDI6469.

[0103] In some embodiments, the tumor or stroma thereof exhibits an immunotherapy resistant signature. In some embodiments, the signature comprises one or more of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and / or Prfl. In some embodiments, the signature In some embodiments, LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl. In some embodiments, the signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl.

[0104] In one embodiment, a method is provided for increasing susceptibility of an immunotherapy resistant tumor in a patient to immunotherapy comprising exposing the tumor or tumor stroma to a binding moiety comprising SEQ ID NO: 1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent. In one embodiment, the tumor or stroma thereof exhibits an immunotherapy resistant signature. In one embodiment, the signature comprises one or more of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcrb, Eomes and / or Prfl. In one embodiment, the signature comprises LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl . In one embodiment, the signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl.

[0105] In one embodiment, the resistance to immunotherapy is resistance to immune checkpoint blockade. In one embodiment, the immune checkpoint is CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN- 15049, CHK 1 and CHK2 kinases, A2aR, and various B-7 family ligands.

[0106] In one embodiment, the tumor and / or stroma thereof expresses LRRC15. In one embodiment, the tumor and / or stroma thereof produces TGF . In one embodiment, the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma. In one embodiment, the prostate cancer is androgen receptor negative or androgen independent. In one embodiment, the tumor is metastatic. In one embodiment, the tumor comprises stroma which comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

[0107] In one embodiment, the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR). In one embodiment, the binding moiety has picomolar affinity for LRRC15. In one embodiment, the binding moiety is an antibody. In oneembodiment, the antibody binds to a mammalian LRRC15. In one embodiment, the antibody is a humanized monoclonal antibody. In one embodiment, the monoclonal antibody is an afucosylated antibody or a low-fucose variant thereof.

[0108] In one embodiment, the cytotoxic agent is a therapeutic radionuclide. In one embodiment, radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. In one embodiment, the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium. In one embodiment, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. In one embodiment, the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine. In one embodiment, the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium. In one embodiment, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

[0109] In one aspect, a method is provided for depleting TFGP expressing cells in a tumor or tumor microenvironment comprising exposing the tumor or tumor microenvironment to a cytotoxic agent associated with or conjugated to a binding moiety comprising SEQ ID NO:1 and SEQ ID NO:2. In one embodiment, the TFGP expressing cells are tumor cells. In one embodiment, the TGFP expressing cells are tumor stoma cells. In one embodiment, the tumor and / or stroma thereof expresses LRRC15. In one embodiment, the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma. In one embodiment, the prostate cancer is androgen receptor negative or androgen independent. In one embodiment, the tumor is metastatic. In one embodiment, the stroma comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

[0110] In one embodiment, the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR). In one embodiment, the binding moiety has picomolar affinity for LRRC15. In one embodiment, the binding moiety is an antibody. In one embodiment, the antibody binds to a mammalian LRRC15. In one embodiment, the antibody is a humanized monoclonal antibody. In one embodiment, the monoclonal antibody is anafucosylated antibody or a low-fucose variant thereof.

[0111] In one embodiment, the cytotoxic agent is a therapeutic radionuclide. In one embodiment, radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. In one embodiment, the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium. In one embodiment, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. In one embodiment, the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine. In one embodiment, the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium. In one embodiment, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

[0112] In one aspect, a bispecific or trispecific targeting molecule is provided that binds to LRRC15 and TGF|3, said targeting molecule comprising at least one occurrence of amino acid sequences SEQ ID NO: 1 and SEQ ID NO:2, and at least one occurrence of a targeting sequence to TGFp. In one embodiment, the targeting molecule comprising amino acid sequences SEQ ID NO: 1 and SEQ ID NO:2, and amino acid sequences SEQ ID NO:3 and SEQ ID NO:4. In one embodiment, the targeting molecule comprising one occurrence of amino acid sequences SEQ ID NO:1 and SEQ ID NO:2, and two occurrences of amino acid sequences SEQ ID NOG and SEQ ID NO:4. In one embodiment, the targeting molecule comprising two occurrences of amino acid sequences SEQ ID NO:1 and SEQ ID NO:2, and one occurrence of amino acid sequences SEQ ID NOG and SEQ ID NOG. In one embodiment, the targeting molecule is humanized. In one embodiment, the targeting molecule is afucosylated or a low-fucose variant thereof.

[0113] In one embodiment, the bispecific or trispecific targeting molecule further comprises a cytotoxic agent. In one embodiment, the cytotoxic agent is a therapeutic radionuclide. In one embodiment, the therapeutic radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof. In one embodiment, the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium. In one embodiment, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium. In one embodiment, the Auger electron emitter is selected from the group consisting oftechnetium, indium, and iodine. In one embodiment, the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium. In one embodiment, the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

[0114] In one aspect, a method is provided for inducing dormancy in a LRRC15-expressing tumor in a patient comprising exposing the tumor or stroma thereof to a beta-emitting radionuclide conjugated to a binding moiety comprising SEQ ID NO:1 and SEQ ID NO:2, optionally in combination with administering an immune checkpoint inhibitor to the patient. In one embodiment, the tumor and / or stroma thereof produces TGF|3. In one embodiment, the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma. In one embodiment, the prostate cancer is androgen receptor negative or androgen independent. In one embodiment, the tumor is metastatic. In one embodiment, the tumor comprises stroma which comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

[0115] In one embodiment, the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR). In one embodiment, the binding moiety has picomolar affinity for LRRC15. In one embodiment, the binding moiety is an antibody. In one embodiment, the antibody binds to a mammalian LRRC15. In one embodiment, the antibody is a humanized monoclonal antibody. In one embodiment, the monoclonal antibody is an afucosylated antibody or a low-fucose variant thereof.

[0116] In one embodiment, the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium.

[0117] In one embodiment, the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF- 05082566, urelumab or MEDI6469.

[0118] In one embodiment, the tumor or stroma thereof exhibits an immunotherapy resistantsignature. In one embodiment, the signature comprises one or more of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and / or Prfl. In one embodiment, the signature comprises LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl. In one embodiment, the signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl.

[0119] As used herein, the terms “comprise”, "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0120] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an enzyme" or "at least one enzyme" may include a plurality of enzymes, including mixtures thereof.

[0121] Throughout this application, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0122] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0123] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the disclosure, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. Each literature reference orother citation referred to herein is incorporated herein by reference in its entirety.

[0124] In the description presented herein, each of the steps of the disclosure and variations thereof are described. This description is not intended to be limiting and changes in the components, sequence of steps, and other variations would be understood to be within the scope of the present disclosure.

[0125] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the disclosure. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0126] Various embodiments and aspects of the present disclosure as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. While certain features of the disclosure have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the disclosure.EXAMPLE 1Anti-LRRC15 Antibody, DUNP19

[0127] As described in PCT / US2021 / 025054, published as WO2021 / 202642, DLTNP19 was developed by humanization of a hybridorna mAb generated from immunizing mice with stably transfected LRRC15-expressing 3T3 cells. Results obtained by flow-cytometry demonstrated that DUNP19 has high in vitro binding specificity to a range of cells with wild-type (wt) and transfected LRRC15 expression, while no binding was observed to negative cells. Liquid chromatography-mass spectrometry (LC-MS) analysis of DUNP19 incubated with LRRC15- expressing cell lines confirmed molecular binding specificity. It was further found that DUNP19 binds to a phylogenetically conserved epitope. DUNP19 binds specifically to 293T cells transfected with rodent or non-human primate LRRC15. LigandTracer (Ridgeview Instruments) was utilized to assess binding interaction between live cells and Lutetium- 177 labeled DUNP19 ([177Lu]DUNP19). These analyses showed that [177Lu]DUNP19 binds targetcells with picomolar affinity (3.29E-10m). Furthermore, confocal imaging revealed that DUNP19 is rapidly internalized by LRRC15-expressing cells. Internalization rate was studied by quantifying emitted fluorescence over time from S AOS2 cells incubated with pH-responsive dye-labeled DUNP19. Identification of colocalization of Alexa-488 labeled DUNP19 with intracellular markers (DAPI and LAMP1) in SAOS2 cells over time was applied to further confirm time of internalization. These studies demonstrated that >50% and 100% of DUNP19 was internalized at 30 minutes and 3 hours, respectively.

[0128] In vivo targeting specificity was evaluated by studying tissue biodistribution of systemically (i.v.) injected [177Lu]DUNP19 in subcutaneous (s.c.) tumor models. Radioconjugate uptake, expressed as percent injected activity per gram (%IA / g), was assessed in 12 different tissues and blood at 24, 48 and 72 hours after injection. Ul i 8 tumors were found to have the highest %IA / g of [177Lu]DUNP19 at all timepoints. Further, uptakes of [177LU]DUNP19 and a non-specific Lutetium-177 labeled mAb were negligible in LRRC15 negative (LNCaP) and U1 18 tumors, respectively.

[0129] In one embodiment, the variable heavy chain of DUNP19 comprises the following sequence:QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQGLEWMGWINPNS DGTNYAQNFLGRVTMTRDTSISTAYMELSRLRSDDSAVYHCVREGRYSTSPFDYWG QGTLVTVSS (SEQ ID NO:1).

[0130] In one embodiment, the variable light chain of DUNP19 comprises the following sequence:DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNRNYLAWYQQKPGQPPKLFIYWSS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGQGTKLEIKR (SEQ ID NO:2).

[0131] Intravenously (i.v.) administered Lutetium-177 labeled DUNP19 ([177Lu]DUNP19) displayed specific and effective uptake in subcutaneous (s.c.) OS (SAOS2) models. Accumulation in normal tissues was very low and target-to-normal tissue ratios rapidly increased throughout the observed 2 weeks. Compared to SAOS2 models, uptake of [177LU]DUNP19 in s.c. U118 GBM models resulted in lower tumor specific accumulation, compatible with LRRC 15 expression levels. In both models, tumors showed the highest uptake of [177LU]DUNP19 after 24-72 hours while rapidly clearing from all other organs. A single injection of [177Lu]DUNP19 resulted in robust therapeutic anti-tumor effects.

[0132] The possibility of using DUNP19 for LRRC15-targeted PET was further investigated. To explore this potential application, DUNP19 was labeled with a positron emitting radionuclide Copper-64 ([64Cu]DUNP19). In vivo kinetics and targeting performance of |MCU |DUNP I 9 was evaluated in LRRC15 expressing osteosarcoma s.c. tumors (SAOS2). Results demonstrated highly specific uptake with excellent tumor to background ratio. Together with data based on other derivatives (e.g. [177Lu]DUNP19, [AF594]DUNP19 and DUNP19-LF), DUNP19 clearly demonstrates impressive versatility as a therapeutic pharmacological agent.

[0133] Internalization of DUNP19 into target cells in vivo after systemic administration was confirmed by confocal microscopy imaging (Leica TCS-SP5). Specifically, 50pm cryosections of SAOS2 s.c. tumors that were harvested from animals 72 hours after i.v. administration of fluorophore AlexaFluor-594 labeled DUNP19 ([AF594]DUNP19) were imaged. To facilitate anatomical localization of [AF594]DUNP19 in relation to subcellular compartments, 50pm sections of snap-frozen OCT-fixed blocks were co-stained for DNA (dapi, blue) and actin ([AF647]phalloidin, red), or lysosomal compartments ([AF488]LAMP1 , yellow). The confocal images clearly demonstrated a majority of [AF594]DUNP19 was in the cytoplasm of the tumor cells, which is in full concordance with the in vitro results.

[0134] A low fucose variant of DUNP19 (DUNP19-LF) was constructed to evaluate the possibility of treating LRRC15-expressing tumors by directing effector cells to recognize cell surface bound DUNP19-LF. Specifically, LRRC 15 -directed ADCC was studied in cell lines derived from hematological malignancies (B-cell acute lymphoblastic leukemia lines Kasumi- 2 and MHH-Call-3) and a solid tumor (Osteosarcoma, SAOS2). The target cells were labeled with luM CFDA-SE and plated at 0.01 x 106cells per well in a 96- well plate. Serial dilutions of 0-100ng / mL of DUNP19-LF or non-specific low fucose isotype control (IgGl-LF) were added to target cells and incubated for 40 minutes at 37 degrees before the addition of effector cells. NK92 transfected CD 16 effector cells were activated with lOOU / mL IL-2 for 24 hours before adding to target cells at 5:1 or 10: 1 effector: target cell ratios. Assay plates were then incubated for 6 hours at 37 degrees and dead cells were stained with fixable viability dye (FVD) 660. Flow cytometry was used to determine all CFDA-SE+ / FVD660+ cells to identify dead target cells killed by antibody dependent cell-mediated cytotoxicity.

[0135] LRRC 15 -targeted ADCC using DUNP19-LF was demonstrated in the LRRC15- expressing tumor cell lines Kasumi-2 and MHH-Call-3 and solid tumor cell line Saos-2. In B cell acute lymphoblastic leukemia lines Kasumi-2 and MHH-Call-3, addition of 2ng / mL to8ug / mL of anti-LRRC15 DUNP19 increased NK92-CD16 mediated cytotoxicity from 25% to 60-80%, indicating that DUNP19 can specifically direct NK92-CD16 cells to initiate ADCC in LRRC15 positive tumor lines. With the addition of isotype control antibody IgGl-LF or effector cells alone, target cell death remained below 30% and is attributable to nonspecific cytotoxicity of NK92-CD16 cells towards tumor cells. In osteosarcoma cell line SAOS2, ADCC was observed with the addition of 0.4ng / mL to lOOng / mL DUNP19, killing between 35-50% of SAOS2 cells. Overall target cell death in both the control and experimental groups was lower than previous ADCC assays and may be due to the varying degrees of cell viability and activation seen in the effector NK92-CD16 cell line. In summary, these results demonstrate that unlabeled DUNP19 has an impressive potential as an LRRC15 targeted immunotherapeutic compound.Example 2ADDITIONAL STUDIES

[0136] DUNP19. DUNP19 comprises the variable heavy chain sequence SEQ ID NO:1 and the variable light chain sequence SEQ ID NO:2. For labeling with Lutetium- 177 and Copper- 64, respectively, DUNP19 was functionalized with benzylisothiocyanate derivatives of the acyclic chelating agent CHX-A”-DTPA (p-SCN-CHX-A”-DTPA) or cyclic NOTA (p-SCN- NOTA), respectively, using amine-reactive chemistry. Radiolabeling was performed as previously described. The average radiochemical yield was 95±5 % with a radiochemical purity >99 % and an average specific activity of 4 MBq / mg. Injection solutions of [177Lu]-DUNP19 were formulated in 2% BSA / PBS containing 0.14 mM EDTA (100-fold molar excess). Binding affinity of the radio-conjugate to life cells was determined using Ligandtracer technology.

[0137] An amine-based protein labeling kit (Invitrogen, #A20173) was used for labeling DUNP19 with AlexaFluor-647 or AlexaFluor-594. Labeling was performed as described by the kit’s recommended protocol.

[0138] Cell culture. U118MG (glioblastoma), U87MG (glioblastoma), RPMI7951 (melanoma), NCI-H196 (small cell lung cancer), HCC1954 (breast cancer), SAOS2 (osteosarcoma; OS), U2OS (OS), Kasumi-2 (leukemia), Calu-1 (non-small-cell lung cancer), RCH-ACV (leukemia), MHH-Call-3 (leukemia), Hs737.T (giant cell sarcoma), HEK293T, LNCaP (prostate cancer), and K7M2 (murine osteosarcoma) were purchased from ATCC. Hu09 (OS) was purchased from the Japanese Cancer Research Resources Bank (Tokyo, Japan). All cell lines were cultured according to the manufacturer’s instructions and frequentlytested for Mycoplasma.

[0139] To overexpress LRRC15, HEK293T and K7M2 cells were transduced with a pLenti- LRRC15-GFP-Puro vector (Origene, NM_130830) with a multiplicity of infection of 5; following selection with puromycin for 14 days, single cell clones were picked.

[0140] Pulldown Assay (Immunoprecipitation - Mass Spectrometry). Pierce Protein G Magnetic Beads (ThermoFisher, #88847) were pre-incubated with 70 ug DUNP19 antibody or hlgGl mAb in PBS at room temperature (RT) for 1 h. Bead-antibody conjugate was recovered using magnetic separation before adding 300 ug protein lysates from U118MG cells (LRRC 15- positive) for 2 h on ice. Beads were washed with PBS and an on-bead digestion was performed with 0.25 % Trypsin for 16 h. MS-MS was run using the Agilent 6530 LC / MS in collaboration with the UCLA Molecular Instrumentation Center according to previously described protocols.

[0141] Flow Cytometry. To assess the binding affinity of DUNP19, cells were blocked (10 % normal goat serum / PBS, 15 minutes, RT) and incubated with serial dilutions of fluorophore- conjugated DUNP19 or hlgGl mAh (0.051 - 1000 ng / mL) in triplicate for 1 h at RT. Cells were washed with 1% BSA in PBS (180 x g, 3 minutes) before adding viability dye per manufacturer’s instructions (Invitrogen, #L34989). Antibody binding capacity of cells was assessed using 1 pg / mL DUNP19 and anti-human TgG Simply Cellular bead standards (Bangs Laboratories, #816). Quantity of LRRC15 surface antigens available for DUNP19 binding was normalized to cell surface area (determined experimentally by confocal microscopy). All flow cytometry experiments were run in collaboration with UCLA’s Jonsson Comprehensive Cancer Center Flow Cytometry Shared Resource using an Attune NxT Flow Cytometer (Invitrogen). Flow cytometry data were analyzed using FlowJo (Version 10, BD Biosciences).

[0142] Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Expression of LRRC15 in cells was determined by Taqman qRT-PCR. Cells were lysed and reverse transcription performed with Cells-to-CT Taqman kit (A25603). LRRC15 was probed with Applied Biosystems Taqman Assay probes (Assay ID Hs00370056_sl) and normalized using GAPDH housekeeping gene (Assay ID Hs02786624_gl). All qPCR assays were run using the ViiA 7 Real-Time PCR system (Applied Biosystems).

[0143] Confocal Microscopy of Cells. For immunocytochemistry, cells were blocked with 10% normal goat serum in PBS for 1 h at room temperature. DUNP19 (20 ug / mL) and anti- PMCA1 antibody (Abeam, ab3528, 1 :100). Primary antibodies were incubated overnight at 4 °C before washing and staining with goat anti-rabbit AlexaFluor647 (1:500, InvitrogenA21235) and goat anti-human AlexaFluor488 (1:500, Invitrogen A11013) secondary antibodies. Cells were fixed with 3.7% paraformaldehyde for 15 minutes at RT before washing and mounting cells with Vectashield mounting media with DAPI (H 1200- 10) onto slides for imaging with a Leica TCS SP8 Digital Light Microscope.

[0144] To confirm internalization, cells (0.0015 x 106cells / well) were seeded in phenol red- free complete media in 384- well u-clear flat bottom black plates (Greiner, #781092) and stained with 1 ug / mL DUNP19-AF647, anti-huLAMPl-A488 antibody (1:250, Invitrogen, 53- 1079-42) and Hoechst 33342 (1 :2000 dilution, Invitrogen) at 4°C for 1 h to prevent antibody internalization while promoting surface binding. Unbound antibody was aspirated and replaced with phenol red-free complete media. Timelapse confocal imaging was performed using a temperature controlled ImageXpress MicroXL High Content Imaging microscope (Molecular Devices). The microscope temperature was set to mimic cell culture conditions (37°C, 5% CO2) and images were taken at lOx magnification (4 sites per well) every 20 minutes for 6-12 h. The plasma membrane signal of DUNP19-AF647 relative to the cytosolic signal (LAMP1) at time 0 (directly after incubation at 4°C) was quantified to calculate the fraction of internalized antibodies over time.

[0145] Subcutaneous Tumor Models. Athymic nude mice (BALB / cAnNRj-Foxnl nu / nu; 6- 8 weeks old, 20-25 g; Janvier) were inoculated with U118MG (5.8 x 106cells), SAOS2 (6 x 106cells), HuO9 (6 x 106cells), HCC1954 (4.9 x 106cells) or LNCaP (5 x 106cells) cells in a 100 pL (1 :1 v / v) mixture of media with Matrigel via subcutaneous injection in the right flank. Tumors developed after 3 to 6 weeks. Tumor volume was estimated with caliper measurements twice weekly (V (mm3) = 0.5 x length x width2).

[0146] Orthotopic Osteosarcoma Model. Nineteen athymic nude mice (BALB / cAnNRj- Foxnlnu / nu; males, 6-8 weeks old, 24.2+1 g; Janvier) were anesthetized, the right knee joint was shaved and the tibia of the right hind limb was punctured. HuO9 cells (1.5 x 106cells in 10 pL media) were injected into the cavity using a microvolume syringe with a 27-gauge needle. Bone wax (Surgical Specialties Corporation, #903) was applied to seal the punctured area and prevent exodus of implanted cells before the area was washed with saline. Tumor development was confirmed using ultra-high resolution CT and [177Lu]-DUNP19 SPECT / CT imaging (nanoScan; Mediso, Budapest, Hungary).

[0147] Imaging. PET: Male mice (B6;129-Rag2tmlFwaII2rgtmlRsky / DwlHsd) (n = 5) bearing s.c. SAOS2 xenografts were intravenously (i.v.) injected with |wCu|-DUNP I 9 (8.75MBq, 100 pg DUNP19 in 100 pL 10 mM ammonium acetate). Dynamic PET images were acquired during the first hour post-injection (p.i.), followed by static scans (20 minutes each) at 12 h, 24 h, and 36 h using a microPET R4 rodent scanner (Siemens). To confirm specificity of the DUNP19 PET-signal, imaging with the bone-seeking PET-probe Fluorine 18-sodium fluoride ([18F]-NaF; 10 MBq i.v.) was performed in mice bearing SAOS2 tumors. SPECT: Mice with intratibial HuO9 xenografts were i.v. injected with [177Lu]-DUNP19 (20 MBq, 30 pg). Mice were scanned for 50-60 minutes under anesthesia (2-3 % isoflurane) using a SPECT / CT device (nanoScan Mediso, Budapest, Hungary).

[0148] [177Lu]Lu-DUNP19 Biodistribution and Kinetics. To investigate the biodistribution of [177LU]-DUNP19, mice with s.c. SAOS2, Hu09, U118MG, HCC1954, or K7M2 tumors, respectively, received 0.5 MBq [177Lu]-DUNP19 (SAOS2, U118MG, K7M2: 16 pg; Hu09, HCC1954: 30 pg) by i.v. injection and were sacrificed at predetermined time points (SAOS2: 6, 24, 48, 72, 168 and 336 hours p.i.; HuO9: 6, 24, 48, 72, 168 and 336 h p.i.; U1 18MG: 24, 48 and 72 hours p.i.; n=4 mice / time point, HCC1954 and K7M2: 72 h p.i.). To confirm targeting specificity, mice bearing both LRRC15+ U118MG (left flank) and LRRC 15-negative LNCaP (right flank) xenografts were i.v. injected with either 0.5 MBq [177Lu]-hIgGl mAh or [177Lu]-DUNP19 (n=4 mice) and sacrificed 48 hours later. To study the effect of the antibody mass amount on the biodistribution and tumor uptake, mice bearing Hu09 tumors (n=5 mice / group) were administered escalating DUNP19 doses (1, 10, 30, 100 and 300 pg / mouse at 0.5 MBq177LU; i.v.) and euthanized 72 h p.i.. In all studies, blood, tumors and several normal tissues were collected, dried and weighed. The radioactivity contained in the respective tissue and reference standards (0.5 MBq |177Lu|-DUNP19) was quantified in a Nal(Tl) automated well counter (1480 WIZARD; Perkin Elmer). Decay-corrected data were expressed as percent injected activity per gram tissue (% lA / g).

[0149] Imaging Quantification. Tumor calcification was analyzed using Imaged software (version 1.53). A brightness threshold of 41 was used to define the area of each tumor sample. The total area of the tumor was then quantified using Imaged ’s built-in area analysis program. Areas of calcification were defined by a brightness threshold of 81 and quantified in the same manner. Percent calcification was calculated by dividing the area of calcification over the area of the entire tumor.

[0150] Confocal Microscopy of Xenografts. Mice with Hu09, SAOS2 and HCC1954 tumors, respectively, were injected with 30 ug DUNP19-AF594 when tumor volume reached between 200-300 mm3. Sections were washed two times with PBS before permeabilizationwith 0.1 % Triton-X / PBS, and blocking for 1 h at RT with 10 % goat serum / PBS. Sections were stained with an anti-LAMPl-A488 antibody (Hu09, SAOS2: anti-hLAMPl, 1:250, Invitrogen, #53-1079-42; HC1954: anti-mLAMPl, 1:250, Invitrogen, #53-1071-82, to detect the murine stroma) overnight at 4°C. The next day, slides were stained with Phalloidin-AF546 for 1 h at RT (1 :2000, Invitrogen, #A22283) and mounted with Vectashield Antifade mounting media with DAPI (Vector Laboratories, H-2000-2). Confocal microscopy was done by the UCLA Advanced Light Microscopy and Spectroscopy Laboratory (ALMS) with a Leica TCS- SP8 microscope at 63x magnification and sequential track imaging for far-red, orange, green and blue emitting dyes. Z-stack of the sections were imaged. Lightning deconvolution and 3D reconstruction (Leica Microsystems) was performed during post-processing.

[0151] Therapy Studies. Average tumor volume at the start of treatment was 181+20 mm3and the average animal weight was 25.2+0.7 g. Mice bearing s.c. HuO9 or U 118MG xenografts with LRRC 15 expression in both tumor cells as well as tumor stroma, and HCC 1954 xenografts with LRRC 15 expression only in the tumor stroma, were randomized into groups of 10-12 animals receiving either [177Lu]-DUNP19 (16-30 pg, i.v.) or no treatment. HCC1954 model: Mice were treated with a single injection of either 10 MBq or 20 MBq [177Lu]-DUNP19. Hu09 model: Animals received three fractionations [177Lu]-DUNP19 at days 0, 32 and 75 resulting in a cumulative administered activity of 50 MBq (group 1 : 10+20+10 MBq; group 2: 20+10+20 MBq). To investigate the impact of pre-therapeutic tumor volume on the efficacy of [177Lu]- DUNP19 in the Hu09 model, mice (n=6-10 mice / group) were administered 30 MBq [177Lu]- DUNP19 (30 pg, i.v.) when tumors reached a volume of 203+64 mm3(group 1) or 504+152 mm3(group 2). U118MG model: Mice were treated with two fractions 1177Lu]-DUNP19 at days 0 and 34 and a cumulative activity of 20 MBq (10+10 MBq) or 30 MBq (20+10 MBq). Treatment efficacy was assessed by measuring tumor growth and time to humane endpoint. Mice survival was analyzed by using a log rank test in GraphPad Prism. P values <0.05 were considered significant statistically. Tumors from a subset of mice were harvested 90-120 days p.i. (except U118-MG: day 155 p.i.) and processed for RNA-sequencing (see below).

[0152] To evaluate the efficacy of |177Lu|Lu-DUNP19 in a clinically relevant orthotopic osteosarcoma model, mice with intratibial Hu09 tumors were randomized (23 days post-tumor engraftment) to receive 20 MBq177Lu-DUNP19 (30 pg, i.v.; n=12) or PBS (n=7). Four days after treatment, [177Lu]Lu-DUNP19 tumor uptake and presence of viable HuO9 tumor was assessed by SPECT / CT imaging. At day 163 post- first injection, mice received an additional 20 MBq [177Lu]-DUNP19 and were re-scanned to detect residual viable HuO9 tumor tissue.Mice were followed up for 190 days after the first [177Lu]-DUNP19 injection.

[0153] Toxicity of [177Lu]-DUNP19. Body weights and hematological toxicity and recovery were monitored in mice treated with [177Lu]-DUNP19. Blood samples were taken before and weekly after injection of [177Lu]-DUNP19 for 4 weeks p.i.. Samples (20 pL) were collected from the tail vein of awake, immobilized mice by piercing the vein with a needle (27G) and collecting blood in a K2EDTA-coated plastic micropipette. Blood cell counts were obtained using an Exigo Veterinary Hematology Analyzer (Boule Medical, Stockholm, Sweden).

[0154] Immunohistochemistry. LRRC15 expression was analyzed on formalin-fixed, paraffin embedded sections using the Dako REAL Peroxidase Detection System (Dako) according to the manufacturer’s instructions. Antigen retrieval was performed by heat-induced epitope retrieval using Tris / EDTA (pH 8.1). Sections were incubated with anti-hLRRC15 antibody [EPR8188(2)] (1:100; #abl50376, Abeam) for 1 h at room temperature.

[0155] Gene Expression Analysis. For RNA-sequencing, tumor tissues were harvested at 90- 120 days (Hu09, HCC1954) or 155 days (U118MG) post-treatment, with the exception of untreated mice (harvested when tumor volume measured greater than 1000 mm3, in accordance with established endpoint protocols). Tumor tissue was preserved in RNA / umr stabilization solution (Invitrogen, AM7020) before RNA isolation with the Qiagen RNeasy kit (#74004). RNA quality control was assayed via TapeStation (Agilent) and stranded mRNA library preparation performed in accordance with Illumina protocols. Samples were sequenced on Illumina’s Novaseq platform to generate 50bp paired-end reads. Library preparation and sequencing was done with the help of UCLA’s Technology Center for Genomics and Bioinformatics (TCGB).

[0156] Raw read count RNA-sequencing data were generated from untreated and [177Lu]- DUNP19 treated Hu09 (untreated n=6, treated n=16), U118MG (untreated n=8, treated n=16) and HCC1954 (untreated n=8, treated n=10) tumors. Paired-end reads were aligned to either human (Hg38) or murine (Mml9) genome using BBSplit, as described in the STAR method. Ambiguous reads were discarded and FastQC analysis was utilized to confirm sequence quality. Low read count filtering was used to remove transcriptomic features for which fewer than 4 samples had at least 5 read counts of a gene, as described by the EdgeR differential analysis user guide. For each tumor model, principal component analysis based on log2 counts in RStudio Version 2023.06.1+524 was plotted. K-Clustering and heat maps were generated on log2-transformed read counts to visualize gene signatures in treated versus untreatedsamples. Differential expression analysis to identify differentially expressed genes was performed using EdgeR (Bioconducter, Version 3.40.2) using quasi-likelihood F-tests within the EdgeR program. A false discovery rate of 5% (adjusted using Benjamini-Hochburg methodology) and absolute log2-fold change >2 were selected as the cutoff for DEGs within this analysis. A positive fold-change represented upregulation and a negative fold change represented downregulation of gene expression in treated tumors. For comparison and visualization of gene expression between clustered samples, z-scores were calculated per gene. Pathway analysis was performed using gene set enrichment analysis and molecular signatures defined using the human and murine Molecular Signature Database (33). Additional analysis was performed using Gene Ontology (GO) Biological Pathways.

[0157] Statistical Analyses. Statistical analyses were conducted using Graphpad Prism software (Version 9.5.1). Data are expressed as mean and standard deviation. Statistical comparisons were performed using one-way ANOVA with Tukey multi-comparison tests and unpaired Student’s t-tests. A P-value of less than 0.05 was considered statistically significant.

[0158] Conjugation and radiolabeling of DUNP19. All buffers were treated with Chelex 100 resin (sodium form, Merck KGaA, Darmstadt, Germany) to remove any metal ions and filtered through a 0.22 pm filter before use. Prior to conjugation, DUNP19 was buffer exchanged using an AMICON Ultra-0.5-centrifugal filter devices with a MWCO 30 kDa (Millipore, Burlington, MA, USA). DUNP19 (500 pg in 500 pl 0.07 M sodium borate, pH 9.3) was mixed with the bifunctional chelator p-SCN-CHX-A”-DTPA (Macrocyclics, Texas, USA) in a 6:1 molar ratio. The mixture was extensively vortexed and incubated overnight at 38°C. The reaction mixture was then centrifuged for 10 minutes at 14000 x g using a 30 kDa filter to remove the excess non-bound chelator. The concentrate containing the CHX-A”-DTPA-DUNP19 conjugate was recovered, and the buffer was adjusted to 0.2 M ammonium acetate (pH 5.5). The conjugate was aliquoted and kept at -20 °C until labeling. For labeling with177Lu, 30-50 pg of the conjugate was mixed with 15-20 MBq177LuCh ( Curium, Sweden) and incubated at 38°C with continuous vortexing for 30 min. Thereafter the radiolabeled conjugate was purified and buffer exchanged using AMICON 30 kDa filter. Radiochemical yield and purity of the radioconjugate were determined using silica-impregnated ITLC strips (150-771 DARK GREEN Tec-Control Chromatography strips, Biodex Medical Systems) eluted with 0.2 M citric acid and measured using the Cyclone Storage Phosphor System (PerkinElmer, Waltham, MA, USA). To reduce radiolysis, the final product was diluted with 2% BSA / -PBS buffer (pH 7.4) and 100X molar excess of EDTA was added to scavenge any free metal ions.

[0159] To determine the maximal attainable specific activity for labeling DUNP19 with177Lu, decreasing amounts of CHX-A”-DTPA-DUNP19 (10, 5, and 2.5 g) were incubated at 38°C for 30 minutes with a fixed quantity of177LuC13 (10 MBq). The radiochemical yield was determined using SG-ITLC, as previously described. Copper-64 was produced at the Washington University in St. Louis School of Medicine Cyclotron facility.<CuCI2 (20 mCi; 25 uL) was diluted with a 10-fold excess 0.1 M ammonium acetate (NH4OAc) ,pH 5.5 and then added to NOTA-conjugated anti-LRRC15 antibody. After mixing for 30 min at room temperature, the antibody conjugate was purified by gel chromatography (PD10) into 0.1 M HEPES buffer in saline. Purity was assessed by radioITLC (Bioscan AR2000 using samples spotted on Whatman paper in a running buffer of 50 mM DTPA (pH 5.5). Quantitative labeling with >99% radiochemical purity was observed.Results

[0160] LRRC15 can be Targeted Utilizing the mAb DUNP19. Initial investigations tested binding specificity of DUNP19. The mAb’s capacity was demonstrated to associate with both human and murine recombinant LRRC15, exhibiting picomolar affinity to both epitopes (Fig. 7A). Specific interaction of DUNP19 with LRRC15 was further substantiated by immunoprecipitation of the LRRC15 protein from a U118MG cell lysate using DUNP19- coated magnetic beads (Fig. 7B). We next characterized the binding of DUNP19 to LRRC15 across a wide range of cancer cell lines from a range of indications including melanoma, glioblastoma and osteosarcoma. DUNP19 demonstrated picomolar affinity for its target antigen (EC50 = 0.125 - 33 ng / mL) (Fig. 1A). Cell lines were selected based on RNA levels in publicly available databases: the EMBL-EBI expression atlas, Harmonizome 3.0, COSMIC, and the Cancer Cell Line Encyclopedia (CCLE) database. Despite selecting cell lines that exhibited high LRRC15 expression, not all were found to have detectable protein on the cell surface. Among those that did, a positive correlation was noted between the number of LRRC15 antigens on the cell surface and LRRC15 mRNA levels (R2= 0.567) (Fig. IB). Specific binding to LRRC15 on the cell surface was further confirmed by confocal microscopy of AlexaFluor647-labeled DUNP19 (Fig. 1C). Next, we studied cellular internalization of DUNP19, a parameter that enhances the retention time of the delivered radionuclide, thereby increasing the radiation dose to the target tissues. Confocal microscopy studies showed that DUNP19 was swiftly internalized by LRRC15 expressing cells (Fig. 1D,E). Interestingly, internalization rates for DUNP19 seemed to be contingent on the quantity of available molecules; faster kinetics were observed in cells with a higher abundance of LRRC15molecules (Hu09: 1.41 x 106antigens, 132.06 ± 10.14 minutes; SA0S2: 0.28 x 106antigens, 145.62 ± 15.18 minutes; Fig. ID). Radiolabeling chemistry did not impact DUNP19; the internalization rate of the CHX- A”-DTP A-conjugate was analogous to that of the unconjugated antibody (Fig. 8A). Furthermore, time-resolved cellular assays indicated that]"Lu- radiolabeling did not affect affinity (U118MG: Kd = 301 + 39 pM; Hu09: 117 + 46 pM; SAOS2: 56 ± 27 pM; RPMI-7951: 25 ± 0.1 pM; Fig. IF).

[0161] DUNP19 In Vivo PET can Monitor Tumor Associated LRRC15 Expression. To evaluate in vivo kinetics of DUNP19 in healthy organs and LRRC 15 -expressing tumors, sequential PET images were acquired of subcutaneous (s.c.) osteosarcoma (SAOS2) bearing mice after intravenous (i.v.) administration of a64Cu-labeled version of the mAb (|<4Cu|- DUNP19). Tumor accumulation was compared to the clinical bone scanning agent [18F]-NaF. |<Cii|-DUNP 19 rapidly and persistently accumulated in LRRC 15+ tumors and decreased from healthy tissue (tumor-to-muscle ratio) over time. In contrast, [18F]-NaF exhibited limited accumulation in osteogenic tumors, with the bladder showing the highest activity due to urinary excretion (Fig. 2A).

[0162] [177Lu]-DUNP19 Exhibits a Favorable Biodistribution.177LU is a clinically relevant beta particle emitter with a half-life of 6.7 days that can be used to deliver ionizing radiation to a target cell, but also to target-null cells in close vicinity via a crossfire effect. Given these characteristics, we hypothesized that [177Lu]-DUNP19 could overcome the heterogeneous expression of LRRC 15 in tumor tissues. First, we evaluated the influence of antibody carrier mass on the biodistribution of [177Lu]-DUNP19. These studies were carried out in s.c. Hu09 tumors and it was determined that a mass of 15-30 pg yielded the optimal tumor-to-tissue uptake ratios. Next, we systematically examined the biodistribution and pharmacokinetic profile of [177Lu]-DUNP19 in a variety of s.c. tumor models originating from diverse malignant tissues. These models encompassed varying levels of LRRC 15 expression and exhibited target expression in distinct tumoral compartments. Evaluations included lesions expressing LRRC 15+ human cancer cells and LRRC 15+ murine CAFs, such as HuO9 and SAOS2 osteosarcomas (OS), and U118MG glioblastoma (GBM). Studies were also conducted in a breast cancer model (HCC1954) characterized by high LRRC15 expression in CAFs, but lacking LRRC15 expression in cancer cells. Lastly, uptake of [177Lu]-DUNP19 was assessed in a murine osteosarcoma model (K7M2AA’AY / 1+), where LRRC 15 is expressed on cancer cells (Figure 2C, 9).

[0163] The accumulation of [177Lu]-DUNP19 in tumors peaked at 72 h post-injection (p.i.)(HCC1954: 12.5 ± 2.8 %IA / g [percent injected activity per gram tissue], U118MG: 13.3 ± 1.1 %IA / g, K7M2“flC75+: 13.6 ± 1.5 %IA / g, SA0S2: 23.1 ± 2.9 %IA / g, HuO9: 43.9 ± 7.9 %IA / g) and remained consistently elevated at all studied time points throughout the time course, up to 336 h p.i. (Figure 2B). Retention of [177Lu]-DUNP19 steadily decreased in blood and healthy organs after injection, and [177Lu]-DUNP19 in blood reflects the expected half-life of a human IgGi in mice, indicating interaction with the murine neonatal fragment crystallizable (Fc) region receptor (FcRn). Of note, murine FcRn demonstrates higher affinity for human IgGi Fc compared to its murine counterpart. Therefore, it is anticipated that blood kinetics would be decreased in translation to the clinic. Retention of [177Lu]-DUNP19 in the liver was representative of typical blood volume and metabolic elimination of antibodies. Taken together, these data indicate a favorable biodistribution profile of [177Lu]-DUNP19 (Fig. 2D,E).

[0164] LRRC15 targeting specificity in vivo was further addressed in s.c. LRRC15+ (U118MG) tumor model by comparing uptake of [177Lu]-DUNP19 to [177Lu]-huIgGi, a human IgGi with non-binding complementary-determining regions (CDRs) that had been radiolabeled with177LU. At 48 h after i.v. administration, tumor uptake of [177Lu]-DUNP19 was significantly higher than [177Lu]-huIgGi, with 14.31 ± 2.01 vs. 4.72 ± 0.82 %IA / g, respectively. Additionally, in LRRC15- s.c. LNCaP tumors, which lack relevant amounts of murine LRRC15 expressing stroma, but are highly vascularized, systemic injection of [177Lu]- DUNP 19 resulted in tumor retention of 6.57 ± 1.28 %IA / g at 48 h p.i. (Fig. 2B). These findings indicate that DUNP19 specifically targets LRRC15-expressing tumor tissue with minimal off- target retention in vivo. The observed accumulation of [177Lu]-DUNP19 in LRRC 15-negative tumors is likely attributed to the enhanced permeability and retention effect, a pathophysiological mechanism wherein the tumor vasculature traps macromolecules >45 kDa.

[0165] Next we investigated the cellular localization of DUNP19 in SAOS2, Hu09, and HCC1954 tumor tissues following systemic administration. Sections from s.c. tumors collected 72 h after i.v. injection of AlexaFluor647-labeled DUNP19 were co-stained for DNA, actin, and lysosomes (LAMP1), and analyzed by confocal microscopy (Fig. 2F,G; Figure 10B). Consistent with our in vitro findings, DUNP19 co-localized with murine LAMP1 in HCC1954 tumors, and with human LAMP1 in Hu09 and SAOS2 tumors. This co-localization suggests cellular internalization of the antibody subsequent to binding with LRRC 15 on the plasma membrane of both cancer and stromal cells.

[0166] Single and Sequential LRRC15-Tar eted Radioimmunotheranostic Applications of [177Lu]-DUNP19 in Osteosarcoma. We investigated the impact of [177Lu]-DUNP19 ontumor volume and overall survival. Following a single systemic administration of 30 MBq [177LU]-DUNP19, significantly reduced tumor growth and prolonged survival were observed in mice bearing s.c. HuO9 tumors. Importantly, treatment with [177Lu]-DUNP19 was even effective in larger tumors (500-600 mm3), albeit to a lesser extent than in smaller (150-200 mm3) ones; this underlines the potential of our RIT to induce antitumor effects in tumors of different sizes (Fig. 3Ai).

[0167] However, irrespective of initial tumor volume, administration of a singular dose of 30 MBq [177LU]-DUNP19 significantly prolonged survival in mice carrying HuO9 s.c. tumors (Fig 3An). Left untreated, the median duration from s.c. inoculation to euthanization was 65 days (range: 58-69 days). All mice subjected to treatment with 30 MBq [177Lu]-DUNP19 at the 150- 200 mm3stage survived through the entirety of the observation period (126 days) compared to 115 days (67-126 days) in the group injected at 500-600 mm3. These findings underscore the substantial impact of a solitary treatment with [177Lu]-DUNP19 on the overall survival of mice, regardless of the tumor size at the initiation of therapy (p < 0.005; Fig. 3 A,B).

[0168] We further studied LRRC15-targeted radio-theranostics in a translationally relevant orthotopic OS model (Fig. 3B, C). HuO9 cells were injected into the left tibia of Balb / c mice and half of the subjects were randomly selected for systemic injection with 30 MBq of [177Lu]- DUNP19 23 days after inoculation. Imaging by SPECT at 72 h after [177Lu]-DUNP19 administration revealed specific accumulation of activity at the tumor site. Within six months of inoculation, all untreated subjects succumbed due to disease-related endpoints. In the treated group, an additional assessment by SPECT imaging was carried out 72 h after injection with a second 30 Mbq dose of [177Lu]-DUNP19. Results from this study revealed that the radiolabeled antibody did not accumulate at the site of Hu09 cell inoculation, or at other anatomical locations (Fig. 3Bi). Based on these results, we concluded that the previously detected LRRC15+ tissue had completely regressed. Further, within the studied time-frame, [177Lu]- DUNP19 were shown to significantly extend survival when compared to untreated animals (Fig. 3Bn).

[0169] Next, we conducted therapy studies using fractionated dosing of [177Lu]-DUNP19. From a translational perspective, this approach is commonly utilized in clinical settings to optimize maximum tolerated dose while reducing dose-limiting toxicities. Rather than adhering to a predetermined activity and treatment schedule, subjects were administered additional therapy doses based on bone marrow toxicity, tumor volume, and their effect on animal weight. Fractionation is also recommended to compensate for the anticipatedheterogeneity in RIT dose distribution, particularly in large poorly vascularized tumors with regions of hypoxia. Mice bearing subcutaneous Hu09 tumors (150-200 mm3) were given cumulative activities of 50 MBq in three fractions over a span of 88 days (Fig. 3Ci). Continuous progression-free survival was sustained in the treated animals throughout the entire study duration, starkly contrasting to untreated animals, which exhibited a median survival of 47 days (Fig. 3Cn).

[0170] Radiation is widely known to induce calcifications in sarcomatous processes. To investigate this phenomenon in animals bearing Hu09 lesions, we quantified tumor radiopacity and the uptake of tumor- associated [177Lu]-DUNP19 using SPECT / CT and gammaspectrometry. Animals treated with [l"Lu]-DUNP19 and untreated mice received an imaging dose of 3.5 MBq [177LU]-DUNP19, and tumors were harvested 72 hours after injection. Treated tumors exhibited significantly lower tumor-associated [177Lu]-DUNP19 activity, coupled with higher levels of tumor calcification (Fig. 3Cin-v).

[0171] [177Lu]-DUNP19 Therapy is Applicable Across a Range of Tumors with Varying Target Expression Patterns. Given the high expression level of LRRC15 on Hu09 cancer cells (Fig. 1), we also sought to understand how the effects of [177Lu]-DUNP19 therapy would change in a tumor with lower LRRC15 expression and of different tissue origin. Mice bearing s.c. U118MG tumors were treated with a cumulative activity of 20 MBq or 30 MBq [177Lu]- DUNP19 in two fractions. [177Lu]-DUNP19 treatment significantly (p<0.0001) extended survival, and therapy prevented further tumor growth as tumor volumes reached a plateau phase around 100-200 mm3. All untreated mice succumbed due to disease-related endpoints by 78 days, whereas 11 / 12 mice (91.67 %) and 12 / 12 mice (100%) were alive at the end of the observation period in the 20 MBq and 30 MBq treatment groups, respectively (Fig. 4A,B).

[0172] Finally, we evaluated the therapeutic efficacy of [177Lu]-DUNP19 in aggressive breast cancer tumors comprising LRRC 15-negative HCC1954 cancer cells and LRRC15+ murine CAFs. In these HCC1954 tumors, a single systemic injection of 20 MBq [177Lu]-DUNP19 effectively suppressed tumor growth and significantly extended median survival compared to untreated mice, where median survival was 30.5 days (p=0.0005; median survival not reached in treated animals). Notably, 80% of the treated mice (8 out of 10) survived until the end of the observation period. (Fig 4C,D).

[0173] Throughout all studies, treatment was well-tolerated as indicated by stable body weights (Fig. 1 1 ). Administration of [177Lu]-DUNP19 resulted in a transient bone marrowsuppression, which recovered to baseline levels within 21 days (Fig. 12).

[0174] LRRCIS-targeted RIT Depletes TGF|J-Driven Signature in Tumors. Having demonstrated the significant potential of our radioimmunotheranostic platform, we aimed to elucidate the molecular effects of LRRC15-targeted RIT on both cancer cells and the tumor microenvironment. Transcripts of bulk RNA-sequencing of tumors harvested at 90- 132 days after [177Lu]-DUNP19 treatment were aligned to human and murine genomes to identify the transcriptomic profiles of human cancer cells and murine stromal cells. Ambiguous reads were subsequently removed (Fig. 5 A).

[0175] In the light of the fractionated dosing regimens employed in our therapy studies (Fig. 3, 4), our first objective was to determine if [177Lu]-DUNP19 injected activity affected the transcriptomic signatures of treated tumors. However, these parameters were not a driver of transcriptomic changes in treated samples within each tumor model. Therefore, we did not separate tumor samples by [177Lu]-DUNP19 dose received in subsequent analyses and compared untreated and treated samples for each model.

[0176] In human cancer cells, RNA-sequencing identified 19,578 protein-coding genes of which 1,985 (Hu09), 1,043 (U118MG), and 23 (HCC1954) were differentially expressed genes (DEGs) following RIT (vs. untreated). In murine stroma, RNA-sequencing identified 663 (Hu09), 319 (U118MG), and 73 (HCC1954) DEGs compared to untreated controls. Tumors containing LRRC15+ cancer cells (HuO9, U118MG) shared 40 DEGs (Fig. 5E,F), several of which had functions similar to the hypothesized roles of LRRC15 (cell migration, invasion, and adhesion) or have been shown to be co-expressed with LRRC15, including COL11A1, FGF13, and CXCL14 (Fig. 5E,F). Gene ontology analysis of DEGs differed between the two models. In U118MG tumors, the most strongly upregulated genes included bone morphogenic protein (BMP2) and inhibin subunit beta A (INHBA), two modulators of TGF0 signaling and epithelial- mesenchymal transition (Fig. 5B,C); gene ontology terms were immune- and epithelial-mesenchymal transition related (response to cortisol, myeloid leukocyte mediated immunity, anatomical structural development) (Fig. 5C). In Hu09 tumors, anti-apoptotic genes (HRK) and genes in the canonical pro-tumorigenic WNT and RAS pathways were downregulated (Fig. 5B,D). Gene ontology analysis further revealed alteration of metabolic and cell cycle pathways in Hu09 treated tumors, including oxidative metabolism, carbohydrate metabolism, and cellular division and response to stress (Fig. 5D) 4.

[0177] To further explore the transcriptomic changes induced by RIT in the tumormicroenvironment, we also conducted a comprehensive analysis of the murine stroma in Hu09, U 118MG, and HCC 1954 tumors. In all three models, RIT induced changes in pathways related to immune activation, including the upregulation of Gzrnk, Cxcr6. and Lek (Fig. 6A-C). Considering prior reports, we aimed to delve deeper into the role of LRRC15 as a driver of immunosuppression. We performed consensus clustering and identified three distinct transcriptional clusters for treated cancer cells in the Hu09 and U118MG models and two clusters in HCC 1954. Intriguingly, similar clustering patterns were also observed in the tumor stroma (Fig. 13).

[0178] Based on these results, we studied whether these changes could be explained by an overall shift in cell types present within the [177Lu]-DUNP19 treated tumor samples (i.e. loss of mesenchymal phenotypes). We employed Syllogist to further assess the proportional distribution of cell types in treated tumors compared to untreated samples. In accordance with the expression of LRRC15 in cancer cells originating from mesenchymal stem cells, HuO9 and U1 18MG tumors displayed a notable overrepresentation of mesenchymal cells across all examined samples, with a significant loss of the mesenchymal cell phenotype in DUNP19- treated Hu09 cancer cells (Fig. 14). No discernible alterations in relative cell composition were observed following treatment with [177Lu]-DUNP19 in other tumor models. Additionally, our observations revealed that HCC 1954 tumor cells predominantly maintained an epithelial phenotype, consistent with the absence of LRRC15 expression in the cancer cells (Fig. 14).

[0179] Overall, and in line with plasma membrane associated LRRC L5 protein levels (Fig. 1B,C), LRRC15 expression was higher in HuO9 than in U118MG cancer cells, and not quantifiable in HCC1954 cancer cells. In contrast, stromal Lrrcl5 expression was 3- and 4-fold higher in HCC1954 tumors than in U118MG and HUO9 tumors, respectively (Fig. 15). Comparison of LRRC15 / Lrrcl5 expression in cancer cells and stroma of Hu09 and U118MG tumors across clusters showed a trend of decreased expression with increasing cluster distance from untreated samples in both cancer cells and stroma (Fig. 6D); the expression of TGFB1, a known regulator of LRRC15 expression, mirrored the LRRC15 / Lrrcl5 expression pattern (Fig. 6D). Interestingly, Lrrcl5 and TGFfil levels in HCC1954 stroma remained constant, while TGFB1 expression was increased in treated LRRC15- HCC 1954 cancer cells (Fig. 15).

[0180] Further analysis of LRRC15+ clusters showed that treatment with [177Lu]-DUNP19 resulted in the progressive loss of a gene signature associated with immune cell exclusion and poor response to immune checkpoint blockade in TGFP-driven, LRRC15+ CAFs (Fig. 6E,F). Consistently, in two mice with HCC 1954 tumors that did not respond to RIT, neither Lrrcl5nor the LRRC15+ CAF gene-signature decreased (Fig. 6H). Taken together, these findings collectively imply that LRRC15 serves as a critical modulator of the immune system within the tumor microenvironment and suggest that targeting LRRC15 with [177Lu]-DUNP19 could remodel the tumor microenvironment, hindering tumor progression.Example 3Combination Treatment of DUNP19 and Immune Checkpoint Inhibitors

[0181] Female Balb / c AnNRj mice (6-8 weeks old, 20-25 g; sourced from Janvier Labs) were utilized for this study. A highly aggressive subcutaneous (s.c.) syngeneic murine triplenegative breast cancer model was established by inoculating 5 x 1034T1 cells, suspended in a 200 pL (1: 1 v / v) mixture of cell culture medium and Matrigel, into the right flank. Tumor volume was assessed biweekly using caliper measurements and calculated with the formula V (mm ) = 0.5xlengthxwidth2. Treatments commenced when tumor volumes reached approximately 100-200 mm3. The 4T1 tumor- bearing mice were randomized into five treatment groups.

[0182] The first group (n=7) received a single intravenous (i.v.) injection of low-fucose anti- LRRC15177Lu-DUNP19 (m!gG2a-LF) (15 MBq). The second group (n=7) received intraperitoneal (i.p.) injections of combined immune checkpoint inhibitor (ICI) treatments: anti-CTLA-4 murine antibody (clone 9H10 Bio X Cell; initial dose of 8 mg / kg, followed by three doses of 4 mg / kg) and anti-PD-1 murine antibody (RMP1-14 ichorbio; 6 mg / kg) administered every 3 days for a total of four injections. The third group (n=8) received a combination treatment consisting of anti-LRRC15177Lu-DUNP19 (m!gG2a-LF) (15 MBq, i.v.) and i.p. ICI treatments (i.e., anti-CTLA-4 and anti-PD-1 murine antibody regimen). The fourth group (n=6) received only PBS (vehicle), serving as the untreated control. Lastly, the fifth group (n=8) received non-labeled low-fucose anti-LRRC15 DUNP19 mIgG2a-LF (300 pg / mouse, administered every 3 days for a total of three injections).

[0183] As shown in Figure 16, mice receiving ICIs alone (squares) or177Lu-DUNP19 treatment alone (circles) exhibited favorable tumor growth kinetics compared to the control (PBS) group (triangles) or the non-labeled anti-LRRC15 DUNP19 group (diamonds). The results further demonstrate that the combination treatment of177Lu-DUNP19 (single 15 MBq dose) and murine ICIs (inverted triangles) provides more advantageous tumor growth kinetics compared to either177Lu-DUNP19 or murine ICIs alone. In summary, these results demonstrate that177LU-DUNP19-RIT significantly decreases immunoresistance by ablating LRRC15-expressing tumor tissue, thereby enhancing the effectiveness of ICI treatment.

Claims

What is claimed is:

1. A method for treating a tumor in a patient comprising administering to the patient a combination of: a. a binding moiety comprising SEQ ID NO:1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent; and b. an immune checkpoint inhibitor.

2. The method of claim 1, wherein the tumor and / or stroma thereof expresses LRRC15.

3. The method of claim 1 wherein the tumor and / or stroma thereof produces TGFp.

4. The method of claim 1, wherein the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma.

5. The binding moiety of claim 4, wherein the prostate cancer is androgen receptor negative or androgen independent.

6. The binding moiety of claim 1 , wherein the tumor is metastatic.

7. The method of claim 1, wherein the tumor comprises stroma which comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

8. The method of claim 1, wherein the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR).

9. The method of claim 1 , wherein the binding moiety has picomolar affinity for LRRC15.

10. The method of claim 1, wherein the binding moiety is an antibody.

11. The method of claim 10, wherein the antibody binds to a mammalian LRRC15.

12. The method of claim 10, wherein the antibody is a humanized monoclonal antibody.

13. The method of claim 10, wherein the monoclonal antibody is an afucosylated antibody or a low- fucose variant thereof.

14. The method of claim 1, wherein the cytotoxic agent is a therapeutic radionuclide.

15. The method of claim 14, wherein the therapeutic radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or acombination thereof.

16. The method of claim 14, wherein the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium.

17. The method of claim 14, wherein the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium.

18. The method of claim 14, wherein the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine.

19. The method of claim 14, wherein the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium.

20. The method of claim 1, wherein the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

21. The method of claim 1 wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD 160, CGEN- 15049, CHK 1 and CHK2 kinases, A2aR, and / or a B-7 family ligand.

22. The method of claim 1 , wherein the inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF- 05082566, urelumab or MEDI6469.

23. The method of claim 1 , wherein the tumor or stroma thereof exhibits an immunotherapy resistant signature.

24. The method of claim 23 wherein the signature comprises one or more of LRRC 15 , Cd3 , Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and / or Prfl.

25. The method of claim 23 wherein the signature consists of LRRC 15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl.

26. The method of any one of claims 1-25, wherein the combination is synergistic in treating the tumor.

27. A method for increasing susceptibility of an immunotherapy resistant tumor in a patient to immunotherapy comprising exposing the tumor or tumor stroma to a binding moiety comprising SEQ ID NO: 1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent.

28. The method of claim 27, wherein the tumor or stroma thereof exhibits an immunotherapy resistant signature.

29. The method of claim 27, wherein the signature comprises one or more of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and / or Prfl.

30. The method of claim 27, wherein the signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl.

31. The method of claim 27, wherein the resistance to immunotherapy is resistance to immune checkpoint blockade.

32. The method of claim 31, wherein the immune checkpoint is CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and / or a B-7 family ligand.

33. The method of claim 27, wherein the tumor and / or stroma thereof expresses LRRC15.

34. The method of claim 27, wherein the tumor and / or stroma thereof produces TGFp.

35. The method of claim 27, wherein the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma.

36. The binding moiety of claim 35, wherein the prostate cancer is androgen receptor negative or androgen independent.

37. The binding moiety of claim 27, wherein the tumor is metastatic.

38. The method of claim 27, wherein the tumor comprises stroma which comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

39. The method of claim 27, wherein the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR).

40. The method of claim 27, wherein the binding moiety has picomolar affinity for LRRC15.

41. The method of claim 27, wherein the binding moiety is an antibody.

42. The method of claim 41, wherein the antibody binds to a mammalian LRRC15.

43. The method of claim 41, wherein the antibody is a humanized monoclonal antibody.

44. The method of claim 41, wherein the monoclonal antibody is an afucosylated antibody or a low- fucose variant thereof.

45. The method of claim 27, wherein the cytotoxic agent is a therapeutic radionuclide.

46. The method of claim 45, wherein the therapeutic radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof.

47. The method of claim 46, wherein the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium.

48. The method of claim 46, wherein the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium.

49. The method of claim 46, wherein the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine.

50. The method of claim 46, wherein the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium.

51. The method of claim 27, wherein the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

52. The method of any one of claims 27-51, wherein the exposing further comprises exposing the tumor or stroma to an immunotherapeutic agent.

53. The method of claim 52, wherein the exposing to the binding moiety and the immunotherapeutic agent is synergistic in increasing susceptibility.

54. The method of claim 52, wherein the immunotherapeutic agent is at least one immune checkpoint inhibitor, T cell therapy, cytokine therapy, chemokine therapy,immunomodulatory drug therapy, monoclonal antibody therapy, or any combination thereof.

55. The method of elaim 54, wherein the at least one immune eheckpoint inhibitor is an inhibitor of CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VIST A, IDO, KIR, 2B4, CD 160, CGEN- 15049, CHK 1 and CHK2 kinases, A2aR, or a B-7 family ligand.

56. The method of claim 54, wherein the at least one immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF-05082566, urelumab, MEDI6469 or any combination thereof.

57. The method of claim 27 or 52, wherein the patient is subsequently treated with an immunotherapeutic agent.

58. The method of claim 57, wherein the immunotherapeutic agent is at least one immune checkpoint inhibitor, T cell therapy, cytokine therapy, chemokine therapy, immunomodulatory drug therapy, monoclonal antibody therapy, or any combination thereof.

59. The method of claim 58, wherein the at least one immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD 160, CGEN- 15049, CHK 1 and CHK2 kinases, A2aR, or various B-7 family ligands.

60. The method of claim 58, wherein the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF-05082566, urelumab, MEDI6469 or any combination thereof.

61. A method for depleting TFGP expressing cells in a tumor or tumor microenvironment comprising exposing the tumor or tumor microenvironment to a binding moiety comprising SEQ ID NO: 1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent.

62. The method of claim 61 , wherein the TFGP expressing cells are tumor cells.

63. The method of claim 61 , wherein the TGFP expressing cells are tumor stoma cells.

64. The method of claim 61, wherein the tumor and / or stroma thereof expresses LRRC15.

65. The method of claim 61, wherein the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma.

66. The binding moiety of claim 65, wherein the prostate cancer is androgen receptor negative or androgen independent.

67. The binding moiety of claim 61, wherein the tumor is metastatic.

68. The method of claim 63, wherein the stroma comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

69. The method of claim 61 , wherein the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR).

70. The method of claim 61 , wherein the binding moiety has picomolar affinity for LRRC15.

71. The method of claim 61 , wherein the binding moiety is an antibody.

72. The method of claim 71, wherein the antibody binds to a mammalian LRRC15.

73. The method of claim 71, wherein the antibody is a humanized monoclonal antibody.

74. The method of claim 73, wherein the monoclonal antibody is an afucosylated antibody or a low- fucose variant thereof.

75. The method of claim 61 , wherein the cytotoxic agent is a therapeutic radionuclide.

76. The method of claim 75, wherein the radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof.

77. The method of claim 76, wherein the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium.

78. The method of claim 76, wherein the beta particle emitter is selected from the groupconsisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium.

79. The method of claim 76, wherein the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine.

80. The method of claim 76, wherein the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium.

81. The method of claim 61, wherein the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

82. The method of any one of claims 61-81, wherein the exposing further comprises exposing the tumor or stroma to an immunotherapeutic agent.

83. The method of claim 82, wherein the combination is synergistic in depleting TFGP expressing cells.

84. The method of claim 82, wherein the immunotherapeutic agent is at least one immune checkpoint inhibitor, T cell therapy, cytokine therapy, chemokine therapy, immunomodulatory drug therapy, monoclonal antibody therapy, or any combination thereof.

85. The method of claim 84, wherein the at least one immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-L1, PD-L2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, or various B-7 family ligands.

86. The method of claim 84, wherein the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF-05082566, urelumab, MEDI6469 or any combination thereof.

87. A method for treating a tumor in a patient comprising administering to the patient a combination of: a. a bispecific or trispecific targeting molecule that binds to LRRC15 and TGF , said targeting molecule comprising at least one occurrence of amino acidsequences SEQ ID NO:1 and SEQ ID N0:2, and at least one occurrence of a targeting sequence to TGFP; and b. an immune checkpoint inhibitor.

88. The method of claim 87, wherein said targeting molecule comprising amino acid sequences SEQ ID NO:1 and SEQ ID NO:2, and amino acid sequences SEQ ID NO:3 and SEQ ID NO:4.

89. The method of claim 87, wherein said targeting molecule comprising one occurrence of amino acid sequences SEQ ID NO:1 and SEQ ID NO:2, and two occurrences of amino acid sequences SEQ ID NO:3 and SEQ ID NO:4.

90. The method of claim 87, wherein said targeting molecule comprising two occurrences of amino acid sequences SEQ ID NO: 1 and SEQ ID NO:2, and one occurrence of amino acid sequences SEQ ID NO:3 and SEQ ID NON.

91. The method of claim 87, wherein the targeting molecule is humanized.

92. The method of claim 87, wherein the targeting molecule is afucosylated or a low-fucose variant thereof.

93. The method of claim 87, wherein the bispecific or trispecific targeting molecule is associated with a cytotoxic agent.

94. The method of claim 93, wherein the cytotoxic agent is a therapeutic radionuclide.

95. The method of claim 94 wherein the therapeutic radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof.

96. The method of claim 95, wherein the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium.

97. The method of claim 95, wherein the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium.

98. The method of claim 95, wherein the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine.

99. The method of claim 95, wherein the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium.

100. The method of claim 93, wherein the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

101. The method of claim 87, wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-L1 , PD-L2, PD-1 , B7-H3, B7-H4, BTLA, HVEM, T1M3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and / or a B-7 family ligand.

102. The method of claim 87, wherein the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781 , IMP321 , lirilumab, IPH2101 , indoximod, NLG 919, INCB024360, PF-05082566, urelumab or MEDI6469.

103. The method of any one of claims 87-102, wherein the combination is synergistic in treating the tumor.

104. A method for inducing dormancy in a LRRC15-expressing tumor in a patient comprising exposing the tumor or stroma thereof to a binding moiety comprising SEQ ID NO:1 and SEQ ID NO:2, wherein the binding moiety is associated with a cytotoxic agent.

105. The method of claim 104, wherein the tumor and / or stroma thereof produces TGF|3.

106. The method of claim 104, wherein the tumor is a pancreatic tumor, ovarian carcinoma, glioblastoma multiforme, osteosarcoma, breast tumor, head and neck tumor, lung tumor, bladder tumor, colorectal tumor, hepatocellular tumor, testicular tumor, endometrial tumor, gastric tumor, renal tumor, prostate tumor, sarcoma, or melanoma.

107. The binding moiety of claim 104, wherein the prostate cancer is androgen receptor negative or androgen independent.

108. The binding moiety of claim 104, wherein the tumor is metastatic.

109. The method of claim 104, wherein the tumor comprises stroma which comprises fibroblasts, mesenchymal cells, epithelial cells, or any combination thereof.

110. The method of claim 104, wherein the binding moiety is a single-chain variable fragment (scFv), a Fv fragment, an antigen binding fragment (Fab), a F(ab’)2 fragment, a bispecific T cell engager, or a chimeric antigen receptor (CAR).

111. The method of claim 104, wherein the binding moiety has picomolar affinity for LRRC15.

112. The method of claim 104, wherein the binding moiety is an antibody.

113. The method of claim 112, wherein the antibody binds to a mammalian LRRC15.

114. The method of claim 112, wherein the antibody is a humanized monoclonal antibody.

115. The method of claim 112, wherein the monoclonal antibody is an afucosylated antibody or a low- fucose variant thereof.

116. The method of claim 104, wherein the cytotoxic agent is a therapeutic radionuclide.

117. The method of claim 116, wherein the therapeutic radionuclide is an alpha particle emitter, a beta particle emitter, an Auger electron emitter, a gamma-ray emitter or a combination thereof.

118. The method of claim 116, wherein the alpha particle emitter is selected from the group consisting of uranium, radium, thorium, and actinium.

119. The method of claim 116, wherein the beta particle emitter is selected from the group consisting of uranium, thorium, actinium, bismuth, thallium, strontium, cesium, lutetium, and zirconium.

120. The method of claim 116, wherein the Auger electron emitter is selected from the group consisting of technetium, indium, and iodine.

121. The method of claim 116, wherein the gamma-ray emitter is selected from the group consisting of uranium, thorium, actinium, cobalt, cesium, and technetium.

122. The method of claim 104, wherein the cytotoxic agent is monomethyl auristatin E, mertansine, maytansanoid, a taxane, streptonigrin, geldanamycin, camptothecin, calicheamicin, duocarmycin, or a derivative or analogue thereof.

123. The method of claim 104, wherein the exposing is in combination with an immune checkpoint inhibitor.

124. The method of claim 123, wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, IDO, KIR, 2B4, CD160, CGEN-15049, CHK 1 and CHK2 kinases, A2aR, and / or a B-7 family ligand.

125. The method of claim 123 wherein the immune checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MEDI0680, AMP-224, AUNP-12, BMS-936559, atezolizumab, durvalumab, avelumab, BMS935559, rHIgM12B7, BMS-986016, GSK2831781, IMP321, lirilumab, IPH2101, indoximod, NLG 919, INCB024360, PF-05082566, urelumab or MEDI6469.

126. The method of any one of claims 123-125, wherein the combination is synergistic in inducing dormancy of the tumor.

127. The method of claim 104, wherein the tumor or stroma thereof exhibits an immunotherapy resistant signature.

128. The method of claim 127 wherein the signature comprises one or more of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcrb, Eomes and / or Prfl.

129. The method of claim 127 wherein the signature consists of LRRC15, Cd3, Cd8, Gimap7, Gzmk, Lat, Lek, Eomes, Cxcr6, Eomes and Prfl.