HK40112947A - Glycoprotein a repetitions predominant (garp)-binding antibodies and uses thereof - Google Patents

Description

Antibodies that bind to glycoprotein A repeat dominant protein (GARP) and their applications

Statement on the Redistribution of Federal Funding

This invention was made with government support under grants from the National Institutes of Health (NIH) Nos. R01AI070603, P01CA186866, R01CA188419, and P30CA138313. The government holds certain rights to this invention.

Merging of sequence lists

The sequence list contained in the file named “103361-134WO1.xml” is 48.7KB in size and was created on October 11, 2022. It is hereby submitted electronically and incorporated by reference.

Cross-references to related applications

This application claims the benefit of U.S. Provisional Application No. 63/254,182, filed October 11, 2021, and U.S. Provisional Application No. 63/402,763, filed August 31, 2022, which are incorporated herein by reference in their entirety.

Technical Field

This disclosure generally relates to the fields of cancer biology, immunology, and medicine. More specifically, it relates to monoclonal antibodies targeting GARP (glycoprotein A repeat dominant protein) for the treatment and detection of cancer, and methods for treating cancer using immunotherapy. Specifically, methods for treating cancer by combining T-cell therapy with antiplatelet agents are provided.

Background Technology

TGF-β is a pleiotropic cytokine widely expressed in most tissues. Abnormalities in its signaling have been associated with a variety of diseases, particularly cancer (Derynck et al., 2001; Massague, 2008). In addition to growth arrest, TGF-β induces various malignant cell phenotypes, including invasion, loss of cell adhesion, epithelial-mesenchymal transition, and metastasis (Bhowmick et al., 2001; Derynck et al., 2001; Oft et al., 1998). Importantly, the role of TGF-β in shaping the tumor microenvironment is a key aspect of its carcinogenic function. For example, TGF-β1 is a potent inducer of angiogenesis (Roberts et al., 1986), directly inducing angiogenesis by inducing VEGF expression (Pertovaara et al., 1994) or by recruiting other cells such as monocytes, which in turn secrete pro-angiogenic molecules to induce angiogenesis (Sunderkotter et al., 1991). TGF-β can also manipulate the tumor microenvironment by altering the effective anti-tumor functions of T cells, NK cells, B cells, or other cells (Kehrl et al., 1986; Kopp et al., 2009), through its direct action and its ability to induce Foxp3 ⁺ regulatory T cells (Li and Flavell, 2008), thereby helping cancer cells evade immune surveillance.

Biochemically, TGF-β exists in at least four different forms: 1) readily soluble active TGF-β; 2) soluble TGF-β associated with latent-related peptides or LAP (forming a TGF-β-LAP complex, referred to as latent TGF-β or LTGF-β); 3) LTGF-β covalently associated with large TGF-β-binding protein (LTBP), forming a TGF-β-LAP-LTBP complex; and 4) the membrane latent form of TGF-β (mTGF-β) (Li and Flavell, 2008; Tran, 2012). Only LAP-free TGF-β is known to be biologically active. Therefore, large amounts of TGF-β are latently isolated in the extracellular matrix and then activated by proteases such as MMP2, MMP9, and plasmin (Lyons et al., 1990; Sato and Rifkin, 1989; Yu and Stamenkovic, 2000), which are then secreted by tumor cells and other cells in the tumor microenvironment. mLTGF-β is expressed by two hematopoietic cell types: platelets and regulatory T cells, and is associated with the transmembrane protein glycoprotein A repeat dominant protein (GARP), also known as leucine-rich repeat protein 32 (LRRC32) (Tran et al., 2009; Wang et al., 2012). In addition to its role as a docking receptor for mLTGF-β, GARP is also crucial for regulating TGF-β activation and bioavailability: GARP enhances proTGF-β maturation and cooperates with integrins in mLTGF-β activation (Wang et al., 2012). This article describes the potential role of GARP in cancer.

Passive immunization via adoptive transfer of large numbers of tumor-reactive lymphocytes (called adoptive cell therapy (ACT)) has shown promising experimental activity in treating patients with metastatic melanoma and is being extensively explored for the treatment of other human cancers. ACT involves the administration of large numbers of highly selective cells with high affinity for tumor antigens. These T cells can be programmed and activated in vitro to exhibit anti-tumor effector functions. Furthermore, patients can be ‘conditioned’ with lymphocyte-clearing chemotherapy or whole-body radiation prior to T cell infusion, which allows for the reduction of immunosuppressive cell types/factors before the infusion of tumor-specific T cells. While ACT appears promising in many respects, much work remains to be done to make the treatment more successful.

The encouraging clinical achievements of ACT face significant obstacles limiting the clinical benefits and wider application of this method. While some inherent difficulties can be attributed to specific methods employed in the isolation, proliferation, or generation of effector lymphocytes, other difficulties, such as the depletion of the proliferative and survival potential of fully differentiated T cells, appear to be more generalized phenomena related to the effector phenotype. Other difficulties stem from extrinsic suppressive mechanisms applied at the tumor site, mediated either through direct cell-to-cell contact with tumor cells, stromal cells, and regulatory T cells (Tregs) or through suppressive cytokines such as TGF-β. As a result, the applied T cells exhibit reduced intratumoral persistence and impaired function, often failing to exert detectable tumor-killing effects. Therefore, methods to evade or overturn these suppressive mechanisms and enhance the therapeutic outcomes of ACT are needed.

Summary of the Invention

This disclosure provides a method for treating cancer. In one aspect, an isolated monoclonal antibody is provided, wherein the antibody specifically binds to GARP. In some aspects, the antibody comprises (a) a first V _H CDR that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to V H CDR1 of humanized PIIO-1 (SEQ ID NO:1) or 5c5 (SEQ ID NO:9); (b) a second V _H CDR that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to V _H CDR2 of humanized PIIO-1 (SEQ ID NO:2) or 5c5 (SEQ ID NO:10); (c) a third V _H CDR that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to V _H CDR3 of humanized PIIO-1 (SEQ ID NO:3) or 5c5 (SEQ ID NO:11); and (d) a V _L CDR that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to V _L CDR3 of humanized PIIO-1 (SEQ ID NO:5) or 5c5 (SEQ ID NO:13). (e) a first V _L CDR that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to CDR1; and (f) a second V L CDR that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to V _L CDR2 of humanized PIIO-1 (SEQ ID NO:6) or 5c5 (SEQ ID NO:14); and (f) a third V _L CDR that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to _{V L} CDR3 of humanized PIIO-1 (SEQ ID NO:7) or 5c5 (SEQ ID NO:15). Therefore, in one aspect disclosed herein is an isolated monoclonal antibody against glycoprotein A repeat dominant protein (GARP), wherein the antibody specifically binds to GARP and comprises i) variable heavy chain (VH) complementarity-determining regions 1 (CDR1), 2, and 3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and ii) variable light chain (VL) complementarity-determining regions 1 (CDR1), 2, and 3 as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; or the antibody comprises i) variable heavy chain (VH) complementarity-determining regions 1 (CDR1), 2, and 3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and ii) variable light chain (VL) complementarity-determining regions 1 (CDR1), 2, and 3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively.

In some respects, the antibody comprises at least 80%, 90%, 95%, 98%, 99%, or 100% of a first V _H CDR identical to SEQ ID NO:1, at least 80%, 90%, 95%, 98%, 99%, or 100% of a second V _H CDR identical to SEQ ID NO:2, at least 80%, 90%, 95%, 98%, 99%, or 100% of a third V _H CDR identical to SEQ ID NO:3, at least 80%, 90%, 95%, 98%, 99%, or 100% of a first V _L CDR identical to SEQ ID NO:5, at least 80%, 90%, 95%, 98%, 99%, or 100% of a second V _L CDR identical to SEQ ID NO:6, and at least 80%, 90%, 95%, 98%, 99%, or 100% of a third V _L CDR identical to SEQ ID NO:7. In specific respects, the antibody comprises the same first V _H CDR as SEQ ID NO:1, the same second V _H CDR as SEQ ID NO:2, the same third V _H CDR as SEQ ID NO:3, the same first V _L CDR as SEQ ID NO:5, the same second V _L CDR as SEQ ID NO:6, and the same third V _L CDR as SEQ ID NO:7.

In other respects, the antibody comprises at least 80%, 90%, 95%, 98%, 99%, or 100% of a first V _H CDR identical to SEQ ID NO:9, at least 80%, 90%, 95%, 98%, 99%, or 100% of a second V _H CDR identical to SEQ ID NO:10, at least 80%, 90%, 95%, 98%, 99%, or 100% of a third V _H CDR identical to SEQ ID NO:11, at least 80%, 90%, 95%, 98%, 99%, or 100% of a first V _L CDR identical to SEQ ID NO:13, at least 80%, 90%, 95%, 98%, 99%, or 100% of a second V _L CDR identical to SEQ ID NO:14, and at least 80%, 90%, 95%, 98%, 99%, or 100% of a third V _L CDR identical to SEQ ID NO:15. In a particular aspect, the antibody comprises the same first V _H CDR as SEQ ID NO:9, the same second V _H CDR as SEQ ID NO:10, the same third V _H CDR as SEQ ID NO:11, the same first V _L CDR as SEQ ID NO:13, the same second V _L CDR as SEQ ID NO:14, and the same third V _L CDR as SEQ ID NO:15.

In other respects, the binding site or epitope is located within the extracellular domain of GARP and may contain, consist substantially of, consist of, or be located within the following: GARP residues 171 to 207 of humanized PIIO-1 (DMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNLSRN; SEQ ID NO:4) and residues 20 to 61 of 5C5 (HQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLSGNQ; SEQ ID NO:8).

In some aspects, the antibody comprises (i) a _VH domain that is at least about 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH domain of humanized PIIO-1 (SEQ ID NO: 18, 19, 20, or 21) and a _VL domain that is at least about 80%, 90%, 95%, 98%, 99%, or 100% identical to the _VL domain of humanized PIIO-1 (SEQ ID NO: 22, 23, or 24); or (ii) a _VH domain that is at least about 80%, 90%, 95%, 98%, 99%, or 100% identical to the _VH domain of 5c5 (SEQ ID NO: 12) and a _VL domain that is at least about 80%, 90%, 95%, 98%, 99%, or 100% identical to _{the VL} domain of 5c5 (SEQ ID NO: 16). In a specific aspect, the antibody contains the same _VH domain as the VH domain of humanized PIIO-1 (SEQ ID NO: 18, 19, 20, or 21) and the same _VL domain as the _VL domain of humanized PIIO-1 (SEQ ID NO: 22, 23, or 24). In another specific aspect, the antibody contains the same _VH domain as the _VH domain of 5c5 (SEQ ID NO: 12) and the same _VL domain as _{the VL} domain of 5c5 (SEQ ID NO: 16). In one specific aspect, the antibody is a humanized PIIO-1 antibody (i.e., HuPIIO-1VH1/L1, HuPIIO-1VH1/L2, HuPIIO-1VH2/L1, HuPIIO-1VH1/L3, HuPIIO-1VH2/L2, HuPIIO-1VH2/L3, HuPIIO-1VH3/L1, HuPIIO-1VH2/L3, HuPIIO-1VH3/L3, HuPIIO-1VH4/L1, HuPIIO-1VH4/L2 and/or HuPIIO-1VH4/L3) or a 5c5 antibody. Therefore, this document also discloses anti-GARP antibodies of any of the foregoing aspects, wherein the antibody comprises at least about 80%, 90%, 95%, 98%, or 99% of the same _VH domain as the humanized PIIO-1 (huPIIO-1) antibody shown in SEQ ID NO:18, 19, 20, or 21, and/or at least about 80%, 90%, 95%, 98%, or 99% of the same _VL domain as the huPIIO-1 antibody shown in SEQ ID NO:22 _, 23, or 24. In some aspects, the antibody comprises the _VH domain shown in SEQ ID NO:18, 19, 20, or 21, and/or the _VL domain shown in SEQ ID NO:22, 23, or ₂₄ . For example, this document discloses anti-GARP antibodies in any of the foregoing aspects, wherein the antibody comprises a _VH domain as shown in SEQ ID NO:20 and a VL domain as shown in SEQ ID NO:23 (VH1VL1), a _VH domain as shown in SEQ ID NO:20 and a VL domain as shown in SEQ ID NO:24 (VH1VL2), a _VH domain as shown in SEQ ID NO:21 and a VL domain as shown in SEQ ID NO:23 (VH1VL1), a _VL domain as shown in SEQ ID NO:20 and as shown in SEQ ID NO:22 (VH1VL3), a _VH domain as shown in SEQ ID NO:21 and a VL domain as shown in SEQ ID NO:24 (VH2VL2), a VH domain as shown in SEQ ID NO:21 and a _VL domain as shown in SEQ ID NO:22 (VH2VL3), a VH domain as shown in SEQ ID NO:19 and a VH domain as shown in SEQ ID NO:23 (VH1VL1), a _VH domain as shown in SEQ ID NO:20 and a _VL domain as shown in SEQ ID NO:24 (VH2VL3), a VH domain as shown in SEQ ID NO:19 and a VH domain as shown in SEQ ID NO:23 (VH1VL1), a _VH domain as shown in SEQ ID NO:20 and a VL domain as shown in SEQ ID NO:23 (VH1VL2), a VH domain as shown in SEQ ID NO:24 (VH2VL2), a _VH domain as shown in SEQ ID NO:25 (VH1VL1), a _VH domain as shown in SEQ ID NO:26 (VH1VL2), a VH domain as shown in SEQ ID NO:27 (VH1VL2), a VH domain as shown in SEQ ID NO:28 (VH1VL2), a _VH domain as shown in SEQ ID The antibody may contain the VH3VL1 domain shown in NO:23, the _VH domain shown in SEQ ID NO:19, the _VH domain shown in SEQ ID NO:24, the _VH domain shown in SEQ ID NO:19, the _VH domain shown in SEQ ID NO:22, the _VH4VL1 domain shown in SEQ ID NO:18, the _{VH domain} shown in SEQ ID NO:24, or _{the VH} domain shown in SEQ ID NO:18 and the _VH4VL3 domain shown in SEQ ID NO:22. In a further aspect, the antibody is _recombinant .

In another aspect, the antibody in any of the foregoing aspects is IgG (such as, for example, IgG1, IgG2, IgG3, or IgG4), IgM, IgA, or an antigen-binding fragment thereof. In some aspects, the antibody is Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, nanobody, or single-domain antibody. In some aspects, the antibody in any of the foregoing aspects can be human, humanized antibody, or deimmunized antibody.

This document also discloses antibodies in any of the foregoing aspects, wherein the antibody is conjugated to platelet-binding agents (such as, for example, cyclooxygenase inhibitors, adenosine diphosphate (ADP) inhibitors (including, but not limited to, clopidogrel, prasugrel, or ticlopidine), phosphodiesterase inhibitors, protease-activated receptor-1 (PAR-1) antagonists, glycoprotein IIB/IIIA inhibitors, adenosine reuptake inhibitors, and thromboxane inhibitors), imaging agents, chemotherapeutic agents, toxins, radionuclides, cytokines, or other therapeutic components. In some aspects, the antibody has at least a second binding specificity, such as bispecific antibodies binding to GARP and a second target.

Not all humanized antibodies disclosed herein exhibit identical behavior. For example, Table G confirms that huPIIO-1VH1VL2 (and also huPIIO-1VH2VL1) is superior to huPIIO-1VH1VL1. Furthermore, Figure 16A/Table H confirms that VH1VL2 has superior homogeneity to the clone huPIIO-1VH2VL1. In addition, huPIIO-1VH1VL2 appears to have superior thermostability compared to the parental 4D3 chimeric antibody, as described in paragraph [00243] and Table F.

This article also discloses polynucleotide molecules containing nucleic acid sequences encoding antibodies for any of the aforementioned aspects.

A further aspect of this disclosure provides a composition comprising an antibody of any of the foregoing aspects and the aspects described herein in a pharmaceutically acceptable carrier. In some aspects, the composition may further comprise an anticancer agent (such as, for example, an immune checkpoint inhibitor, including but not limited to, antibodies that block: PD-1 (such as, for example, nivolumab (BMS-936558 or MDX1106), pembrolizumab, CT-011, AMP-224, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559)). MPDL3280A or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, ipilimumab (MDX-010), trimemumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, avorimab), B7-H4, B7-H3, T-cell immune receptors with Ig and ITIM domains (TIG). IT (such as, for example, BMS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuating factors (BTLA), T cell activation V-domain Ig repressor (VISTA) (such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR) -022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013 and Immutep).

In another aspect, this disclosure provides an isolated polynucleotide molecule comprising a nucleic acid sequence encoding an antibody of any of the foregoing aspects or other aspects described herein. For example, this disclosure discloses a recombinant polypeptide comprising: an antibody _VH domain comprising CDRs 1, 2, and 3 of the VH domain of the huPIIO-1 antibody as shown in SEQ ID NO:1, 2, and 3, or CDRs 1, 2, and 3 of the _VH domain of 5c5 as shown in SEQ ID NO:9, 10, and 11, respectively; and/or an antibody _VL domain comprising CDRs 1, 2, and 3 of the _VL domain of huPIIO-1 as shown in SEQ ID NO:5, 6, and 7, respectively _, or CDRs 1, 2, and 3 of the _VL domain of 5c5 as shown in SEQ ID NO:13, 14, and 15, respectively.

In one aspect, this document discloses an isolated polynucleotide molecule comprising a nucleic acid sequence encoding an antibody or polypeptide of any of the foregoing aspects. For example, this document discloses an isolated polynucleotide molecule wherein the nucleic acid comprises SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and/or SEQ ID NO:31.

In yet another embodiment, this disclosure provides a host cell comprising one or more polynucleotide molecules encoding an antibody or a recombinant polypeptide of any of the foregoing aspects, or an isolated nucleic acid of any of the foregoing aspects. In some aspects, the host cell is a mammalian cell, yeast cell, bacterial cell, ciliated cell, or insect cell.

This article also discloses methods for treating, inhibiting, reducing, lowering, improving, and/or preventing cancer and/or metastases in subjects with cancer (such as, for example, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, hematologic malignancies, clear cell renal cell carcinoma, head/neck squamous cell carcinoma, lung squamous cell carcinoma, melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, small cell lung cancer (SCLC), triple-negative breast cancer, acute lymphoblastic leukemia (ALL). The method involves administering to the subject a therapeutically effective amount of any of the aforementioned antibodies or compositions. In some aspects, the cancer is a GARP-positive cancer. This includes acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or small lymphocytic lymphoma (SLL).

One aspect disclosed herein is a method for treating, inhibiting, reducing, lowering, improving, and/or preventing cancer and/or metastasis, wherein the antibody is in a pharmaceutically acceptable composition. In some specific aspects, the antibody is administered systemically. In other aspects, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

This document also discloses methods for treating, inhibiting, reducing, lowering, improving, and/or preventing cancer and/or metastasis in any of the foregoing aspects, wherein the method further comprises administering an anticancer therapy to a subject and/or administering an anticancer agent to a subject (such as, for example, i) a TGFβ inhibitor, including but not limited to LY2157299, trabedersen, fresolimumab, LY2382770, lucanix, or PF-03446962, and/or ii) an antiplatelet agent, including but not limited to cyclooxygenase inhibitors, adenosine diphosphate (ADP) inhibitors (such as, for example, clopidogrel, prasugrel, or ticlopidine), phosphodiesterase inhibitors, protease-activated receptor-1 (PAR-1) antagonists, glycoprotein IIB/IIIA inhibitors, and adenosine reuptake inhibitors. Take inhibitors and thromboxane inhibitors, and/or iii) immune checkpoint inhibitors (such as antibodies that block, for example, the following: PD-1 (such as, for example, nivolumab (BMS-936558 or MDX1106), pembrolizumab, CT-011, AMP-224, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, ipilimumab (MDX-010), trimemumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, avorimab), B7-H4). B7-H3, T-cell immune receptors with Ig and ITIM domains (TIGIT) (such as, for example, BMS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuating factors (BTLA), T-cell activation V-domain Ig repressor (VISTA) (such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016) The anticancer therapy may include LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep. In some of these aspects, the second anticancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, targeted therapy, immunotherapy (such as, for example, adoptive cell transfer therapy), or cytokine therapy. In some aspects, immunotherapy is administered before, concurrently with, or after the antiplatelet agent. In some aspects, the method may further include lymphocyte depletion of the subject prior to administration of T-cell therapy (such as, for example, via administration of cyclophosphamide and/or fludarabine). In certain aspects, the antiplatelet agent is any of the anti-GARP antibodies of any of the foregoing aspects or a fragment thereof.

In one aspect, this document discloses methods for treating, inhibiting, reducing, lowering, improving, and/or preventing cancer and/or metastasis in any of the foregoing aspects, wherein adoptive cell transfer therapy includes the transfer of T cells (including but not limited to tumor-infiltrating lymphocytes (TILs), chimeric antigen receptor (CAR) T cells, CD8 ⁺ T cells, and/or CD4 ⁺ T cells), chimeric antigen receptor (CAR) T cells, B cells, natural killer (NK) cells, CAR NK cells, CAR macrophages (CARMA), and/or NK T cells. In some aspects, the T cells are tumor-specific.

This document also discloses methods for treating, inhibiting, reducing, lowering, improving, and/or preventing cancer and/or metastasis in any of the foregoing aspects, wherein tumor-specific T cells are engineered to express T cell receptors (TCRs) or chimeric antigen receptors (CARs) that are antigen-specific to tumor antigens such as, for example, tEGFR, Her2, CD19, CD20, CD22, mesothelin, CEA, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, FBP, MAGE-A1, MUC1, NY-ESO-1, and/or MART-1. In some aspects, the CAR comprises an intracellular domain of a co-stimulatory molecule selected from the group consisting of CD28, CD27, 4-IBB, OX40ICOS, and combinations thereof.

In some respects, this article discloses methods for treating, inhibiting, reducing, lowering, improving and/or preventing cancer and/or metastasis in any of the foregoing aspects, wherein the cells subsequently metastasized are autologous.

Another embodiment of this disclosure provides a method for detecting cancer in a subject, comprising obtaining a potentially cancerous tissue sample from the subject and testing the tissue sample for the presence of increased GARP (including, but not limited to, soluble GARP or cells expressing GARP) levels relative to a non-cancer control. In some aspects, the detection of GARP is achieved by using an anti-GARP antibody from any of the foregoing aspects. In some aspects, the method is further defined as an in vitro or ex vivo method.

In one aspect, this document discloses a method for stimulating T cells and/or B cells in a subject with cancer, comprising administering to the subject an effective amount of any of the aforementioned anti-GARP antibodies. For example, this document discloses a method for stimulating T cells (such as, for example, Th1 CD4+ T cells, Th2 CD4+ T cells, effector CD8+ T cells (CD25+, CD45RA-+, CD45RO-, and CD127-) and/or effector memory CD8+ T cells (CD25-, CD45RA-, CD45RO+, and CD127+)) and/or B cells (including, but not limited to, T cells and B cells in the tumor microenvironment) in a subject with cancer, comprising administering to the subject an effective amount of an anti-GARP antibody (such as, for example, an anti-GARP antibody comprising heavy chain CDR1, CDR2, and CDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; such as, for example, as shown in SEQ ID NO:18, 19, 20, or 21). Heavy chain variable domains) and/or light chain CDR1, CDR2, and CDR3 as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 (such as, for example, light chain variable domains as shown in SEQ ID NO:22, 23, and 24). Such antibodies may include, but are not limited to, HuPIIO-1VH1/L1, HuPIIO-1VH1/L2, HuPIIO-1VH2/L1, HuPIIO-1VH1/L3, HuPIIO-1VH2/L2, HuPIIO-1VH2/L3, HuPIIO-1VH3/L1, HuPIIO-1VH2/L3, HuPIIO-1VH3/L3, HuPIIO-1VH4/L1, HuPIIO-1VH4/L2, and/or HuPIIO-1VH4/L3.

In one aspect, the T cells stimulated by any of the foregoing methods are endogenous tumor-infiltrating lymphocytes (TILs). This document also discloses methods for stimulating T cells in any of the foregoing aspects, wherein the CD8 T cells are TILs or chimeric antigen receptor (CAR) T cells administered to the subject as a component of immunotherapy.

This article also discloses a method for stimulating transfected donor T cells (such as, for example, Th1CD4+ T cells, Th2CD4+ T cells, effector CD8+ T cells (CD25+, CD45RA-+, CD45RO-, and CD127-) and/or effector memory CD8+ T cells (CD25-, CD45RA-, CD45RO+, and CD127+)) in the tumor microenvironment of a subject, comprising administering T cells and an anti-GARP antibody (such as, for example, an anti-GARP antibody containing heavy chain CDR1, CDR2, and CDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, or heavy chain variable domains as shown in, for example, SEQ ID NO:18, 19, 20, or 21) and/or light chain variable domains as shown in, for example, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively). Chain CDR1, CDR2, and CDR3 (such as, for example, the light chain variable domains shown in SEQ ID NO: 22, 23, 24). Such antibodies may include, but are not limited to, HuPIIO-1VH1/L1, HuPIIO-1VH1/L2, HuPIIO-1VH2/L1, HuPIIO-1VH1/L3, HuPIIO-1VH2/L2, HuPIIO-1VH2/L3, HuPIIO-1VH3/L1, HuPIIO-1VH2/L3, HuPIIO-1VH3/L3, HuPIIO-1VH4/L1, HuPIIO-1VH4/L2, and/or HuPIIO-1VH4/L3. In one aspect, the anti-GARP antibody may be administered before, simultaneously with, or after the transfer of donor T cells. In one aspect, the T cells are TILs or chimeric antigen receptor (CAR) T cells administered to the subject as a component of immunotherapy.

In one aspect, this document discloses a method for inducing the proliferation of T cells or B cells in a subject with cancer, comprising administering to the subject an effective amount of any of the aforementioned anti-GARP antibodies (such as, for example, anti-GARP antibodies comprising heavy chain CDR1, CDR2, and CDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, (such as, for example, heavy chain variable domains as shown in SEQ ID NO:18, 19, 20, or 21) and/or light chain CDR1, CDR2, and CDR3 as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively). DR3 (such as, for example, the light chain variable domain as shown in SEQ ID NO: 22, 23, 24). Such antibodies may include, but are not limited to, HuPIIO-1VH1/L1, HuPIIO-1VH1/L2, HuPIIO-1VH2/L1, HuPIIO-1VH1/L3, HuPIIO-1VH2/L2, HuPIIO-1VH2/L3, HuPIIO-1VH3/L1, HuPIIO-1VH2/L3, HuPIIO-1VH3/L3, HuPIIO-1VH4/L1, HuPIIO-1VH4/L2, and/or HuPIIO-1VH4/L3.

This article also discloses a method for inducing the proliferation of T cells or B cells in subjects with cancer, which includes administering an effective amount of any of the aforementioned anti-GARP antibodies to the subject.

In one aspect, this article discloses a method for blocking T cell consumption of CD8+ T cells, comprising contacting CD8+ T cells with an effective amount of any of the aforementioned anti-GARP antibodies. In some aspects, CD8+ T cells are contacted with anti-GARP antibodies in vitro. In other aspects, CD8+ T cells are located in the tumor microenvironment.

This article also discloses a method for inhibiting Tregs in the tumor microenvironment of a subject, which includes administering a therapeutically effective amount of any of the foregoing anti-GARP antibodies to the subject.

In one aspect, this article discloses a method for blocking the formation of the GARP-LTGFβ1 complex in cancer, which includes contacting the cancer with a therapeutically effective amount of any of the aforementioned anti-GARP antibodies.

This article also discloses a method for increasing the efficacy of immune checkpoint blockade (ICB) therapy in subjects, which includes administering a therapeutically effective amount of any of the aforementioned anti-GARP antibodies to subjects receiving ICB therapy.

In one aspect, this article discloses a method for activating T cells or B cells contained in a subject suffering from cancer, which includes administering an effective amount of any of the aforementioned anti-GARP antibodies to the subject. For example, this article discloses a method for activating T cells (such as, for example, Th1 CD4+ T cells, Th2 CD4+ T cells, effector CD8+ T cells (CD25+, CD45RA-+, CD45RO-, and CD127-) and/or effector memory CD8+ T cells (CD25-, CD45RA-, CD45RO+, and CD127+)) or B cells contained in a subject with cancer, comprising administering to the subject an effective amount of an anti-GARP antibody (such as, for example, an anti-GARP antibody containing heavy chain CDR1, CDR2, and CDR3 (such as, for example, heavy chain variable domains as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively) and/or as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively) and/or as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively) to the subject. 5. Light chains CDR1, CDR2, and CDR3 as shown in SEQ ID NO:6 and SEQ ID NO:7 (such as, for example, light chain variable domains as shown in SEQ ID NO:22, 23, and 24). Such antibodies may include, but are not limited to, HuPIIO-1VH1/L1, HuPIIO-1VH1/L2, HuPIIO-1VH2/L1, HuPIIO-1VH1/L3, HuPIIO-1VH2/L2, HuPIIO-1VH2/L3, HuPIIO-1VH3/L1, HuPIIO-1VH2/L3, HuPIIO-1VH3/L3, HuPIIO-1VH4/L1, HuPIIO-1VH4/L2, and/or HuPIIO-1VH4/L3. In one aspect, T cells and/or B cells are located in the tumor microenvironment.

This article also discloses a method for assessing the sensitivity of cancer to immune checkpoint blockade (ICB) therapy, comprising obtaining a cancerous tissue sample and measuring GARP expression in the sample; wherein elevated GARP expression relative to a non-cancer control indicates cancer resistance to ICB therapy, and lower or equivalent GARP expression relative to a non-cancer control indicates cancer sensitivity to ICB therapy. In some aspects, GARP expression levels can be obtained by measuring any of the anti-GARP antibodies described in any of the foregoing embodiments.

In one aspect, this article discloses a method for sensitizing cancer cells to immune checkpoint blockade (ICB) therapy, which includes contacting ICB-resistant cancer cells with anti-GARP from any of the foregoing aspects.

Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples illustrate certain embodiments of the invention, they are given by way of illustration only, as various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art based on this detailed description.

Attached Figure Description

The following drawings form part of and are included within this specification to further illustrate certain aspects of this disclosure. A better understanding of this disclosure can be achieved by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the official authority will provide a copy of this patent or patent application publication with color drawings.

Figures 1A through 1F show the association between GARP upregulation and poor prognostic significance in cancer. (Figure 1A) Summary of studies on GARP alterations across cancers. Data obtained from the response to the GARP gene LRRC32 query on November 16, 2015, at www.cbioportal.org. (Figure 1B) Specificity analysis of hGARP antibodies in pre-B EV and pre-B leukemia cells expressing hGARP. (Figure 1C) Patient-matched unaffected breast cancer and primary breast cancer. Representative images and IHC GARP scores are shown. (Figure 1D) Representative images of GARP IHC (black areas) in normal tissue and cancer. Scale bar: 20 μm. (Figure 1E) Expression intensity of GARP-positive cells. (Figure 1F) Correlation between GARP expression and overall survival in colon and lung cancer (left and middle panels) and Gleason score in prostate cancer (right panel). The sample size (n) is indicated. Kaplan-Meier curves are shown in Figure 1F for lung and colon cancer, where p-values were calculated using a log-rank test. Two-sample t-tests were used to compare group differences in Figures 1C and 1E and prostate cancer in Figure 1F. HR represents hazard ratio. *P<0.05. **P<0.01. ***P<0.001. ****P<0.0001.

Figures 2A through 2F show the shedding of membrane-bound GARP from cancer cells and its significance as a potential cancer biomarker. (Figure 2A) GARP lysis in the post-ER compartment occurs only in the presence of gRP94. N-terminal FLAG-labeled GARP is stably expressed in WT or pre-gRP94 B KO cells. Whole-cell lysates were treated with Endo H or PNGase F and then immunoblotted with FLAG antibodies. (Figure 2B) The lower fragment protein is GARP, as determined by immunoreactivity and mass spectrometry analysis. The peptide sequence of GARP identified by mass spectrometry is indicated (SEQ ID NO: 17). Figure (2C) Soluble GARP in the serum of prostate cancer patients and healthy controls. (Figure 2D) Correlation analysis between GARP positivity and PSA1 levels (left panel) and GARP positivity and metastatic status of prostate cancer (right panel). (Figure 2E) Quantification of the GARP-TGF-β1 complex in the serum of prostate cancer patients and healthy controls by sandwich ELISA. (Figure 2F) ELISA levels of active TGFβ from purified recombinant soluble GARP-Fc. Differences in distribution in Figure 2D were calculated using a chi-square test. Group differences in Figure 2E were compared using a two-sample t-test. *P<0.05. ***P<0.001.

Figures 3A to 3J show how forced GARP expression on normal mammary epithelial cells enhances TGF-β signaling and drives epithelial-mesenchymal transition (EMT) and invasion. (Figure 3A) NMuMG cells were transfected to stably express membrane-bound GARP, followed by Western blotting against E-cadherin, vimentin, and phosphorus-SMAD-2/3. (Figure 3B) NMuMG cells were treated with recombinant human TGF-β1, soluble GARP, and an allotype antibody control, or left untreated in serum-free medium for 24 hours, followed by morphological analysis. (Figure 3C) NMuMG cells were treated with soluble GARP-Fc (sGARP) in serum-free medium, with duration indicating time. Vimentin upregulation was detected by Western blotting analysis. (Figure 3D) NMuMG cells were treated with an increased dose of soluble GARP, followed by Western blotting against vimentin. (Figure 3E) Western blotting of GARP, TGFβ, and β-actin controls. (Fig. 3F) ELISA quantification of soluble GARP in conditioned medium of NMuMG EV, GARP, and GARP-Fc cells. (Fig. 3G) In vitro scratch assay indicating differences in 24-hour time interval closure. (Fig. 3H) Summary of three independent scratch assays. (Fig. 3I) In vivo imaging of fluorescein-enhanced bioluminescence in mice after injection of GARP, GARP-Fc, and control NMuMG cells at weeks 3 and 6. (Fig. 3J) Histological analysis of NMuMG-GARP tumors by H&E, and histological analysis of vimentin and E-cadherin expression by IHC. Scale bar: 20 μm. Two-sample t-tests were used to compare group differences in Fig. 3H. *P<0.05. **P<0.01. Two independent experiments were performed, and the results were similar.

Figures 4A through 4G show the blocking effect of GARP silencing on breast cancer growth and metastasis. (Figure 4A) ShRNA knockdown of GARP mRNA in NMuMG* cells. Cells treated with out-of-order shRNA (SCR) served as controls. (Figure 4B) Flow cytometry analysis of GARP expression on the cell surface of GARP KD and SCR NMuMG* cells. (Figure 4C) Immunoblot analysis of total GARP and TGF-β levels in GARP KD and SCR NMuMG cells. (Figure 4D) MTT assay comparing growth kinetics of NMuMG*-SCR and NMuMG*-GARP-KD cells. (Figures 4E through 4G) NMuMG*SCR and NMuMG*-GARP KD cells were injected into NOD-Rag1 ^-/- mice, and tumor growth kinetics (Figure 4G) and tumor metastasis (Figures 4F and 4G) were monitored. Tumor growth differences in Figures 4D and 4E were calculated using a two-factor ANOVA. Two-sample t-tests were used to compare group differences in Figures 4F and 4G. **P<0.01.

Figures 5A to 5J show how GARP upregulation promotes TGF-β activation, tumor growth, metastasis, and immune tolerance in mouse breast cancer cells. (Figure 5A) Immunoblotting of GARP, TGF-β, and β-actin controls in 4T1 cells stabilized to express GARP, GARP-Fc, or control EVs. (Figure 5B) Quantification of active TGF-β1 in 72-hour conditioned medium from 4T1 EV, GARP, and GARP-Fc cells by ELISA. (Figure 5C) Naïve CD4+ T cells stimulated with anti-CD3 and anti-CD ^- 28 mAbs in the presence of 50% 3-day conditioned medium from 4T1-EV, 4T1-GARP, and 4T1-GARP-Fc cells. Foxp3 expression was analyzed by flow cytometry on day 3. (Figure 5D) Female BALB/c mice were injected into the fourth mammary fat pad indicating the tumor. Tumor volume was measured every 3 days. (Figure 5E) (Figure 5D) Tumor weight at the endpoint, in grams. (Fig. 5F) Lung tissue was isolated and embedded in paraffin. The number of tumor nodules in the lung was counted. (Fig. 5G) Tumor tissue was isolated after 3 weeks and embedded in OCT. Fresh frozen sections were stained against p-SMAD-2/3 mAb. Scale bar: 100 μm. (Fig. 5H) Summary statistics of p-SMAD-2/3 staining intensity as defined independently by the research pathologist. (Figs. 5I to 5J) Tumor-infiltrating lymphocytes were isolated and the number of CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Tregs was counted by flow cytometry. (5I) Representative flow cytometry plots. (Fig. 5J) Summary of the percentage of Tregs in the tumor microenvironment. Tumor growth differences in Fig. 5D were calculated using a two-factor ANOVA. Two-sample t-tests were used to compare group differences in other figures. *P<0.05. **P<0.01. ***P<0.001.

Figures 6A through 6G show the effect of GARP upregulation in B16 mouse melanoma tumors attenuating adoptive T-cell immunotherapy. (Figure 6A) Experimental protocol. (Figure 6B) Mean tumor growth kinetics of B16-GARP-Fc and B16-EV (n=6). (Figure 6C) Survival differences between the two experimental groups as shown. (Figure 6D) Representative FACS plot of antigen-specific donor T cells in peripheral blood indicated by CD8 ⁺ CD90.1 ⁺ surface markers. (Figure 6E) Frequency of donor T cells in peripheral blood of tumor-bearing mice at different time points after ACT. (Figure 6F) Representative FACS plot of intracellular IFNγ staining in response to homologous gp100 peptide stimulation of peripheral blood antigen-specific donor T cells. (Figure 6G) Quantification of the frequency of IFNγ-producing donor T cells in peripheral blood of mice receiving B16-GARP-Fc or B16-EV. The p-values in Figure 6C were calculated using a log-rank test. Two-sample t-tests were used to compare group differences in the other figures. *P<0.05. ***P<0.001.

Figures 7A to 7F show the crucial role of platelet-intrinsic GARP in the generation of active TGFβ. (Figure 7A) Platelet consumption leads to a complete loss of active and total TGFβ. (Figures 7B to 7D) Expression of GARP and LAP in indicator mouse models. Platelets from Plt-Tgfβ1KO mice expressed similar levels of the surface GARP-TGFβ1 complex compared to WT platelets. (Figure 7E) Measurement of active TGFβ in mice. In WT mice, serum active TGFβ was elevated compared to plasma. (Figure 7F) Measurement of total TFGβ in mice. Serum levels of total latent TGFβ were decreased in Plt-Tgfβ1KO mice, but not in Plt-gp96KO or Plt-GARPKO mice.

Figures 8A through 8D demonstrate the efficacy of adoptive T-cell therapy in melanoma in WT, Plt-Tgfβ1KO, and Plt-GARPKO recipient mice. (Figure 8A) Tumor growth was more effectively controlled in Plt-GARPKO mice compared to WT mice. (Figure 8B) Peripheral blood Pmel cells in Plt-GARPKO mice showed enhanced persistence and (Figure 8C) enhanced function. (Figure 8D) Platelets in Plt-Tgfβ1KO mice expressed GARP and were still able to activate TGFβ, but tumor control was not improved.

Figures 9A to 9H show that the platelet-derived GARP-TGFβ complex attenuates anti-tumor T cell immunity. (Figures 9A to 9C) Tumor size (9A) and total survival in WT and Plt-GARPKO mice. MC38 growth was significantly reduced in Plt-GARPKO mice compared to WT mice. (Figure 9D) Plt-GARPKO mice carrying MC38 had reduced serum levels of active TGFβ. (Figures 9E to 9F) Immunohistochemical staining of p-Smad2/3 in MC38 tumor sections showed significantly reduced TGFβ signaling in MC38 cells of Plt-GARPKO mice. (Figure 9G) Systemic bone marrow-derived suppressor cells (Figure 9H) and tumor-infiltrating regulatory T cells were reduced in Plt-GARPKO mice.

Figures 10A through 10D show the enhancement of cancer-fighting adoptive T-cell therapy by antiplatelet pharmacologists. (Figure 10A) Effects of Cy and AP on tumor growth (left). Antiplatelet agents plus adoptive T-cell transfer were highly effective for B16-F1, with most mice achieving relapse-free survival exceeding 3 months (right). (Figure 10B) Antigen-specific T cells in the blood, inguinal lymph nodes (ILN), and spleen of mice receiving simultaneous antiplatelet therapy and ACT were maintained in high numbers. (Figure 10C) Antiplatelet agents provided no benefit when the transferred T cells lacked IFN-γ (Figure 10D) or when anti-IFN-γ neutralizing antibodies were administered.

Figure 11 shows the determination of binding affinity and thermal stability.

Figure 12 shows the ELISA assessment of nonspecific antibody binding to baculovirus.

Figures 13A and 13B show the CE-SDS results for reduced (Figure 13A) and non-reduced (Figure 13B).

Figure 14 shows the variable region sequences of the candidate heavy chain for humanized PIIO-1. For huPIIO-1VH1, the nucleic acid is SEQ ID NO:27 and the amino acid sequence is SEQ ID NO:20. For huPIIO-1VH2, the nucleic acid is SEQ ID NO:28 and the amino acid sequence is SEQ ID NO:21. For huPIIO-1VH3, the nucleic acid is SEQ ID NO:26 and the amino acid sequence is SEQ ID NO:19. For huPIIO-1VH4, the nucleic acid is SEQ ID NO:25 and the amino acid sequence is SEQ ID NO:18.

Figure 15 shows the variable region sequences of the humanized candidate light chain of PIIO-1 (the top three) and the leader sequences of the heavy and light chains (bottom sequences; the nucleic acid is SEQ ID NO:32, and the amino acid is SEQ ID NO:37). For huPIIO-1VL1, the nucleic acid is SEQ ID NO:30, and the amino acid sequence is SEQ ID NO:23. For huPIIO-1VL2, the nucleic acid is SEQ ID NO:31, and the amino acid sequence is SEQ ID NO:24. For huPIIO-1VL3, the nucleic acid is SEQ ID NO:29, and the amino acid sequence is SEQ ID NO:22.

Figure 16 shows the human κ constant light region sequence (top; nucleic acid is SEQ ID NO:33; amino acid is SEQ ID NO:34) and the human IgG1 constant region heavy chain sequence (bottom; nucleic acid is SEQ ID NO:35; amino acid is SEQ ID NO:36).

Figures 17A to 17E show the characterization of anti-GARP monoclonal antibodies. 17A. Cell specificity of surface GARP and α-GARP mAbs on human platelets and Tregs, as detected by flow cytometry. 17B. Specificity of anti-GARP Ab clones determined by flow cytometry using 293T cells transfected with hGARP (free GARP) or hGARP and TGFβ (GARP-LAP complex). 17C. 293T cells expressing mGARP/hGARP chimeras examined against anti-GARP antibody recognition. 17D. Pre-B-hGARP cells incubated with or without human LTGFβ (huLTGFβ) in the presence of anti-GARP or an allotype control. Cells were stained against surface hLTGFβ (hLAP) to determine the ability of the Abs to block the binding of hLTGFβ to GARP. 17E.Jurkat-hGARP cells were treated with 2H4 anti-GARP Ab (20 μg/ml) for 24 hours, and then immunoblotting was performed targeting the pSMAD3 level in total cell lysates.

Figures 18A to 18F show the generation of GARP humanized mice. A. Construct design scheme. mLrrc32 indicates the mouse allele. ^hLrrc32KI indicates the human Lrrc32 knock-in allele. B. PCR confirmation of mouse genotype. HO, homozygous. C. Flow cytometry confirmation of GARP expression on CD41 ⁺ platelets using species-specific GARP antibody. D. Binding specificity of our hGARP monoclonal antibody to platelets (left) and CD4 ⁺ CD25 ⁺ Treg cells (right) from mouse peripheral blood (PB). E. Toxicity studies with F. huPIIO-1. ^hLrrc32KI mice (n=5/group) were injected intraperitoneally with 200 μg of mIgG1 isotype or huPIIO-1 antiGARP antibody twice weekly (n=5/group) for a total of 6 doses. Body weight or PB platelet levels were measured.

Figures 19A–19E demonstrate the efficacy of the humanized PIIO-1 and anti-PD1 combination therapy against CMT167 lung cancer and remodeled tumor-infiltrating CD8 ⁺ T cell compartments. Figure 19A shows the tumor volume 18 days after sc injection of ¹ x 10⁵ CMT-167 cells. Mice were treated with four injections of the indicator antibody (days 8, 11, 14, and 17). Figure 19B shows the frequency of tumor-infiltrating CD8 ⁺ T cells in the tumor at day 18 (left – representative flow cytometry plot gated to CD45 ⁺ cells; right – data quantification). Figure 19C shows the reduced UMAP size of the multicolor T cell consumption plot gated to live CD45 ⁺ CD3 ⁺ CD8 ⁺ T cells, with secondary sampling of 5000 cells per sample. Unsupervised clustering analysis was performed using the FlowSOM algorithm, with elbow method used for digit identification. The left panel shows all cell clusters of tandem CD8 ⁺ TILs. The middle and right panels show clusters 3 and 10 only in the indicator treatment group. Figure 19D shows high accumulation of clusters 3 and 10 in the combination group. Analysis was performed by EdgeR between the anti-PD1 group and the combination group. Figure 19E shows a heatmap of the expression of indicator markers for all CD8 ⁺ T cell subclusters. N = 5–7/group. *p<0.05, **p<0.01; A, Two-way repeated measures ANOVA with multiple comparisons. B, Two-tailed independent Student's t-test. Data = mean +/- SEM.

Figures 20A to 20F show the impact of LRRC32 gene expression on the immune landscape and ICB responsiveness in human cancers. A-C. TCGA analysis. A. Correlation between LRRC32 expression levels and indicative immune pathways in various human cancer types. The value in each cell indicates the t-statistic comparing the top 1/3 and bottom 1/3 LRRC32 expression groups. B. Correlation between LRRC32 expression and immune subtypes in non-small cell lung cancer. (C1. Wound healing. C2. IFN-γ predominance. C3. Inflammation. C4. Lymphocyte depletion. C6. TGF-β predominance) C. Box plot comparing the enrichment of relevant immune pathways in human lung cancer between high and low LRRC32 gene expression groups. Statistical significance was determined using t-tests for A and C and Fisher's exact test for B. Significance codes: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. D-F. Batch RNA-seq data analysis of pre-treatment tumor samples from 167 patients with metastatic urothelial carcinoma (mUC) who received atezolizumab in the phase 2 trial (IMvigor210). D. Box plots comparing the expression of LRRC32 gene (left) and LRRC32-TGFB-related features (right, as defined in methods) in all types, immune desert, immune exclusion, and immune inflammatory tumors from 167 patients from IMvigor210 between responders (CR/PR, red) and non-responders (SD/PD, blue). CR, complete response; PR, partial response; SD, stable disease; PD, disease progression. E-F. Kaplan-Meier survival plots comparing overall survival probability (y-axis) and follow-up time (months, x-axis) from IMvigor210 in all types, immune desert, immune exclusion, and immune inflammatory tumors. Groups were divided based on high (red) or low (green) expression levels of the LRRC32 gene (E) and LRRC32-TGFB related features (F). Significance was determined using a log-rank test. *p<0.05; **p<0.01.

Figures 21A to 21E show the in vitro characterization of the anti-GARP antibody PIIO-1. A. GARP expression on human regulatory T cells and platelets was assessed by flow cytometry using 10 μg/ml PIIO-1. B. 293 cell lines were transfected with empty vector (EV), human GARP-only (hGARP), or co-transfected with hGARP and latent TGFβ1. GARP expression on the indicator cell lines was detected by flow cytometry using 10 μg/ml PIIO-1. C. Human GARP sequences were sequentially replaced with mouse GARP as shown in the diagram to generate chimeric constructs of human and mouse GARP. Transfection efficiency was assessed by HA tag expression on the constructs. All constructs were transfected into 293 cells. D. Crystal structure of the GARP (green)-LTGFβ (gray) complex (PDB DOI: 10.2210/pdb6GFF/pdb). The PIIO-1 recognition region is highlighted in orange. E. Jurkat cell lines overexpressing hGARP were incubated with LTGFβ1 and an indicated concentration of an isotype control or PIIO-1 at 37°C for 30 min. Human LAP expression levels were detected by flow cytometry. All experiments represented 2 to 6 independent trials.

Figures 22A to 22I show the antitumor efficacy of PIIO-1-enhanced anti-PD-1 ICB in GARP+ triple-negative breast cancer. A. Experimental protocol. ¹ x 10⁵ 4T1-hGARP breast tumor cells were injected into the mammary fat pads of BALB/c mice, followed by intraperitoneal injection of 100 μg/mouse of PIIO-1 antibody and/or 150 μg/mouse of anti-PD-1 every three days. B. Primary tumor growth curve. C. Overall survival of mice in the four groups. D. Summary of tumor-free mouse incidence in each group. E. Lungs were collected and sectioned at the experimental endpoint and stained with H&E. Representative images from each group of mice are shown. Scale bar, 20 μm. The number of visible lung metastatic nodules was plotted and compared. F. Summary of metastasis incidence in each group. G. Tumors were collected at the endpoint and stained with IHC for pSMAD3 and α-SMA. Representative images of tumor tissue from the four groups of mice are shown (left). Scale bar, 50 μm. Quantitative analysis of IHC staining (right). H. Serum was collected at the endpoint for each mouse. Total serum TGFβ activity was assessed by ELISA. I. Mice in the combined group whose tumors had regressed were monitored for 300 days and then rechallenged with 5 x ^10⁵ wild-type 4T1 mammary tumors from the contralateral mammary fat pad. Untreated BALB/c mice not previously exposed to tumors were used as controls. Overall survival of mice in both groups. *p<0.05;**,p<0.005;***,p<0.001. Tumor curve analysis was performed using repeated measures two-way ANOVA. Overall survival was analyzed by the log-rank (Mantel-Cox) test. Paired t-tests were used to analyze plots E and G based on tumor collection time points. Other data were analyzed using GraphPad Prism with two-tailed Student's t-tests. Turkey program was used to correct for multiple tests in plots B and C. All data are presented as mean ± SEM.

Figures 23A to 23G show the effect of PIIO-1 monotherapy on regulating CD8+ T cells in the TME and conferring cancer protection in hLRRC32KI mice. A. ¹ x 10⁵ MB-49 bladder cancer cells were subcutaneously injected into the right flank of hLRRC32KI mice. PIIO-1 (200 μg/mouse, ip) was delivered every 3 days starting on day 4 for a total of 4 treatments. Representative tumor curves. B. 1 x ^10⁵ MB-49 bladder cancer cells were subcutaneously injected into the right flank of hLRRC32KI mice. PIIO-1 (200 μg/mouse, ip) was delivered on days 6 and 9. Tumors were collected on day 10 and subjected to flow cytometry. Frequency of CD8+ T cells as a percentage of viable CD45+ lymphocytes (left). 1 x ^10⁵ MB-49 bladder cancer cells were subcutaneously injected into the right flank of hLRRC32KI mice. PIIO-1 (200 μg/mouse, ip) was administered every three days starting on day 6, for a total of 6 treatments. Tumors were collected on day 22 and subjected to flow cytometry. Comparison of CD8+ T cells between ISO and PIIO-1 (right). C. Frequency of CD25+Foxp3+ Tregs in CD4+ tumor-infiltrating T cells (left). Frequency of CTLA4+VISTA+ Tregs in tumors (right). D. Differential expression analysis of CD8+ TIL cluster frequencies between ISO and PIIO-1. UMAP size of tumor-infiltrating CD8+ T cells from B decreased after staining with 33 markers and spectral flow cytometry analysis. Data of gated live CD45+CD3+CD8+ T cells are shown, with secondary sampling of 5000 cells per sample. Unsupervised clustering analysis was performed using the FlowSOM algorithm, with the elbow method used to determine the number of clusters. E. Heatmap of D showing the expression levels of indicator markers for each cluster. AE.N = 4–6/group, data (mean +/- SEM) represent two independent experiments. F. Differential expression analysis of CD8+ TIL-produced cytokines between ISO and PIIO-1 treated tumors. ¹ x 10⁵ MB-49 bladder cancer cells were subcutaneously injected into the right flank of hLRRC32KI mice. PIIO-1 (200 μg/mouse, ip) was administered every 3 days starting on day 5 for a total of 4 treatments. Tumors were collected on day 17. Intracellular staining for 17 cytokines was performed, followed by spectral flow cytometry and CD45+CD3+CD8+ T cell analysis. G. Cytokine levels in Figure F, indicated by a heatmap, showing the cytokine expression intensity of each CD8+ T cell cluster. *p<0.05, **p<0.01; Tumor curve analysis was performed using repeated measures two-way ANOVA. Cluster differences were measured using a two-tailed Student's t-test.

Figures 24A to 24D show the preclinical activity of PIIO-1 enhancing anti-PD-1 antibody against bladder cancer. A. Experimental protocol. ¹ x 10⁵ MB-49 bladder cancer cells were subcutaneously injected into the right flank of hLRRC32KI mice. PIIO-1 (200 μg/mouse, ip) and anti-PD-1 antibody (100 μg/mouse, ip) were delivered every 3 days. PIIO-1 was administered on day 4 for a total of 6 doses, and anti-PD-1 antibody was administered on day 10 for a total of 4 doses. B. Representative overall survival of mice treated with isotype control antibody (n=5), PIIO-1 (n=6), anti-PD-1Ab (n=10), or a combination of anti-PD-1Ab and PIIO-1 (n=10). C. Summary of therapeutic efficacy based on complete response. D. PIIO-1 and anti-PD-1 generated a better anti-tumor memory response. Mice with tumor-eliminating tumors were re-challenged subcutaneously with live MB-49. Overall survival was compared. Tumor-free untreated mice were used as controls. *p<0.05;**,p<0.005;***,p<0.001. Overall survival was analyzed using the log-rank (Mantel-Cox) test. The Turkey program was used to correct for multiple tests in plots B and D. All p-values are presented as mean ± SEM.

Figures 25A-25E show how PIIO-1 attenuates the typical TGFβ pathway in tumor-infiltrating immune cells and restores antitumor immunity in hLRRC32KI mice. A. ¹ x 10⁵ MB-49 bladder cancer cells were subcutaneously injected into the right flank of hLRRC32KI mice. PIIO-1 (200 μg/mouse, ip) was administered on days 18 and 20, for a total of two doses. Tumors were collected on day 21. TILs were then isolated and stained for intracellular pSMAD2/3 and cell lineage markers on the cell surface, followed by flow cytometry analysis. B. Quantification of Figure A. ¹ x 10⁵ MB-49 bladder cancer cells were subcutaneously injected into the right flank of hLRRC32KI mice. PIIO-1 (200 μg/mouse, ip) was delivered on days 6 and 9, for a total of two doses. Tumors were collected on day 10. Single-cell suspensions were prepared and RNA was isolated, followed by batch RNA sequencing. C. Volcano plot of gene expression. Differential gene expression is shown in red (upregulated) or blue (downregulated). Representative transcripts such as Ccl3, Ccl9, Cxcl14, Cxcl15, Il6, and Tnfrsf25 are indicated. D. Gene set enrichment analysis of differentially expressed genes between PBS- and PIIO-1-treated tumors. E. Comparison of TILs between PBS- and PIIO-1-treated tumors based on deconvolution of batch RNA sequencing data. *p<0.05, **p<0.01; other data were analyzed using a two-tailed Student's t-test, and data are presented as mean +/- SEM.

Figures 26A to 26L show the antitumor activity promoted by PIIO-1 in a CD8+ T cell and CXCR-3-dependent manner. A and B. CD8-dependent antitumor activity. A. Experimental protocol. B. Tumor growth curves in mice treated with the indicator condition (isotype, n=5; PIIO-1, n=5; anti-CD8, n=3; combination, n=5). C-F. The antitumor activity of PIIO-1 depends on the active outflow of lymphocytes from secondary lymphoid tissues. C. Experimental protocol. D. Tumor growth curves in mice treated with the indicator condition (isotype, n=4; PIIO-1, n=4; FTY720, n=6; combination, n=6). E. Frequency of CD8+ and CD4+ T cells in peripheral blood of the indicator group mice. F. Absolute number of CD8+ T cells in tumors. G. Effect of PIIO-1 on CXCR3 expression and CD8+ T cell number in drained lymph nodes. hLRRC32KI mice carrying MB-49 were treated with either PIIO-1 or ISO for two cycles, and then CXCR3 expression on CD8+ T cells in the drained lymph node (LN) was analyzed. H-L. The antitumor effect of PIIO-1 requires CXCR3. H. Experimental protocol. I. Tumor growth curves of mice treated with the indicated conditions (isotype, n=5; PIIO-1, n=5; FTY720, n=7; combination, n=7). J. Tumor weight on day 17. K. Absolute number of CD8+ T cells in the drained lymph node (dLN). L. Absolute number of CD8+ T cells in the tumor. *p<0.05, **p<0.01; Tumor curve analysis was performed using repeated measures two-way ANOVA. Other data were analyzed using a two-tailed Student's t-test. Plots B, D, and I were corrected for multiple tests using the Sidak program. Data are presented as mean +/- SEM.

Figures 27A–27F. Systemic administration of PIIO-1 to hLRRC32KI mice to increase peripheral lymphocyte activity, including CD8+ T cells and their function. A. hLRRC32KI mice were intravenously injected with 200 μg/mouse of PIIO-1 or mIgG1 every 48 hours for a total of 3 injections. Mice were sacrificed 24 hours after the third PIIO-1 injection and peripheral lymph nodes were harvested. B. Absolute number of live cells from peripheral lymph nodes. C–E. Flow cytometry analysis of peripheral lymph nodes, examining the frequencies of C. CD3+CD8+ T cells, D. Ki67+CD8+ T cells, and E. Foxp3+ regulatory T cells. F. Percentage of CD8+ T cells producing IFNγ and TNFα obtained by intracellular staining. N = 3/group, data represent two independent experiments. Statistical analysis was performed using a two-tailed Student's t-test. Data are presented as mean ± SEM. *p<0.05, **p<0.01.

Figures 28A and 28B show the CD8+ T cell phenotype in the TME altered by GARP expression. A. Subcluster analysis of tumor-infiltrating CD8+ T cells in MB-49 tumors overexpressing EV and hGARP. ¹ x 10⁵ MB-49-EV or hGARP cells were sub-injected into C57BL/6 mice and tumors were harvested on day 18. The UMAP size of tumor-infiltrating CD8+ T cells decreased after staining with 33 markers and spectral flow cytometry analysis. Data for gating live CD45+CD3+CD8+ T cells are shown, with 5000 cells subsampled per sample. Unsupervised clustering analysis was performed using the FlowSOM algorithm, with the elbow method used to determine the number of clusters. B. A heatmap showing the expression levels of indicator markers for each cluster. Cluster differences were measured using a two-tailed Student's t-test. Data are presented as mean +/- SEM. ***p < 0.001.

Figures 29A–29D show the alterations in CD8+ T cell infiltration and clustering by PIIO-1. A. Cell density analysis of tumor-infiltrating CD8+ T cells in MB-49 tumors treated with mIgG1 or PIIO-1. 1 × 10⁵ MB-49 cells were injected s.c. into the right flank of male hLRRC32KI mice. PIIO-1 or ISO (200 μg/mouse, i.p.) was delivered on days 6 and 9. Tumors were collected on day 10 and histological samples were subjected to multiple IF analysis. (Left) Histological samples were stained with CD45, CD8, SMA, and DAPI. (Top right) Tumor regions defined for computational analysis are shown. The boundary at α = 1 represents the boundary between the stroma and the tumor region. This boundary was reduced by α to create additional tumor regions (see Supplementary Methods for more details). (Bottom right) Quantification of CD8+ T cell density in the regions defined in (A) for ISO and PIIO-1 treatment. Compared to ISO, PIIO-1 treatment increased CD8+ T cell density in the intermediate II region. B. The interdependence of SMA+ and CD8+ T cell densities in the internal region as defined in (A). Densities obtained from slides from different mice are shown with different symbols. When the tumor was treated with PIIO-1 (Corr = -0.62), the negative correlation between SMA+ and CD8+ T cells in ISO (Corr = -0.86) decreased. C. The core steps used to calculate the two-point correlation function, in which the CD8+ T cell density in a ring region of radius r and thickness δ corresponding to the CD8+ T cells at the center is calculated (see Supplementary Methods for more details). D. The two-point correlation function C(r) of CD8+ T cells in the internal (left) and intermediate II (right) tumor regions of ISO and PIIO-1 treated mice varies with distance r. Multiple curves of the same color show C(r) data obtained from different slides from different mice (ISO or PIIO-1 treated). Data showed that C(r) in PIIO-1 treatment had a larger peak at r≈7 μm compared to ISO in the middle II region. This indicates an increase in CD8+ T cell grouping within a 7 μm length range in the middle II region when treated with PIIO-1. Cell density differences were measured by Welch t-test. Data are presented as mean +/- SEM.

Figures 30A to 30C show PIIO-1 overcoming resistance to PD-1 blockers and promoting CD8+ T cell infiltration in LLC and CMT-167 models. A. Summary of the number of mice with uncontrolled tumors (>115 mm² on day 17) in each treatment group. ⁵ x 10⁵ LLC cells were subcutaneously injected into the right flank of hLRRC32KI female mice, followed by treatment with ISO, PIIO-1, PD-1, or a combination thereof. Treatments were delivered on day 8 post-tumor inoculation and every 3 days thereafter, for a total of 4 doses. B. Tumor volume 18 days after subcutaneous injection of ¹ x 10⁵ CMT-167 cells. Mice were treated with 4 injections of the indicator antibody (days 8, 11, 14, and 17). C. Frequency of tumor-infiltrating CD8+ T cells in CMT-167 tumors on day 18 (left - representative flow cytometry plot with CD45+ cells gated; right - data quantification). Data from A were analyzed using a two-tailed Fisher exact test. Other data were analyzed using a two-tailed Student's t-test. All data are presented as mean ± SEM. *p<0.05, ****p<0.0001.

Figures 31A-31C show that PIIO-1 attenuates the typical TGFβ pathway in immune cells and primarily targets Tregs in dLNs. Figure 31A shows the subcutaneous injection of ¹ x 10⁵ MB-49 cells into the right flank of male hLRRC32KI mice. Humanized PIIO-1 (200 μg/mouse, ip) was administered on days 18 and 20. dLNs were collected on day 21, then isolated and stained for intracellular pSMAD2/3 using cell lineage markers (see Supplementary Methods for more details), followed by flow cytometry analysis. The expression level of pSMAD2/3 in cells derived from dLNs is shown. Figure 31B shows the quantification of Figure 31A. Figure 31C shows the subcutaneous injection of ¹ x 10⁵ MB-49 cells into the right flank of male hLRRC32KI mice. Humanized PIIO-1 (200 μg/mouse, ip) was administered on day 18. Tumor dLNs, tumors, and spleens were collected on day 19. Humanized PIIO-1 was detected using anti-human Fc antibody. Cells co-expressing humanized PIIO-1 and LAP were gated and their cell identity was further analyzed. Data were analyzed using a two-tailed Student's t-test and presented as mean +/- SEM. *p<0.05, **p<0.01.

Figure 32 shows that the presence or absence of anti-CXCR3 with or without anti-GARP antibody PIIO-1 did not alter the number of Tregs in the tumor mesothelioma (TME). ¹ x 10⁵ MB-49 cells were subcutaneously injected into the right flank of male hLRRC32KI mice. Humanized PIIO-1 and anti-CXCR3 antibody (200 μg/mouse, ip) were administered every 3 days starting on day 5 for a total of 4 treatments. The absolute number of Tregs in the tumor was then quantified by flow cytometry based on the live-gated TIL phenotype: CD45+CD3+CD4+CD25+Foxp3+. Data are presented as mean +/- SEM. No significant differences were observed between groups according to a two-tailed Student's t-test.

Figures 33A and 33B show the characterization of anti-human GARP antibodies used to identify cell surface GARP and block GARP-LTGFβ interaction. Figure 33A shows the detection of GARP expression in the Jurkat-hGARP cell line by flow cytometry using an indicated concentration of anti-GARP antibody. The geometric mean fluorescence intensity (gMFI) of human GARP is plotted. Figure 33B shows the incubation of a Jurkat cell line stably expressing hGARP with recombinant LTGFβ1 and an indicated concentration of isotype control or anti-GARP antibody at 37°C for 30 min. The expression level of human LTGFβ1 was detected by flow cytometry.

Detailed Implementation

This study demonstrates that membrane-bound and soluble GARP are widely expressed in human cancer cells but less so in normal epithelial cells, and that GARP expression is consistently associated with advanced stages of cancer and poor prognosis. Furthermore, GARP itself was found to possess transformative potential, imbuing normal breast epithelial cells with tumorigenicity. GARP expression in cancer cells was observed to induce increased TGF-β activity, possibly due to its ability to concentrate cis- and trans-LTGF-β, thereby contributing to cancer invasiveness and metastasis. GARP expression in the tumor microenvironment promotes the induction of regulatory T cells and thus weakens the function of effector T cells against cancer. However, neutralizing GARP by blocking its ability to bind to TGF-β can produce anticancer activity even without chemotherapy. Specifically, novel antibody molecules capable of effectively binding to and neutralizing GARP are provided herein: humanized PIIO-1 antibodies HuPIIO-1VH1/L1, HuPIIO-1VH1/L2, HuPIIO-1VH2/L1, HuPIIO-1VH1/L3, HuPIIO-1VH2/L2, HuPIIO-1VH2/L3, HuPIIO-1VH3/L1, HuPIIO-1VH2/L3, HuPIIO-1VH3/L3, HuPIIO-1VH4/L1, HuPIIO-1VH4/L2, HuPIIO-1VH4/L3, and a 5c5 antibody. Therefore, the antibodies of these embodiments can be used in methods for treating cancer and enhancing immune responses (e.g., in combination with adoptive T-cell therapy).

While T-cell therapy has the potential to treat cancer by recognizing and attacking tumor cells, the tumor microenvironment can weaken the ability of adopted effector T cells to control cancer by inducing regulatory T cells to evade the immune system. Therefore, embodiments of this disclosure overcome the challenges associated with the prior art by providing a method for treating cancer comprising a combination of T-cell therapy and an antiplatelet agent. In this method, the antiplatelet agent can enhance adoptive T-cell therapy on the tumor because soluble factors secreted by activated platelets have been shown to suppress T cells. For example, latent TGFβ and GARP secreted by platelets have been shown to induce resistance in cancer cells to adoptive T-cell therapy. Therefore, antiplatelet factors such as anti-GARP monoclonal antibodies (which can block TGFβ binding) can be used in combination with T-cell therapy to overcome this resistance and treat cancer. Furthermore, other immunotherapies such as immune checkpoint inhibitors can be used in combination with T-cell therapy and antiplatelet agents to enhance the immune response.

I. Definition

As used herein, "substantially free of" with respect to a specified component means that the specified component was not intentionally formulated into the composition and/or is present only as a contaminant or in trace amounts. Therefore, the total amount of the specified component due to any accidental contamination of the composition is well below 0.05%. Most preferably, the composition in which the amount of the specified component is undetectable using standard analytical methods is preferred.

As used herein in the specification and claims, “a” (or “an”) may mean one or more. As used herein in the specification and claims, when used with the word “comprising,” the word “a” may mean one or more. As used herein in the specification and claims, “another” or “additionally” may mean at least a second or more.

As used herein in the specification and claims, the term “about” is used to indicate that the value includes inherent error variations with respect to the apparatus or method used to determine the value, or variations that exist between the objects under study.

"Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject or the procedure or manner in which a subject is treated for the purpose of obtaining therapeutic benefit from a disease or health-related condition. For example, treatment may include the administration of a pharmaceutically effective amount of an antibody that inhibits GARP signaling. In another instance, treatment may include the administration of T-cell therapy and a pharmaceutically effective amount of an antiplatelet agent (e.g., an antibody that inhibits GARP signaling).

"Subject" and "patient" refer to human or non-human beings, such as primates, mammals, and vertebrates. In a particular embodiment, the subject is a human.

An “increase” can refer to any change that results in a greater quantity of symptoms, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in symptoms, activity, or composition by a statistically significant amount. Therefore, an increase can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, as long as the increase is statistically significant.

"Reduction" can refer to any change that results in a smaller amount of symptoms, disease, composition, condition, or activity. When the genetic output of a gene product utilizing a substance is lower than the output of a gene product not utilizing the substance, the substance should also be understood as reducing the genetic output of the gene. For example, reduction can be a change in the symptoms of a disease that results in fewer symptoms than previously observed. Reduction can be any individual, median, or average reduction in symptoms, activity, or composition at a statistically significant amount. Therefore, the reduction can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, provided that the reduction is statistically significant.

"Inhibit," "inhibiting," and "inhibition" refer to a reduction in activity, response, symptom, disease, or other biological parameters. This can include, but is not limited to, complete ablation of activity, response, symptom, or disease. It can also include, for example, a 10% reduction in activity, response, symptom, or disease compared to natural or control levels. Therefore, the reduction compared to natural or control levels can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction in between.

The word "reducing" or other terms such as "reducing" and "reduction" refer to a reduction in an event or characteristic (e.g., tumor growth). It should be understood that this is usually related to a standard or expected value; in other words, it is relative, but it is not always necessary to cite a standard or relative value. For example, "reducing tumor growth" means reducing the rate of tumor growth relative to a standard or control.

The term "prevent" or other forms of the term (such as "preventing" or "prevention") means to prevent a particular event or characteristic in order to stabilize or delay its development or progression, or to minimize the likelihood of its occurrence. Prevention does not require comparison with a control, as it is generally more absolute than, for example, reduction. As used herein, something can be reduced but not prevented, but something reduced can also be prevented. Similarly, something can be prevented but not reduced, but something prevented can also be reduced. It should be understood that, unless otherwise expressly stated, the use of other terms is also explicitly disclosed in the context of reduction or prevention.

The term "treatment" refers to the medical management of a patient aimed at curing, improving, stabilizing, or preventing a disease, pathological condition, or ailment. This term includes active treatment, which is treatment specifically aimed at improving a disease, pathological condition, or ailment, and also includes etiological treatment, which is treatment aimed at eliminating the cause of the related disease, pathological condition, or ailment. Furthermore, the term includes palliative treatment, which is treatment aimed at relieving symptoms rather than curing a disease, pathological condition, or ailment; preventative treatment, which is treatment aimed at minimizing or partially or completely suppressing the development of a related disease, pathological condition, or ailment; and supportive treatment, which is treatment used to complement another specific therapy aimed at improving a related disease, pathological condition, or ailment.

As used throughout this application, the terms "treatment benefit" or "treatment effectiveness" mean anything that promotes or enhances the health of a subject in relation to the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of the disease. For example, cancer treatment may involve, for instance, a reduction in tumor size, a reduction in tumor invasiveness, a reduction in cancer growth rate, or prevention of metastasis. Cancer treatment may also refer to prolonging the survival of a subject with cancer.

The “effective amount” of a drug refers to the amount of drug sufficient to provide the desired effect. The “effective” amount of drug will vary from subject to subject, depending on many factors such as the subject’s age and general condition, the specific drug or one or more, etc. Therefore, it is not always possible to specify a quantitative “effective amount.” However, those skilled in the art can determine the appropriate “effective amount” for any subject’s situation using routine experiments. Furthermore, as used herein and unless otherwise expressly stated, the “effective amount” of a drug may also refer to an amount covering both therapeutic and prophylactic effective amounts. The “effective amount” of a drug necessary to achieve a therapeutic effect can vary depending on factors such as the subject’s age, sex, and weight. Dosing regimens can be adjusted to provide the optimal therapeutic response. For example, several separate doses may be administered daily, or the dose may be reduced proportionally, as indicated by the urgency of the treatment situation.

The “therapeutic effective amount” or “therapeutic effective dose” of a composition (e.g., a composition comprising a pharmaceutical agent) refers to the amount that effectively achieves the desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is control of type 1 diabetes. In some embodiments, the desired therapeutic outcome is control of obesity. The therapeutically effective amount of a given therapeutic agent will generally vary depending on factors such as the type and severity of the condition or disease being treated, and the subject’s age, sex, and weight. The term may also refer to the amount of therapeutic agent or the rate of delivery of the therapeutic agent (e.g., an amount that varies over time) that effectively promotes the desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary depending on the condition being treated, the subject’s tolerance, the pharmaceutical agent and/or pharmaceutical formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of the pharmaceutical agent in the formulation, etc.), and various other factors as understood by those skilled in the art. In some cases, the desired biological or medical response has been achieved after administering multiple doses of the composition to a subject over a period of time of days, weeks, or years.

"Anti-cancer" agents can have negative effects on a subject's cancer cells/tumors, for example, by promoting the killing of cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting immune responses to cancer cells or tumors, preventing or inhibiting cancer progression, or increasing the lifespan of subjects with cancer.

The term “antibody” is used in the broadest sense herein and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, for example, individual antibodies comprising that population are identical except for possible mutations (e.g., naturally occurring mutations) that may be present in small amounts. Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies. In some embodiments, such monoclonal antibodies typically comprise an antibody containing a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a method comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select a unique clone from a plurality of clones (such as a hybridoma clone library, phage clones, or recombinant DNA clones). It should be understood that the selected target-binding sequence can be further modified, for example, to improve affinity for the target, humanize the target-binding sequence, improve its production in cell cultures, reduce its immunogenicity in vivo, create multispecific antibodies, etc., and antibodies containing modified target-binding sequences are also monoclonal antibodies of the present invention. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are generally not contaminated by other immunoglobulins.

The phrase "pharmaceutically or pharmacologically acceptable" means a molecular entity and composition that, when administered to animals (such as humans) where appropriate, will not produce adverse, allergic, or other adverse reactions. According to this disclosure, the preparation of pharmaceutical compositions comprising antibodies or other active ingredients is known to those skilled in the art. Furthermore, for administration to animals (e.g., humans), it should be understood that the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA's Office of Biostandards.

As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol/aqueous solutions, salt solutions, parenteral media (such as sodium chloride, Ringer's glucose, etc.)), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters (such as ethyl oleate)), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic reagents, absorption delay agents, salts, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids and nutritional supplements, similar materials, and combinations thereof. The pH and precise concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

The term "unit dose" or "dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of therapeutic composition calculated to produce the desired response discussed above associated with its administration (i.e., appropriate route and treatment regimen). The amount to be administered, depending on both the number of treatments and the unit dose, depends on the desired effect. The actual dose of the composition of this embodiment administered to a patient or subject can be determined by physical and physiological factors such as the subject's weight, age, health status, and sex, the type of disease being treated, the extent of disease penetration, prior or concurrent therapeutic interventions, the patient's idiopathic condition, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, the dose may also include administrations of about 1 μg/kg/body weight to about 1000 mg/kg/body weight (such ranges include intermediate doses) or more, and any ranges that can be derived therefrom. In non-limiting examples of ranges that can be derived from the figures listed herein, ranges such as about 5 μg/kg/body weight to about 100 mg/kg/body weight, about 5 μg/kg/body weight to about 500 mg/kg/body weight, etc., may be administered. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject.

When applied to cells, the terms "contact" and "exposure" are used herein to describe the process of delivering a therapeutic construct and a chemotherapeutic or radiotherapeutic agent to or directly juxtaposed with target cells. For example, to achieve cell killing, two agents are delivered to cells in combined amounts that effectively kill cells or prevent their division.

The term "immune checkpoint" refers to molecules, such as proteins, in the immune system that provide inhibitory signals to their components to balance the immune response. Known immune checkpoint proteins include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD1) and its ligands programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), as well as LAG-3, lymphocyte activation gene 3 (LAG-3), B- and T-lymphocyte attenuation factor (BTLA), B7 homolog 3 (B7H3), B7 homolog 4 (B7H4), T-cell immunoglobulin and mucin domain 3 (Tim-3), and cytotoxic immunoglobulin-like receptor (KIR). The pathways involving LAG3, B- and T-lymphocyte attenuating factors (BTLA), T-cell activation V-domain Ig repressor (VISTA), B7H3, B7H4, TIM3, T-cell immune receptors with Ig and ITIM domains (TIGIT), and KIR are recognized in the art as constituting immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (see, for example, Pardoll, 2012, Nature Rev Cancer 12:252-264; Mellman et al., 2011, Nature 480:480-489).

"Immune checkpoint inhibitors" refer to any compound that inhibits the function of immune checkpoint proteins. Inhibition includes both reduction and complete blockage of function. Specifically, immune checkpoint proteins are human immune checkpoint proteins. Therefore, immune checkpoint protein inhibitors are, in particular, inhibitors of human immune checkpoint proteins. Examples of checkpoint inhibitors include, but are not limited to, anti-PD-1 (such as, for example, nivolumab (BMS-936558 or MDX1106), pembrolizumab, CT-011, MK-3475), anti-PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A or MSB0010718C), such as, for example, PD-L2 (rHIgM12B7), anti-CTLA-4 (such as, for example, ipilimumab (MDX-010), trimemumab (CP-675,206)), anti-IDO, anti-B7-H3 (such as, for example, MGA271, MGD009, avorimab), anti-B7-H4, and anti-PD-L1 (such as, for example, atezolizumab, MGD009, pembrolizumab), and anti-PD-L2 (such as, for example, atezolizumab, MGD009, pembrolizumab), and anti-PD-L2 (such as, for example, atezolizumab, MGD009, pembrolizumab), and anti-PD-L3 ... TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), anti-TIGIT (such as, for example, BMS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A or PVSRIPO), anti-BTLA, anti-LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013 and Immutep).

II. Antibodies in Examples

In some embodiments, antibodies or fragments thereof are considered that bind to at least a portion of the GARP protein and inhibit GARP signaling and cancer cell proliferation. As used herein, the term "antibody" is intended to broadly refer to any immune binding agent, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG, as well as polypeptides containing an antibody CDR domain that retains antigen-binding activity. Antibodies may be selected from the group consisting of chimeric antibodies, affinity-matured antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, or antigen-binding antibody fragments or natural or synthetic ligands. Preferably, the anti-GARP antibody is a monoclonal antibody or a humanized antibody.

Therefore, polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs (including engineered forms of any of the foregoing) specific to GARP proteins, one or more of their corresponding epitopes, or conjugates thereof can be created by known methods and as described herein, regardless of whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of natural compounds.

Examples of antibody fragments suitable for this embodiment include, but are not limited to: (i) Fab fragments consisting of _VL , _VH , _CL , and _CH1 domains; (ii) “Fd” fragments consisting of _VH and _CH1 domains; (iii) “Fv” fragments consisting of the _VL and _VH domains of a single antibody; (iv) “dAb” fragments consisting of the _VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (“scFv”) wherein the _VH and _VL domains are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bispecific single-chain Fv dimers (see U.S. Patent No. 5,091,513); and (ix) bispecific antibodies, multivalent or multispecific fragments constructed by gene fusion (U.S. Patent Publication 20050214860). Fv, scFv, or bisomatic antibody molecules can be stabilized by incorporating disulfide bridges linking the _VH and _VL domains. Small bodies containing scFv linked to the CH3 domain can also be prepared (Hu et al., 1996).

Antibody-like binding peptide mimics were also considered in the examples. Liu et al. (2003) described “antibody-like binding peptide mimics” (ABiP), which are peptides that act as thrombosing antibodies and have some advantages such as a longer serum half-life and a less cumbersome synthesis method.

Animals can be inoculated with antigens, such as the extracellular domain (ECD) protein of GARP, to produce antibodies specific to the GARP protein. Typically, the antigen binds to or conjugates with another molecule to enhance the immune response. As used herein, a conjugate is any peptide, polypeptide, protein, or non-protein substance that binds to an antigen used to elicit an immune response in an animal. Antibodies produced in animals in response to antigen inoculation comprise a variety of distinct molecules (polyclonal antibodies) made by various B lymphocytes that produce individual antibodies. Polyclonal antibodies are a mixed population of antibody species, each capable of recognizing different epitopes on the same antigen. Under the correct conditions for the production of polyclonal antibodies in an animal, most antibodies in the animal's serum will recognize common epitopes on the antigenic compound against which the animal has been immunized. This specificity is further enhanced by affinity purification to select only those antibodies that recognize the target antigen or epitope.

Monoclonal antibodies are monospecies antibodies, where each antibody molecule recognizes the same epitope because all antibody-producing cells originate from a single B lymphocyte cell line. Methods for generating monoclonal antibodies (MAbs) generally begin along the same route as methods for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to generate monoclonal antibodies. In some embodiments, rabbit, sheep, or frog cells are used to generate monoclonal antibodies. The use of rats is well-known and can offer certain advantages. Mice (e.g., BALB/c mice) are routinely used and typically produce a high proportion of stable fusions.

Hybridoma technology involves fusing a single B lymphocyte from a mouse previously immunized with the GARP antigen with an immortalized myeloma cell (typically a mouse myeloma). This technology provides a method for propagating a single antibody-producing cell indefinitely, enabling the production of an unlimited number of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity.

Plasma B cells (CD45 ⁺ CD5 ^- CD19 ⁺ ) were isolated from freshly prepared rabbit peripheral blood mononuclear cells from immunized rabbits and further selected for GARP-binding cells. After enriching antibody-producing B cells, total RNA was isolated and cDNA was synthesized. DNA sequences from the antibody variable regions of both the heavy and light chains were amplified, constructed into phage display Fab expression vectors, and transformed into *E. coli*. GARP-specific binding Fabs were selected through multiple rounds of enrichment panning and sequencing. Selected GARP-binding targets were expressed as full-length IgG in rabbit and rabbit/human chimeric forms using a mammalian expression vector system in human embryonic kidney (HEK293) cells (Invitrogen) and purified using a protein G resin and rapid protein liquid chromatography (FPLC) separation unit.

In one embodiment, the antibody is a chimeric antibody, such as an antibody containing an antigen-binding sequence derived from a non-human donor, which is grafted onto a heterologous non-human, human, or humanized sequence (e.g., frame and/or constant domain sequences). Methods have been developed to replace the light and heavy chain constant domains of monoclonal antibodies with similar domains derived from humans, while keeping the variable regions of the exogenous antibody intact. Alternatively, “fully human” monoclonal antibodies are produced in transgenic mice with human immunoglobulin genes. Methods have also been developed to convert the variable domains of monoclonal antibodies into more human forms by recombinantly constructing antibody variable domains with rodent (e.g., mouse) and human amino acid sequences. In “humanized” monoclonal antibodies, only the hypervariable CDR is derived from mouse monoclonal antibodies, while the frame and constant regions are derived from human amino acid sequences (see U.S. Patent Nos. 5,091,513 and 6,881,557). It is believed that replacing rodent-specific amino acid sequences in antibodies with amino acid sequences found at corresponding positions in human antibodies reduces the likelihood of adverse immune responses during therapeutic use. Hybridomas or other cells that produce antibodies may also undergo gene mutations or other changes, which may or may not alter the binding specificity of antibodies produced by hybridomas.

Methods for generating polyclonal antibodies in various animal species, and for generating monoclonal antibodies of various types (including humanized, chimeric, and fully human), are well known in the art and are highly predictable. For example, feasible descriptions of such methods are provided in the following U.S. patents and patent applications: U.S. Patent Application Nos. 2004/0126828 and 2002/0172677; and U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149; 4,277,437. 4,366,241；4,469,797；4,472,509；4,606,855；4,703,003；4,742,159；4,767,720；4,816,567；4,867,973；4,938,948；4,946,778；5,021,236；5,164,296；5,196,0 66；5,223,409；5,403,484；5,420,253；5,565,332；5,571,698；5,627,052；5,656,434；5,770,376；5,789,208；5,821,337；5,844,091；5,858,657；5,861,155；5,87 1,907; 5,969,108; 6,054,297; 6,165,464; 6,365,157; 6,406,867; 6,709,659; 6,709,873; 6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; and 6,891,024. All patents, patent application publications, and other publications cited herein are hereby incorporated by reference.

Antibodies can be produced from any animal source, including birds and mammals. Preferably, antibodies are from sheep, mice (e.g., mice and rats), rabbits, goats, guinea pigs, camels, horses, or chickens. Furthermore, newer technologies allow for the development and screening of human antibodies from human combinatorial antibody libraries. For example, phage antibody expression technology allows for the production of specific antibodies without animal immunization, as described in U.S. Patent No. 6,946,546, which is incorporated herein by reference. These technologies are further described in the following literature: Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).

Antibodies against GARP are fully expected to have the ability to neutralize or counteract GARP effects, regardless of the animal species, monoclonal cell line, or other source of the antibody. For the production of therapeutic antibodies, certain animal species may be less preferred because they may be more likely to elicit an allergic response due to activation of the complement system via the antibody's "Fc" portion. However, intact antibodies can be enzymatically digested into an "Fc" (complement-binding) fragment, along with an antibody fragment having a binding domain or CDR. Removal of the Fc portion reduces the likelihood of the antigen-antibody fragment triggering an undesirable immunological response, and therefore, antibodies without the Fc may be preferentially used for prophylactic or therapeutic treatment. As mentioned above, antibodies can also be constructed as chimeric antibodies or partially or fully human antibodies to reduce or eliminate adverse immunological consequences resulting from the administration of antibodies produced in other species or having sequences from other species to animals.

Substitutional variants typically involve the exchange of one amino acid for another at one or more sites within a protein and can be designed to modulate one or more properties of the polypeptide while sacrificing or retaining other functions or properties. Substitutions can be conserved, meaning that one amino acid is replaced by an amino acid having a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions can be non-conservative, affecting the function or activity of the peptide. Non-conservative changes typically involve replacing residues with chemically different residues, such as replacing polar or charged amino acids with nonpolar or uncharged amino acids, and vice versa.

The protein can be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also anticipated that bacteria containing such variants can be used in the composition and method. Therefore, protein isolation is not required.

It is anticipated that the composition contains approximately 0.001 mg to approximately 10 mg of total polypeptides, peptides, and/or proteins per ml. Therefore, the protein concentration in the composition can be approximately, at least approximately, or at most approximately 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derived therefrom). Among them, approximately, at least approximately or at most approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 5 2%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% can be antibodies that bind to GARP.

Antibodies, or preferably the immunological portion of antibodies, may be chemically conjugated to or expressed as fusion proteins with other proteins. For the purposes of this specification and the appended claims, all such fusion proteins are included in the definition of antibodies or the immunological portion of antibodies.

Examples provide anti-GARP antibodies and antibody-like molecules, polypeptides and peptides linked to at least one reagent to form antibody conjugates or payloads. To enhance the efficacy of antibody molecules as diagnostic or therapeutic agents, at least one desired molecule or moiety is typically linked, covalently bound, or conjugated. Such molecules or moiety can be, but are not limited to, at least one effector or reporter molecule. Effector molecules include molecules with desired activities, such as cytotoxic activity. Non-limiting examples of effector molecules attached to antibodies include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, etc. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabeled molecules, haptens, fluorescently labeled molecules, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photosynthetic molecules, colored particles, or ligands such as biotin.

Several methods for linking or conjugating antibodies to their conjugate portions are known in the art. Some attachment methods involve the use of metal chelating complexes, such as organic chelating agents like diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3-,6-diphenylglyuron-3 attached to the antibody. Monoclonal antibodies can also be reacted with enzymes in the presence of conjugating agents such as glutaraldehyde or periodate. Conjugates with fluorescein-labeled molecules are prepared in the presence of these conjugating agents or by reaction with isothiocyanates.

III. T-cell therapy

Certain embodiments of this disclosure relate to obtaining T cells and administering them to a subject as an immunotherapy targeting cancer cells. Over the past two decades, several fundamental methods for the derivation, activation, and expansion of functional anti-tumor effector T cells have been described. These include: autologous cells, such as tumor-infiltrating lymphocytes (TILs); T cells activated in vitro using autologous dendritic cells (DCs), lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells separated by capturing the target cell membrane; allogeneic cells naturally expressing anti-host tumor T-cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells that have been genetically reprogrammed or “redirected” to express tumor-responsive TCRs or chimeric TCR molecules, exhibiting antibody-like tumor recognition capabilities known as “T-bodies.” These methods have led to numerous protocols for T-cell preparation and immunization that can be used in the methods of this disclosure.

A. T cell preparation

In some embodiments, T cells are derived from blood, bone marrow, lymph, or lymphoid organs. In some aspects, the cells are human cells. Cells are typically primary cells, such as those isolated directly from the subject and/or isolated from and frozen from the subject. In some embodiments, cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4 ⁺ cells, CD8 ⁺ cells, and their subpopulations, such as those defined by function, activation state, maturation, differentiation potential, expansion, recycling, localization and/or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. For the subject to be treated, the cells may be allogeneic and/or autologous. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from the subject as described herein, preparing, processing, culturing, and/or engineering them, and reintroducing them into the same patient before or after cryopreservation.

The subtypes and subsets of T cells (e.g., CD4 ⁺ and/or CD8 ⁺ T cells) are naive T ( _TN ) cells, effector T cells ( _TEFF ), memory T cells and their subtypes (such as stem cell memory T ( _TSCM ), central memory T ( _TCM ), effector memory T ( _TEM ), or terminally differentiated effector memory T cells), tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated non-variant T (MAIT) cells, naturally occurring and adopted regulatory T (Treg) cells, and helper T cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α/β T cells, and δ/γ T cells).

In some embodiments, cells that are positive for or negative for specific markers such as surface markers are enriched or consumed in one or more T cell populations. In some cases, such markers are those that are absent or expressed at relatively low levels in some T cell populations (e.g., non-memory cells) but present or expressed at relatively high levels in some other T cell populations (e.g., memory cells). In one embodiment, cells that are positive for or express high levels of CD45RO, CCR7, ^CD28 , CD27, ^CD44 , CD127, and/or CD62L are enriched (i.e., positively selected) and cells that are positive for or express high levels of CD45RA are consumed (e.g., negatively selected). In some embodiments, cells that are positive for or express high levels of CD122, CD95, CD25, CD27, and/or IL7-Ra (CD127) are enriched or consumed. In some instances, CD8 ⁺ T cells were enriched in cells that were positive for CD45RO (or negative for CD45RA) and positive for CD62L.

In some embodiments, T cells are isolated from PBMC samples by negative selection of markers expressed on non-T cells (such as B cells, monocytes, or other leukocytes such as CD14). In some aspects, CD4 ⁺ or CD8 ⁺ selection steps are used to isolate CD4 ⁺ helper cells and CD8 ⁺ cytotoxic T cells. Such CD4 ⁺ and CD8 ⁺ populations can be further sorted into subpopulations by positive or negative selection of markers expressed or expressed at relatively high levels on one or more initial, memory, and/or effector T cell subsets.

In some embodiments, initial, central memory, effector memory, and/or central memory stem cells are further enriched or depleted in CD8 ⁺ cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulations. In some embodiments, enrichment for central memory T ( _TCM ) cells is performed to increase efficacy, such as improving long-term survival, expansion, and/or engraftment after administration, which is particularly robust in some respects in such subpopulations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, a combination of _TCM -enriched CD8 ⁺ T cells and CD4 ⁺ T cells further enhances efficacy.

In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a single-cell suspension is obtained. The single-cell suspension can be obtained in any suitable manner, for example, mechanically (using, for example, a gentleMACS ^™ dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., with collagenase or DNase). The single-cell suspension of the enzymatically digested tumor is cultured in interleukin-2 (IL-2). The cells are cultured until confluence (e.g., about 2 × ^10⁶ lymphocytes), for example, from about 5 to about 21 days, preferably from about 10 to about 14 days. For example, cells can be cultured from 5 days, 5.5 days, or 5.8 days to 21 days, 21.5 days, or 21.8 days, such as from 10 days, 10.5 days, or 10.8 days to 14 days, 14.5 days, or 14.8 days.

The cultured T cells can pool and rapidly expand. Rapid expansion provides an increase of at least about 50-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more) in the number of antigen-specific T cells over a period of about 10 to about 14 days, preferably about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold or more) over a period of about 10 to about 14 days, preferably about 14 days.

Expansion can be accomplished by any of a variety of methods known in the art. For example, T cells can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15) (preferably IL-2). Nonspecific T cell receptor stimulants may include approximately 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Raritan, N.J.). Alternatively, T cells can be rapidly expanded in vitro by stimulating peripheral blood mononuclear cells (PBMCs) with one or more cancer antigens (including their antigenic portions, such as epitopes or cells), optionally expressed from a vector (such as a human leukocyte antigen A2 (HLA-A2) binding peptide) in the presence of T cell growth factors (such as 300 IU/ml IL-2 or IL-15 (preferably IL-2)). In vitro induced T cells can be rapidly expanded by restimulating HLA-A2-expressing antigen-presenting cells with a pulse. Alternatively, T cells can be restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

Autologous T cells can be modified to express T cell growth factors that promote autologous T cell growth and activation. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable modification methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain aspects, modified autologous T cells express T cell growth factors at high levels. T cell growth factor coding sequences, such as those for IL-12, are readily available in the art, as are promoters, and their operative linking to T cell growth factor coding sequences promotes high-level expression.

B. Genetically engineered antigen receptors

T cells can be genetically engineered to express antigen receptors such as engineered TCRs and/or chimeric antigen receptors (CARs). For example, autologous T cells can be modified to express T cell receptors (TCRs) that are antigen-specific to cancer antigens. Suitable TCRs include, for example, those that are antigen-specific to melanoma antigens, such as gp100 or MART-1. Suitable modification methods are known in the art. See, for example, Sambrook and Ausubel, ibid. For example, T cells can be transduced to express T cell receptors (TCRs) that are antigen-specific to cancer antigens using transduction techniques described below: Heemskerk et al., HumGene Ther. 19:496-510 (2008) and Johnson et al., Blood 114:535-46 (2009).

In some embodiments, T cells comprise one or more nucleic acids encoded with one or more antigen receptors, introduced through genetic engineering, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not typically present in the cell or in samples obtained from the cell, such as samples obtained from another organism or cell, for example, which are not typically found in engineered cells and/or organisms derived from such cells. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acids not found in nature (e.g., chimeric nucleic acids).

In some embodiments, the CAR contains an extracellular antigen recognition domain that specifically binds to the antigen. In some embodiments, the antigen is a protein expressed on the cell surface. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, recognizes on the cell surface in the context of major histocompatibility complex (MHC) molecules.

Exemplary antigen receptors (including CARs and recombinant TCRs) and methods for engineering and introducing receptors into cells, including, for example, those described in International Patent Application Publications WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061, U.S. Patent Application Publications US2002131960, US2013287748, US20130149337, and U.S. Patents 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6, 410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118 and those described in European Patent Application No. EP2537416, and/or those described by Sadelain et al., Cancer Discov. April 2013; 3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., October 2012; 24(5):633-39; Wu et al., Cancer, March 2012 18(2):160-75. In some respects, genetically engineered antigen receptors include CARs as described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO/2014055668 A1.

In some respects, the tumor antigen is human telomerase reverse transcriptase (hTERT), survivin, mouse double microsome 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), activin, alpha-fetoglobulin (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, or cyclin (D1). For example, the target antigen is hTERT or survivin. In some respects, the cancer antigen is CD38. In other respects, the target antigen is CD33 or TIM-3. In still other respects, it is CD26, CD30, CD53, CD92, CD148, CD150, CD200, CD261, CD262, or CD362. In some embodiments, the engineered immune cells may contain antigens targeting one or more other antigens. In some embodiments, one or more other antigens are tumor antigens or cancer markers. Other antigens include: orphan tyrosine kinase receptor ROR1, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, κ light chain, Lewis Y, Ll-cell adhesion molecule, MAGE-A1, mesothelin, and MUC. 1. MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gplOO, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, liver glycoprotein B2, CD123, CS-1, c-Met, GD-2 and MAGE A3, CE7, Wilms' tumor 1 (WT-1), cyclins (such as cyclin A1 (CCNA1)) and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other pathogens.

1. Chimeric antigen receptor

In some embodiments, engineered antigen receptors include chimeric antigen receptors (CARs), including activating or stimulatory CARs, co-stimulatory CARs (see WO2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215)(2013). CARs typically include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some respects via a linker and/or transmembrane domain. Such molecules typically mimic or approximate signaling via natural antigen receptors, signaling via such receptors in combination with co-stimulatory receptors, and/or signaling via co-stimulatory receptors alone.

In some embodiments, a CAR is constructed to be specific to a particular antigen (or marker or ligand), such as an antigen expressed in a specific cell type to be targeted by adoptive therapy, for example a cancer marker, and/or an antigen intended to induce an inhibitory response, such as an antigen expressed on normal or disease-free cell types. Therefore, a CAR typically includes one or more antigen-binding molecules in its extracellular portion, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, a CAR includes one or more antigen-binding portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from a variable heavy (VH) and variable light (VL) chain of a monoclonal antibody (mAb).

In some aspects, antigen-specific binding or recognition components are linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to an extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some cases, transmembrane domains are selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

In some embodiments, the transmembrane domain is derived from a natural or synthetic source. In some aspects, when the source is natural, the domain may be derived from any membrane-binding or transmembrane protein. Transmembrane regions include those derived from (i.e., transmembrane regions containing at least) the following: α, β, or ζ chains of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD5, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain primarily comprises hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

CARs typically comprise at least one or more intracellular signaling components. In some embodiments, a CAR comprises an intracellular component of the TCR complex, such as a TCR CD3 ⁺ chain mediating T cell activation and cytotoxicity, for example, a CD3ζ chain. Thus, in some aspects, the antigen-binding molecule is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and/or other CD transmembrane domains. In some embodiments, a CAR further comprises a portion of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, a CAR comprises a chimeric molecule between CD3-ζ (CD3-Q) or Fc receptor γ and CD8, CD4, CD25, or CD16.

2. T cell receptor (TCR)

In some embodiments, genetically engineered antigen receptors include recombinant T-cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells. A “T-cell receptor” or “TCR” refers to a molecule containing a variable α-chain and β-chain (also referred to as TRa and TCRp, respectively) or a variable γ-chain and δ-chain (also referred to as TCRy and TCR5, respectively) and capable of specifically binding to antigenic peptides that bind to MHC receptors. In some embodiments, TCRs are in αβ form. Typically, TCRs existing in αβ and γδ forms are structurally similar, but T cells expressing them may have different anatomical locations or functions. TCRs can be present on the cell surface or in a soluble form. TCRs are typically present on the surface of T cells (or T lymphocytes) and are generally responsible for recognizing sites that bind to major histocompatibility complex (MHC) molecules. In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immunune System in Health and Disease, 3rd ed., Current Biology Publications, 4:33, 1997). For example, in some aspects, each chain of the TCR may possess an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term “TCR” should be understood to encompass its functional TCR fragments. The term also encompasses complete or full-length TCRs, including TCRs in αβ or γδ form.

Therefore, for the purposes of this document, reference to TCR includes any TCR or functional fragment, such as the antigen-binding moiety of a TCR that binds to a specific antigenic peptide (i.e., an MHC-peptide complex) bound to an MHC molecule. The terms "antigen-binding moiety" or "antigen-binding fragment" for TCR are used interchangeably and refer to a molecule containing a portion of a TCR domain but binding to an antigen (e.g., an MHC-peptide complex) that binds to the intact TCR. In some cases, the antigen-binding moiety contains variable domains of the TCR, such as variable α-chains and variable β-chains of the TCR, sufficient to form binding sites for binding to a specific MHC-peptide complex, such as typically where each chain contains three complementarity-determining regions.

In some embodiments, the variable domains of the TCR chain associate to form loops or complementarity-determining regions (CDRs) similar to those of immunoglobulins, which confer antigen recognition and peptide specificity by forming binding sites on the TCR molecule. Typically, like immunoglobulins, CDRs are separated by frame regions (FRs) (see, for example, Jores et al., Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; also see Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the primary CDR responsible for recognizing processed antigens, although CDR1 of the α-chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the β-chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β-chain may contain a further hypervariable (HV4) region.

In some embodiments, the TCR chain contains a constant domain. For example, like immunoglobulins, the extracellular portion of the TCR chain (e.g., α-chain, β-chain) may contain two immunoglobulin domains: a variable domain at the N-terminus (e.g., _Va or Vp; generally amino acids 1 to 116, based on Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest," USDept. Health and Human Services, Public Health Service, National Institutes of Health, 1991, 5th edition), and a constant domain adjacent to the cell membrane (e.g., α-chain constant domain or Ca _). The β-chain constant domain (or Cp, typically amino acids 117 to 259, based on Kabat) is generally composed of amino acids 117 to 295 (based on Kabat). For example, in some cases, the extracellular portion of a TCR formed by two chains contains two distal membrane constant domains and two distal membrane variable domains containing CDRs. The constant domains of the TCR contain short linker sequences in which cysteine residues form disulfide bonds, creating a link between the two chains. In some embodiments, the TCR may have additional cysteine residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domain.

In some embodiments, the TCR chain may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules such as CD3. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.

Generally, CD3 is a multiprotein complex that in mammals can possess three distinct chains (γ, δ, and ε) as well as a ζ-chain. For example, in mammals, the complex can contain a homodimer of CD3γ, CD3δ, two CD3ε, and CD3ζ chains. CD3γ, CD3δ, and CD3ε chains are highly correlated cell surface proteins of the immunoglobulin superfamily, each containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a characteristic feature that enables these chains to associate with positively charged T-cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif called an immunoreceptor tyrosine-based activation motif, or ITAM, while each CD3ζ chain has three of these conserved motifs. Generally, ITAMs are involved in the signal transduction capabilities of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating signals from the TCR into the cell. The CD3- and ζ- chains, together with the TCR, form the so-called T-cell receptor complex.

In some embodiments, a TCR can be a heterodimer of two chains, α and β (or optionally γ and δ), or it can be a single-chain TCR construct. In some embodiments, a TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) linked by one or more disulfide bonds.

In some embodiments, a TCR for a target antigen (e.g., a cancer antigen) is identified and introduced into cells. In some embodiments, the nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of a publicly available TCR DNA sequence. In some embodiments, the TCR is obtained from a biological source, such as from cells, such as from T cells (e.g., cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, T cells can be obtained from cells isolated in vivo. In some embodiments, high-affinity T cell clones can be isolated from a patient, and the TCR can be isolated. In some embodiments, the T cells can be cultured T cell hybridomas or clones. In some embodiments, the TCR clone for the target antigen has been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system or HLA). See, for example, tumor antigens (see, for example, Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J. Immunol. 175:5799-5808). In some embodiments, the phage displays a TCR for isolating the target antigen (see, for example, Varela-Rohena et al. (2008) Nat. Med. 14:1390-1395 and Li (2005) Nat. Biotechnol. 23:349-354). In some embodiments, the TCR or its antigen-binding portion can be synthesized based on knowledge of the TCR sequence.

IV. Treatment Methods

Certain aspects of this embodiment can be used to prevent or treat diseases or disorders associated with GARP signaling. GARP signaling can be reduced by any suitable drug to prevent cancer cell proliferation. Preferably, such a substance is an anti-GARP antibody.

In some embodiments, this document provides methods for treating an individual's cancer or slowing its progression, methods comprising administering to the individual an effective amount of an antiplatelet agent and T-cell therapy. Examples of cancers to be treated include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, precancerous lesions in the lungs, colon cancer, melanoma, and bladder cancer.

In some embodiments, an individual has cancer that is resistant to one or more anticancer therapies (has been shown to be resistant to one or more anticancer therapies). In some embodiments, resistance to anticancer therapies includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer in the original or new site after treatment. In some embodiments, resistance to anticancer therapies includes progression of cancer during treatment with anticancer therapies. In some embodiments, the cancer is in an early or late stage.

In some embodiments of the methods disclosed herein, activated CD4 and/or CD8 T cells in an individual are characterized by γ-IFN-producing CD4 and/or CD8 T cells and/or enhanced cytolytic activity relative to those prior to administration of the combination. γ-IFN can be measured by any method known in the art, including, for example, intracellular cytokine staining (ICS) involving cell fixation, permeabilization, and staining with anti-γ-IFN antibodies. Cytolytic activity can be measured by any method known in the art, such as a cell-killing assay using a mixture of effector and target cells.

T-cell therapy can be administered before, during, or after antiplatelet agents, or in various combinations. The intervals between administrations can range from simultaneous to minutes to days to weeks. In embodiments where T-cell therapy and antiplatelet agents are administered separately to the patient, it is typically ensured that no significant time interval is exceeded between each delivery, allowing both compounds to still exert a beneficial combined effect on the patient. In such cases, it is anticipated that antibody therapy and anticancer therapy can be administered to the patient approximately 12 to 24 or 72 hours apart, and more specifically, approximately 6 to 12 hours apart. In some cases, a significantly longer treatment duration may be required, where several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapse between the respective administrations.

In some embodiments, a non-myeloablative lymphoablative chemotherapy may be administered to the subject prior to T-cell therapy. The non-myeloablative lymphoablative chemotherapy can be any suitable such therapy, which can be administered via any suitable route. Non-myeloablative lymphoablative chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is potentially metastatic melanoma. An exemplary route of administration of cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In a particular aspect, approximately 60 mg/kg of cyclophosphamide is administered for two days, followed by approximately 25 mg/ ^m² of fludarabine for five days.

In some embodiments, a T-cell growth factor that promotes the growth and activation of autologous T cells is administered to the subject simultaneously with or after autologous T cells. The T-cell growth factor can be any suitable growth factor that promotes the growth and activation of autologous T cells. Examples of suitable T-cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2. IL-12 is a preferred T-cell growth factor.

T-cell therapy and antiplatelet agents can be administered via the same route of administration or via different routes. In some embodiments, T-cell therapy and/or antiplatelet agents are administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracardiaclysm, or intranasally. Effective amounts of T-cell therapy and antiplatelet agents can be administered to prevent or treat disease. The appropriate dosage of T-cell therapy and antiplatelet agents is determined based on the type of disease to be treated, the severity and duration of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the attending physician's discretion.

Intratumoral injection or injection into the tumor vascular system is particularly considered for discrete, solid, accessible tumors. Local, regional, or systemic administration may also be appropriate. For tumors >4 cm, the volume to be administered will be approximately 4 to 10 ml (especially 10 ml), while for tumors <4 cm, a volume of approximately 1 to 3 ml (especially 3 ml) will be used. Multiple injections as a single dose delivery consist of volumes of approximately 0.1 to approximately 0.5 ml.

A. Pharmaceutical Composition

When clinically applying a therapeutic composition containing an inhibitory antibody, it is often advantageous to prepare a pharmaceutical or therapeutic composition suitable for the intended application. In some embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise between about 2% and about 75% by weight, or, for example, between about 25% and about 60%, and any range thereof.

This article also provides pharmaceutical compositions and formulations comprising T-cell therapy, antiplatelet agents, and pharmaceutically acceptable carriers.

The therapeutic compositions of this embodiment are advantageously administered as liquid solutions or suspensions in the form of injectable compositions; they can also be prepared in solid forms suitable for dissolving or suspending in a liquid prior to injection. These formulations can also be emulsified.

The active compound can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as liquid solutions or suspensions; they can also be prepared in solid forms suitable for adding liquid prior to injection to prepare solutions or suspensions; and the formulation can also be emulsified.

Suitable pharmaceutical dosage forms for injectable applications include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be a fluid readily injectable. It should be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi.

Protein compositions can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed from free carboxyl groups can also be derived from inorganic bases (such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide) and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.).

Pharmaceutical compositions may include solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate flowability can be maintained, for example, by using coatings (such as lecithin), in the case of dispersions, by maintaining the desired particle size, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. Prolonged absorption of injectable compositions can be provided by using delayed-absorption agents in the composition, such as aluminum monostearate and gelatin.

The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (such as an antibody or peptide) of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 22nd edition, 2012) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexachlorobenzyl quaternary ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10) The following are pharmaceutically acceptable carriers: polypeptides (residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans such as chondroitinase.

B. Antiplatelet agents

Examples of this method relate to antiplatelet agents. The phrase "antiplatelet agent" refers to any compound that inhibits platelet activation, aggregation, and/or adhesion, and is intended to include all pharmaceutically acceptable salts, prodrugs (e.g., esters), and solvates (including hydrates) of compounds having one or more chiral centers that may exist as racemic mixtures, racemic mixtures, and as individual diastereomers or enantiomers, including all such isomers and mixtures thereof, and is intended to include any crystalline polymorphs, eutectic, and amorphous forms.

Non-limiting examples of antiplatelet agents that can be used in the oral dosage forms disclosed herein include adenosine diphosphate ( _ADP ) antagonists or _P2Yi2 antagonists, phosphodiesterase (PDE) inhibitors, adenosine reuptake inhibitors, vitamin K antagonists, heparin, heparin analogs, direct thrombin inhibitors, glycoprotein IIB/IIIA inhibitors, antithrombin, and pharmaceutically acceptable salts, isomers, enantiomers, polymorphic crystalline forms (including amorphous forms), solvates, hydrates, cocrystals, complexes, active metabolites, active derivatives and modifications, prodrugs, etc.

ADP antagonists or _{P2Y12 antagonists} block ADP receptors on platelet cell membranes. These _P2Y12 receptors are important in platelet aggregation, involving platelet cross _- linking via fibrin. Blocking this receptor inhibits platelet aggregation by blocking activation of the glycoprotein Ilb/ _IIIa pathway. In an exemplary embodiment, the antiplatelet agent is an ADP antagonist or a _P2Y12 antagonist. In another exemplary embodiment, the antiplatelet agent is thienopyridine. In yet another exemplary embodiment, the ADP antagonist or _{P2Y12 antagonist} is thienopyridine.

In another exemplary embodiment, the ADP antagonist or _{P2Y12 antagonist} is a member selected from the group consisting of: sulfinpyrazone, ticlopidine, clopidogrel, prasugrel, R-99224 (the active metabolite of prasugrel, supplied by Sankyo), R-1381727, R-125690 (Lilly), C-1330-7, C-50547 (Millennium Pharmaceuticals), INS-48821, INS-48824, INS-446056, INS-46060, INS-49162, INS-49266, INS-50589 (Inspire Pharmaceuticals), and Sch- ₅₇₂₄₂₃ (Schering Plough). In another exemplary embodiment, the ADP antagonist or _P2Y12 antagonist is ticlopidine hydrochloride (TICLID ^™ ). In another exemplary embodiment, the ADP antagonist or _{P2Y1 2} antagonist is a member selected from the group consisting of: sulfadiazine, ticlopidine, AZD6140, clopidogrel, prasugrel, and mixtures thereof. In another exemplary embodiment, the ADP antagonist or _{P2Y1 2} antagonist is clopidogrel. In another exemplary embodiment, the therapeutically effective dose of clopidogrel is about 50 mg to about 100 mg. In another exemplary embodiment, the therapeutically effective dose of clopidogrel is about 65 mg to about 80 mg. In another exemplary embodiment, the ADP antagonist or _{P2Y1 2} antagonist is a member selected from the group consisting of: clopidogrel bisulfate (PLA VIX ^™ ), clopidogrel bisulfate, clopidogrel hydrobromide, clopidogrel mesylate, cangrelore tetrasodium (AR-09931MX), ARL67085, AR-C66096, AR-C126532, and AZD-6140 (AstraZeneca). In another exemplary embodiment, the ADP antagonist or _{P2Y12 antagonist} is prasugrel. In another exemplary embodiment, the therapeutically effective dose of prasugrel is about 1 mg to about 20 mg. In another exemplary embodiment, the therapeutically effective dose of clopidogrel is about 4 mg to about 11 mg. In another exemplary embodiment, the ADP antagonist or _P2Y12 antagonist is a member selected from the group consisting of clopidogrel, ticlopidine, sulfinpyrazone, AZD6140, prasugrel, and mixtures _thereof .

In some embodiments, the antiplatelet agent is clopidogrel or a pharmaceutically acceptable salt, solvate, polymorph, cocrystal, hydrate, enantiomer, or prodrug thereof. In another embodiment, clopidogrel or a pharmaceutically acceptable salt, solvate, polymorph, cocrystal, hydrate, enantiomer, or prodrug thereof is a powder.

A PDE inhibitor is a drug that blocks one or more of the five isoforms of phosphodiesterase (PDE), thereby preventing the inactivation of the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) by the corresponding one or more PDE isoforms. In an exemplary embodiment, the antiplatelet agent is a PDE inhibitor. In an exemplary embodiment, the antiplatelet agent is a selective cAMP PDE inhibitor; in an exemplary embodiment, the PDE inhibitor is cilostazol (Pletal ^™ ).

Adenosine reuptake inhibitors prevent cells from reuploading adenosine into platelets, erythrocytes, and endothelial cells, thereby increasing extracellular adenosine concentrations. These compounds inhibit platelet aggregation and induce vasodilation. In an exemplary embodiment, the antiplatelet agent is an adenosine reuptake inhibitor. In an exemplary embodiment, the adenosine reuptake inhibitor is dipyridamole (Persantine ^™ ).

Vitamin K inhibitors are used to prevent thrombosis (the inappropriate clotting of blood in blood vessels). This is useful for primary and secondary prevention of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke in susceptible patients. In an exemplary embodiment, the antiplatelet agent is a vitamin K inhibitor, which is selected from the group consisting of: acetocoumarin, chlorindion, dicumarol (dihydroxycoumarin), diphenylindion, ethyl dicumarol, phenylpropromin, phenylindion, thiaclocoumarin, and warfarin.

Heparin is a biological substance, typically made from pig intestines. It works by activating antithrombin III, which prevents thrombin from clotting blood. In an exemplary embodiment, the antiplatelet agent is heparin or a heparin prodrug. In an exemplary embodiment, the antiplatelet agent is a heparin analog or a heparin analog prodrug. In an exemplary embodiment, the heparin analog is a member selected from: antithrombin III, bemiparin, dalteparin, danalteparin, enoxaparin, fondaparin (subcutaneous), nadroparin, parhexaparin, revelhexaparin, sulodide, and tinzaparin.

Direct thrombin inhibitors (DTIs) are a class of drugs that act as anticoagulants (delaying blood clotting) by directly inhibiting thrombin. In an exemplary embodiment, the antiplatelet agent is a DTI. In another exemplary embodiment, the DTI is monovalent. In another exemplary embodiment, the DTI is bivalent. In an exemplary embodiment, the DTI is selected from the following: hirudin, bivalirudin (IV), lepirudin, disiludin, argatroban (IV), dabigatran, dabigatran etexilate (oral formulation), melagatan, cimetidine (oral formulation, but with liver complications), and their prodrugs.

In an exemplary embodiment, the antiplatelet agent is selected from the group consisting of: aloprolin, beraprost, carbaspirin calcium, clocromenine, defibrinolytic acid, ditazobium, epoprostol, indobufen, iloprost, piscotamide, rivaroxaban (oral FXa inhibitor), treprostacyclin, trifluralin, or a prodrug thereof.

In some embodiments, the antiplatelet agent is an antibody or fragment thereof that binds to at least a portion of the GARP protein. As used herein, the term "antibody" is intended to broadly refer to any immunobinding agent, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG, as well as polypeptides containing an antibody CDR domain that retains antigen-binding activity. Antibodies can be selected from the group consisting of chimeric antibodies, affinity-matured antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, or antigen-binding antibody fragments or natural or synthetic ligands. Preferably, the anti-GARP antibody is a monoclonal antibody or a humanized antibody. Thus, polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs (including engineered forms of any of the foregoing) specific to the GARP protein, its corresponding epitopes, or conjugates thereof, can be created by known methods and as described herein, regardless of whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of natural compounds.

Examples of antibody fragments suitable for use in this embodiment include, but are not limited to: (i) Fab fragments consisting of _VL , _VH , _CL , and _CH1 domains; (ii) “Fd” fragments consisting of _VH and _CH1 domains; (iii) “Fv” fragments consisting of the _VL and _VH domains of a single antibody; (iv) “dAb” fragments consisting of the _VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (“scFv”) wherein the _VH and _VL domains are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bispecific single-chain Fv dimers (see U.S. Patent No. 5,091,513); and (ix) bispecific antibodies, multivalent or multispecific fragments constructed by gene fusion (U.S. Patent Application Pub. 20050214860). Fv, scFv, or bisomatic antibody molecules can be stabilized by incorporating disulfide bridges linking the _VH and _VL domains. Small bodies containing scFv linked to the CH3 domain can also be prepared (Hu et al., 1996).

C. Other treatments

In some embodiments, the compositions and methods of this embodiment involve antibodies or antibody fragments against GARP in combination with a second or additional therapy to inhibit its activity in cancer cell proliferation. Such therapies can be applied to treat any disease associated with GARP-mediated cell proliferation. For example, the disease could be cancer.

In some embodiments, the compositions and methods of this embodiment involve a combination of T-cell therapy and antiplatelet agents with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., mastectomy and lumpectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

This method and composition (including combination therapies) enhance therapeutic or protective effects and/or increase the therapeutic effect of another anticancer or antiproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in combined amounts to effectively achieve the desired effects (such as killing cancer cells and/or inhibiting excessive cell proliferation). The process may involve contacting cells with both an antibody or antibody fragment and a second therapy. Tissues, tumors, or cells can be contacted with one or more compositions or pharmacological preparations containing one or more of the following agents (i.e., antibodies or antibody fragments or anticancer agents), or with two or more different compositions or preparations, wherein one composition provides 1) an antibody or antibody fragment, 2) an anticancer agent, or 3) both an antibody or antibody fragment and an anticancer agent. Furthermore, such combination therapies are anticipated to be used in conjunction with chemotherapy, radiotherapy, surgery, or immunotherapy.

When applied to cells, the terms "contact" and "exposure" are used herein to describe the process of delivering a therapeutic construct and a chemotherapeutic or radiotherapeutic agent to or directly juxtaposed with target cells. For example, to achieve cell killing, two agents are delivered to cells in combined amounts that effectively kill cells or prevent their division.

Inhibitory antibodies can be administered before, during, or after anticancer treatment, or in various combinations. The intervals between administrations can range from simultaneous to minutes to days to weeks. In embodiments where antibodies or antibody fragments are administered separately from the anticancer agent, it is typically ensured that no significant time interval exists between each delivery, allowing both compounds to still exert a beneficial combined effect on the patient. In such cases, it is anticipated that antibody therapy and anticancer therapy can be administered to the patient approximately 12 to 24 or 72 hours apart, and more specifically, approximately 6 to 12 hours apart. In some cases, a significantly longer treatment duration may be required, where several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapse between the respective administrations.

In some embodiments, the treatment course will last from 1 to 90 days or longer (such range includes intermediate days). It is anticipated that one agent may be administered on any day from day 1 to day 90 (such range includes intermediate days), or any combination thereof, and another agent may be administered on any day from day 1 to day 90 (such range includes intermediate days), or any combination thereof. One or more administrations of the agent may be given to the patient within a single day (24-hour period). Furthermore, after a course of treatment, a period of non-treatment with anticancer therapy is anticipated. This period may last from 1 to 7 days and/or 1 to 5 weeks and/or 1 to 12 months or longer (such range includes intermediate days), depending on the patient's condition, such as their prognosis, intensity, health status, etc. Treatment cycles are expected to be repeated as needed.

In some embodiments, an additional therapy is the administration of small molecule enzyme inhibitors or anti-transfer agents. In some embodiments, an additional therapy is the administration of side effect limiters (e.g., agents designed to reduce the occurrence and/or severity of treatment side effects, such as antinausea agents). In some embodiments, an additional therapy is radiation therapy. In some embodiments, an additional therapy is surgery. In some embodiments, an additional therapy is a combination of radiation therapy and surgery. In some embodiments, an additional therapy is gamma irradiation. In some embodiments, an additional therapy is a therapy targeting the PBK/AKT/mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemopreventive agent. Additional therapies may be one or more of the chemotherapeutic agents known in the art.

Various combinations can be used. In the following example, antibody therapy or T-cell therapy and antiplatelet agents are "A", and anticancer therapy is "B":

Administering any compound or therapy of this embodiment to a patient will follow the general protocol for administering such compounds, taking into account the toxicity of the reagent (if any). Therefore, in some embodiments, there are steps for monitoring toxicity attributable to the combination therapy.

1. Chemotherapy

According to this embodiment, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapy agent" is used to refer to compounds or compositions administered in cancer treatment. These agents or drugs are classified according to their patterns of activity within cells, such as whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, insert into DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, indomethacin, and piperazine; acridines such as benzepam, carboquinone, metoprolol, and urotepam; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylo-melamine; acetyl proteins (especially pozzolanic acid and bruolinone); camptothecin ( Including synthetic analogs such as topotecan; bryostatin; carlistatin; CC-1065 (including its synthetic analogs adorelasin, calcelacin, and bisacrylacin); nostocin (especially nostocin 1 and nostocin 8); dolasstatin; docamycin (including synthetic analogs KW-2189 and CB1-TM1); eleutheroside; hygroscopicin; styracidium; spongiformin; nitrogen mustard, such as chlorambucil, naphthalenemus, and chlorambucil. Phosphoramides, estradiol, ifosfamide, dichloroethyl methylamine, oxynitrogen mustard hydrochloride, melphalan, neo-embezzin, benzyl mustard cholesterol, prednisolone, trofenoxam, and uramustine; nitrosoureas, such as carmustine, pyriprethroid nitrosourea, formustine, lomustine, nimustine, and ramustine; antibiotics, such as enediyne antibiotics (e.g., chachimycin, especially chachimycin γI and chachimycin ΩI); danendomycin, including danendomycin A; bisphosphonates, such as clodrophosphonate; esperamycin; and new tumor suppressor chromophores and related chromophores, arubicin, actinomycin, azoserine, bleomycin, actinomycin C, carabinin, erythromycin, carcinomatin, chromomycin, actinomycin D, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, doxorubicin (including morpholino-doxorubicin) Star, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, maseromycin, mitomycin (such as mitomycin C), mycophenolic acid, nopramine, olivomycin, pepromycin, methylmitomycin, puromycin, triamcinolone acetonide, rodorubicin, streptomycin, strepzotocin, tretinoin, ubenmex, fenestrated statin and zolrubicin; antimetabolites, such as ammonia Metoprolol and 5-fluorouracil (5-FU); folic acid analogs, such as folate, aminopterin, and trimethyltroxa; purine analogs, such as fludarabine, 6-mercaptopurine, thioimide, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and aziridine; androgens, such as calotestosterone, drotalesterone propionate, and cyclothionol. Androstane and testosterone; anti-adrenergic agents such as mitotane and triostane; folic acid supplements such as frolinic acid; glucuronolactone; aldehyde phosphoramide glycoside; aminolevulinic acid; dihydropyrimidine dehydrogenase inactivator; acridine; amustine; bisulfite; ethanodehydropterin; defofamine; colchicine; seriquinone; efornithine base hydrochloride; erythritol; epothilone Etogluconol; Gallium nitrate; Hydroxyurea; Lentinus polysaccharide; Clonidamine; Maytansin alkaloids, such as maytansin and anthraquinone; Mitoxanthraquinone; Mitoxanthraquinone; Moperadol; Diamine nitrazepam; Pentostatin; Methamidomus acid; Pirarubicin; Loxoanthraquinone; Podophyllotoxin; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razosen; Rhizomycin; Cizonan; Germanium spiroamine; Alternaria spp. ketoacid; Triaminequinone; 2,2',2”- Trichlorotriethylamine ; trichothecotoxins (especially T-2 toxin, lacryma-jobi A, lacryma-jobi A, and serpentin); amino acid esters; vinblastine; dacarbazine; mannitol mustard hydrochloride; dibromomannitol; dibromoeusine; guanethidine; carbendazim; ara-C; cyclophosphamide; taxanes, such as paclitaxel and docetaxel; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); Ifosfamide; mitoxantrone; vincristine; vinorelbine; noscholine; teniposide; edaraxacum; donomycin; aminopterin; capecitabine; ibandronate sodium; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, purcamycin, gemcitabine, isovinblastine, farnesyltransferase inhibitors, antiplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

2. Radiation therapy

Other factors that cause DNA damage and have been widely used include what are commonly referred to as gamma rays, X-rays, and/or the targeted delivery of radioactive isotopes to tumor cells. Other forms of DNA damage factors have also been considered, such as microwaves, proton beam irradiation (US Patents 5,760,395 and 4,870,287), and UV irradiation. All of these factors are likely to cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from long-term (3 to 4 weeks) daily doses of 50 to 200 roentgens to single doses of 2,000 to 6,000 roentgens. Radioactive isotope dose ranges vary considerably and depend on the isotope's half-life, the intensity and type of emitted radiation, and the uptake by tumor cells.

3. Immunotherapy

Those skilled in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapy typically relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab is such an example. Immune effectors can be, for example, antibodies specific to certain markers on the surface of tumor cells. An antibody alone can act as an effector of the therapy, or it can recruit other cells to actually influence cell killing. Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) to act as a target. Alternatively, effectors can be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

Antibody-drug conjugates have emerged as a breakthrough approach for developing cancer therapies. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) consist of monoclonal antibodies (MAbs) covalently linked to cytotoxic drugs. This approach combines the high specificity of the MAb against its antigenic target with a highly potent cytotoxic drug, resulting in an “armed” MAb that delivers the payload (drug) to tumor cells with enriched antigen levels (Carter et al., 2008; Teicher 2014; Leal et al., 2014). Targeted drug delivery also minimizes its exposure in normal tissues, thereby reducing toxicity and improving the therapeutic index. The FDA’s approval of two ADC drugs—brentuximab vedotin in 2011 and trastuzumab bemtansine (T-DM1) in 2013—validates this approach. Currently, more than 30 ADC candidates are in various phases of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and adaptor-payload optimization become increasingly sophisticated, the discovery and development of new ADCs increasingly rely on the identification and validation of novel targets suitable for this approach (Teicher 2009) and the generation of targeting MAbs. Two criteria for ADC targets are upregulated/high-level expression in tumor cells and robust internalization.

In one aspect of immunotherapy, tumor cells must carry some easily targeted markers, i.e., markers not present on most other cells. Many tumor markers exist, and any of these markers can be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialic acid Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is combining anticancer effects with immunostimulatory effects. Immunostimulatory molecules also exist, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, and γ-IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (US Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); and cytokine therapies, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., ...). References include: (1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent 5,824,311). It is anticipated that one or more anticancer therapies can be used in conjunction with the antibody therapies described herein.

In some embodiments, immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints are molecules in the immune system that either upregulate (e.g., co-stimulatory molecules) or downregulate. Inhibitory checkpoint molecules that immune checkpoint blockade may target include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuation factor (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and T cell activation V domain Ig inhibitor (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.

Immune checkpoint inhibitors can be pharmaceuticals, such as small molecules, recombinant forms of ligands or receptors, or, in particular, antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both incorporated herein by reference). Known immune checkpoint protein inhibitors or analogues thereof can be used, particularly chimeric, humanized, or human antibodies. As those skilled in the art will appreciate, alternative and/or equivalent names may be used for certain antibodies mentioned in this disclosure. Such alternative and/or equivalent names are interchangeable in the context of this invention. For example, pembrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. Specifically, the PD-1 ligand-binding partner is PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. Specifically, the PDL1 binding partner is PD-1 and/or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. Specifically, the PDL2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. US8735553, US8354509, and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists used in the methods provided herein are known in the art, such as those described in U.S. Patent Applications Nos. US20140294898, US2014022021 and US20110008369, all of which are incorporated herein by reference.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, is an anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, pembrolizumab, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.

Another immune checkpoint that can be targeted using the methods presented in this paper is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off” switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily, expressed on the surface of helper T cells, and delivers inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 on antigen-presenting cells, also known as B7-1 and B7-2, respectively. CTLA4 delivers inhibitory signals to T cells, while CD28 delivers stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and is likely important for their function. T cell activation via T cell receptors and CD28 leads to increased expression of the inhibitory receptor CTLA-4 of the B7 molecule.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

The anti-human CTLA-4 antibody (or its VH and/or VL domains) suitable for this method can be generated using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, the anti-CTLA-4 antibodies disclosed in the following literature can be used in the methods disclosed herein: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as trimemumab; formerly known as tesimumab, US Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncolog y 22(145): Abstract No. 2505 (Antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304. The teachings of each of the foregoing publications are incorporated herein by reference. Antibodies that compete with any of these antibodies recognized in the art for binding to CTLA-4 may also be used. For example, humanized CTLA-4 antibodies are described in International Patent Applications Nos. WO2001014424, WO2000037504 and US Patent No. US8017114; all of which are incorporated herein by reference.

Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and) or its antigen-binding fragments and variants (see, for example, WOO 1/14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes with the aforementioned antibody for binding to the same epitopes on the CTLA-4 antibody and/or binds to the same epitopes on the CTLA-4 antibody as the aforementioned antibody. In yet another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the aforementioned antibody (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

Other molecules used to modulate CTLA-4 include CTLA-4 ligands and receptors, such as those described in all of the U.S. Patent Nos. US5844905, US5885796 and International Patent Application Nos. WO1995001994 and WO1998042752, which are incorporated herein by reference; and immune adhesion, for example, as described in U.S. Patent No. US8329867, which is incorporated herein by reference.

4. Surgical procedures

Approximately 60% of cancer patients will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgeries. Curative surgeries include resection, in which all or part of the cancerous tissue is physically removed, excised, and/or destroyed, and can be used in combination with other therapies, such as the treatments described herein, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and/or replacement therapy. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments also include laser surgery, cryosurgery, electrosurgery, and microsurgical control surgery (Moore's procedure).

After the removal of some or all of the cancerous cells, tissue, or tumor, a cavity may form in the body. Treatment can be performed by perfusion, direct injection, or application of additional anticancer therapy to a localized area. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The dosage of these treatments may also vary.

5. Other medicines

Other agents are anticipated to be used in combination with certain aspects of this embodiment to improve therapeutic efficacy. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cell inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of overproliferating cells to apoptosis-inducing agents, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions will increase the anti-overproliferative effect on neighboring overproliferating cell populations. In other embodiments, cell inhibitors or differentiation agents may be used in combination with certain aspects of this embodiment to improve the anti-overproliferative efficacy of the treatment. Cell adhesion inhibitors are expected to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Other agents that increase the sensitivity of overproliferating cells to apoptosis, such as antibody C225, are also anticipated to be used in combination with certain aspects of this embodiment to improve therapeutic efficacy.

V. Products or reagent kits

In various aspects of the embodiments, a kit containing a therapeutic agent and/or other therapeutic agents and delivery agents is contemplated. In some embodiments, this disclosure contemplates a kit for preparing and/or administering the therapies of the embodiments. The kit may include one or more sealed vials containing any of the pharmaceutical compositions of the embodiments. The kit may include, for example, at least one GARP antibody and reagents for preparing, formulating and/or administering the components of the embodiments or performing one or more steps of the methods of the invention. In some embodiments, the kit may also include suitable containers that do not react with the components of the kit, such as Eppendorf tubes, assay plates, syringes, bottles or tubes. The containers may be made of sterilizable materials, such as plastic or glass.

In some embodiments, products or kits comprising adoptive T cells are provided, and antiplatelet agents (e.g., anti-GARP antibodies) are also provided herein. Products or kits may further include a packaging insert containing instructions on using adoptive T cells in combination with an antiplatelet agent to treat an individual's cancer or delay cancer progression in an individual or enhance the immune function of an individual with cancer. Either the adoptive T cells and/or antiplatelet agent described herein may be included in the product or kit. In some embodiments, the adoptive T cells and antiplatelet agent are located in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers may be formed from a variety of materials, such as glass, plastics (such as polyvinyl chloride or polyolefins), or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the container contains the formulation, and a label on or associated with the container may indicate instructions for use. Products or kits may further include other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another reagent (e.g., a chemotherapeutic agent and an antitumor agent). Suitable containers for one or more reagents include, for example, bottles, vials, bags, and syringes.

The kit may further include a specification outlining the procedural steps of the methods described herein, and will follow substantially the same procedures as those described herein or known to those skilled in the art. The illustrative information may be in a computer-readable medium containing machine-readable instructions that, when executed using a computer, cause a real or virtual process to demonstrate the delivery of an effective amount of the therapeutic agent.

VI. Examples

The following examples are included to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques that the inventors have discovered that work well in the practice of the invention, and therefore can be considered as preferred modes of practice. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention and still obtaining the same or similar results.

Example 1 - GARP expression in cancer cells

Recent studies, including the Cancer Genome Atlas (TCGA) project, have shown that the GARP gene LRRC32 is amplified in up to 30% of patients with many human cancer types, including ovarian, lung, breast, and head and neck cancer (Figure 1A). To examine GARP protein expression, immunohistochemistry (IHC) was performed on human tumor microarrays from archived human tumors, and subsequently, it was determined whether GARP expression had any prognostic significance. The specificity of anti-human GARP antibodies was determined by their staining on pre-B leukemia cell lines stably transfected with human GARP (Figure 1B). Given the amplification of LRRC32 in human breast cancer (Szepetowski et al., 1992), GARP expression was first assessed in breast cancer patients using IHC. Results were read and scored in a double-blind manner by clinicopathologists. IHC analysis of patient-matched unaffected breast tissue versus primary breast cancer (n=16) indicated significantly increased GARP expression in cancer tissue in 9 of the 16 patients (Figure 1C). By RT-PCR, GARP mRNA expression was ≥2-fold higher in 28.5% of patients with breast cancer (n=42) than in normal breast tissue. IHC was then performed on cancer samples, which included 55 colon cancer samples, 55 adjacent normal tissues, and 11 corresponding lymph nodes and adjacent normal tissues (Fig. 1D). Normal epithelial samples did not show significant GARP positivity (Fig. 1D and 1E). However, for GARP, staining of primary cancer (colon and lung cancer) and lymph node (LN) metastatic tissues showed varying degrees of positivity (all isotype control antibodies were negative) (Fig. 1E). The percentage of GARP-positive cells in primary cancers was 26.1% (p=8.6 x ^10⁻⁹ ) compared to undetectable levels (defined as 0) in normal tissues, and the percentage of GARP-positive cells in LN metastases was 25% (p=0.008). Within the range of 0 to 4, the GARP intensity score ranged from 0 to 3, with a mean of 0.78 in primary colorectal cancer (p = 1.1 x ^10⁻⁸ ) and a mean of 1.18 in LN metastases (p = 0.003) (Figure 1E). Similarly, significantly increased GARP levels were found in primary lung and prostate cancer (Figure 1E). More importantly, GARP levels were negatively correlated with overall survival in patients with colorectal and lung cancer, regardless of tumor pathological grade or lymph node status (Figure 1F). High GARP expression was also associated with high Gleason scores in prostate cancer (p = 0.035) (Figure 1F). These results demonstrate for the first time that GARP is widely expressed in human cancers and that expression levels are associated with disease aggressiveness.

In vitro biochemical studies have confirmed that GARP also exists in a soluble form, secreted as a complex with latent TGF-β1 from Treg cells (Gauthy et al., 2013). Studies have also shown that GARP folds and is expressed on the cell surface in dependence of the molecular chaperone grp94 in the endoplasmic reticulum (Zhang et al., 2015). To determine whether GARP secretion is a Treg cell-specific event or an inherent phenomenon of GARP, N-terminal hemagglutinin (HA)-labeled GARP was expressed in mouse pre-B cells with and without grp94, and GARP expression in cell lysates and conditioned medium was then analyzed. Three GARP bands were observed only in gp96 ⁺ (WT) cells: molecular weights of approximately 75, 45, and 30 kDa, respectively (Figure 2A). The 45 and 30 kDa fragments appear to be post-translational cleavage products of the full-length cell surface GARP (75 kDa), as they are more resistant to Endo H compared to PNGase F and were found in WT cells but not in grp94 KO cells. If so, the 30 kDa N-terminal GARP fragment should be released from the cell surface into the culture medium. In fact, gel extraction and sequencing of the 30 kDa protein in the culture medium by mass spectrometry confirmed that it originated from the N-terminal fragment of GARP (Figure 2B).

Next, the presence of soluble GARP (sGARP) in the serum of cancer patients was determined, and whether serum sGARP levels had any prognostic significance. Serum was collected from male normal controls (n=7) and prostate cancer patients (n=48) and analyzed for GARP using ELISA. sGARP was found to be present in the serum of both normal individuals and prostate cancer patients (Figure 2C). Further analysis revealed that higher GARP levels were associated with increased prostate cancer-specific antigen (PSA) levels and metastasis (Figure 2D). Furthermore, the presence of the sGARP-TGF-β1 complex in the serum of prostate cancer patients and normal controls was assessed using a GARP-TGF-β1 sandwich ELISA. As predicted, the levels of the soluble GARP and TGF-β1 complex in the serum of cancer patients were higher than in normal subjects (Figure 2E). To further understand the function of soluble GARP, a fusion protein was prepared consisting of the N-terminal extracellular domain of GARP (GARP-Fc) linked to the Fc domain of IgG. This construct was expressed in Chinese hamster ovary (CHO) cells. The GARP fusion protein was then purified from conditioned medium. As measured by active TGF-β ELISA, a direct association between GARP-Fc and active TGF-β1 was found (Fig. 2F), indicating the presence of the GARP-Fc-TGF-β1 complex.

Example 2 - GARP and TGF-β

Forced GARP expression in normal mouse mammary epithelial cells upregulates TGF-β expression and drives tumorigenesis. In normal mouse mammary epithelial (NMuMG) cells, TGF-β exerts both growth-inhibiting and epithelial-mesenchymal transition (EMT) responses (Xie et al., 2003). Therefore, NMuMG cells have been widely used to study TGF-β signaling and biology (Xu et al., 2009). Given that GARP modulates the bioavailability of TGF-β, NMuMG cells were used for bioassays to investigate the effects of both membrane-bound and soluble GARP on epithelial cells. Stable GARP expression was found to induce Smad-2/3 phosphorylation and vimentin expression, but downregulate E-cadherin, consistent with increased canonical TGF-β signaling (Fig. 3A). Furthermore, NMuMG cells stimulated with soluble GARP-Fc transformed from their typical polygonal and flattened epithelial cell morphology to a spindle-shaped morphology within 24 hours (Fig. 3B), accompanied by time- and dose-dependent upregulation of vimentin (Figs. 3C and 3D). As expected, NMuMG cells stably expressing GARP or GARP-Fc exhibited higher levels of active TGF-β1 (Fig. 3E) and soluble GARP expression (Fig. 3F) compared to cells transduced with empty vector (EV). An in vitro “scratch” assay was performed to measure the migration properties of GARP-expressing cells. The gap closure rate (generated by scraping culture plates) of GARP-expressing cells was significantly increased, indicating increased acquired migration ability (Figs. 3G and 3H). The ability of forced GARP expression to induce tumor formation in vivo by NMuMG cells was also examined. For this purpose, GARP-expressing ^NMuMG cells or EV control cells, all of which were also engineered to co-express luciferase, were injected into the fourth mammary fat pad of female immunodeficient NOD-Rag1 -/- mice. In vivo imaging with bioluminescence revealed that the bioactive substance was formed only in mice receiving GARP ⁺ or GARP-Fc ⁺ NMuMG cells, but not in mice receiving EV-transduced cells (Fig. 3I). Histological examination confirmed that cells expressing GARP formed tumors (Figure 3J). Overall, these results indicate that GARP possesses transformative properties via upregulation of TGF-β, thus identifying GARP as a potential novel oncogene.

Silencing GARP delays tumor growth. A variant of the normal mouse mammary epithelial cell line (NMuMG*) in which the RNA-binding protein hnRNPE1 is knocked down via RNA interference has recently been described as capable of forming tumors in nude mice (Howley et al., 2015). Interestingly, these cells were found to express significant levels of endogenous GARP (Fig. 4A–4C), suggesting the possibility that enhanced TGF-β biogenesis, in addition to silencing the TGF-β-mediated translational repressor complex, also drives breast cancer in this model. To test this hypothesis, short hairpin RNA (shRNA) knockdown (KD) of GARP was performed in NMuMG* cells (Fig. 4A–4C). As determined by MTT assay, GARP silencing did not affect the in vitro proliferation of NMuMG* cells (Fig. 4D). Notably, silencing GARP alone in NMuMG* cells significantly attenuated their in vivo growth (Fig. 4E). Furthermore, the ability of these GARPKD cells to metastasize to the lungs and liver was impaired (Fig. 4F and 4G).

Upregulation of GARP in mouse breast cancer cells promotes TGF-β activation, tumor growth, metastasis, and immune tolerance. LRRC32 was initially described as a gene that frequently amplifies in breast cancer (Ollendorff et al., 1994), and TGF-β signaling has been shown to promote breast cancer invasion and metastasis (Massague, 2008; Padua et al., 2008; Siegel et al., 2003). However, the ongoing investigation into the biology of TGF-β in cancer focuses on its extrinsic role in cancer through the regulation of host immune responses (Li and Flavell, 2008). Therefore, the effects of GARP on cancer growth and metastasis in an endogenic immune-sufficient environment were examined in a highly invasive and metastatic 4T1 breast cancer model in BALB/c mice (Pulaski and Ostrand-Rosenberg, 2001). Similar to the NMuMG system, overexpression of GARP or GARP-Fc in 4T1 cells led to increased production of active TGF-β (Figs. 5A and 5B). One of the key mechanisms by which TGF-β suppresses tumor-specific immunity is through the induction of Foxp3 ⁺ Tregs. To this end, purified naïve CD4 ⁺ T cells were cultured in vitro for 3 days in conditioned medium derived from 4T1-GARP, 4T1-GARP-Fc, and empty vector (EV) control cells in the presence of a polyclonal T cell activator. The conditioned medium derived from GARP-expressing cells was 2 to 3 times more efficient at inducing Treg differentiation than the medium derived from control cells (Fig. 5C). 4T1-EV, 4T1-GARP, and 4T1-GARP-Fc cells were orally injected into the fourth right mammary fat pad of 6- to 8-week-old female BALB/c mice. GARP-expressing cells were found to be more aggressive, as indicated by both increased primary tumor growth kinetics (Figs. 5D and 5E) and increased lung metastases (Fig. 5F). This aggressiveness was also found to be associated with enhanced TGF-β signaling in the tumor microenvironment, such as the increase in p-Smad-2/3 in cancer cells (Fig. 5G and 5H) and the expansion of tolerant Treg cells (Fig. 5I and 5J).

Case Study 3 - Melanoma Research

Studies using the 4T1 tumor model raised the question of whether GARP inhibits the function of tumor-specific T cells. To explore this possibility, a B16 melanoma model with well-defined antigen specificity and CD8 ⁺ T cell receptor (TCR) transgenic mice (Pmel) with T cells specific to the melanoma-associated antigen gp100 were used (Muranski et al., 2008; Overwijk et al., 2003). B16-F1 cells were prepared with or without GARP-Fc and then subcutaneously injected into C57BL/6 mice. The tumor-bearing mice were then lymphaticly depleted with cyclophosphamide (CY) before adoptive cell transfer (ACT) of the activated Pmel cells in vitro (Rubinstein et al., 2015) (Fig. 6A). GARP-Fc expression in B16 cells was found to induce increased resistance to ACT (Figs. 6B and 6C), which was associated with a decrease in the number of antigen-specific Pmel cells in the recipient mice, particularly during the first four weeks of tumor growth when the tumor surface area was less than 100 ^mm² (Figs. 6D and 6E). Similarly, in mice carrying GARP-Fc ⁺ B16 melanoma, the ability of Pmel CD8 ⁺ T cells to produce IFNγ in response to antigen stimulation was also impaired (Figs. 6F and 6G). Example 4 - GARP as a novel therapeutic target for cancer

The study described in this article has demonstrated that GARP is aberrantly expressed in various human cancers, and that GARP expression in mouse tumors is associated with increased TGF-β bioavailability, cancer invasiveness, and T cell tolerance. Next, an antibody-based strategy was used to determine whether GARP could serve as a novel therapeutic target for cancer. To generate anti-GARP monoclonal antibodies (mAbs), mice were immunized with recombinant human GARP and then boosted with irradiated whole myeloma SP2/0 cells stably expressing human GARP, with the aim of generating conformationally specific mAbs against GARP.

Platelets not only produce and store high levels of TGFβ intracellularly, but are also the only known cellular entities constitutively expressing the cell surface docking receptor GARP for TGFβ. Therefore, platelets may promote systemic TGFβ levels through active secretion and GARP-mediated capture from other cells or the extracellular matrix. This study investigated the extent and method by which platelets contribute to the physiological TGFβ pool. Baseline serum was obtained from wild-type (WT) mice, followed by administration of platelet-consuming antibodies. These mice were then subjected to exsanguination, and serum TGFβ was quantified by ELISA. Platelet consumption resulted in a complete loss of activity and total TGFβ, which rebounded effectively once platelet counts recovered (Figure 7A). These experiments demonstrate that platelets make a major contribution to circulating TGFβ levels.

By focusing on the role of platelet GARP in the production of active TGFβ, this study experimentally explored the biology of platelet-derived TGFβ in cancer immunity. In addition to the platelet-specific Hsp90b1 KO mouse, two other mouse models were generated: one with selective deletion of GARP in platelets (Pf4-cre-Lrrc32flox/flox or Plt-GARPKO), and the other with platelet-restricted TGFβ1 knockout (Pf4-cre-Tgfb1flox/flox or Plt-Tgfβ1KO). Since gp96 is also a specific partner of GARP, platelets from both Plt-gp96KO and Plt-GARPKO mice did not express the cell surface GARP-TGFβ complex. However, compared with WT platelets, platelets from Plt-Tgfβ1KO mice expressed similar levels of surface GARP-TGFβ1 complex (Figs. 7B to 8D), indicating that the GARP-TGFβ1 complex can be formed in the absence of autocrine TGFβ1.

The levels of active and latent TGFβ in plasma and serum were then measured in WT and knockout mice (Figs. 7E–7F). In WT mice, serum active TGFβ was elevated compared to plasma, indicating the role of platelets and/or the coagulation cascade in TGFβ activation (Fig. 7E). Importantly, serum active TGFβ was almost absent in Plt-gp96KO and Plt-GARPKO mice, confirming the importance of platelet-inherent GARP in converting latent TGFβ to its active form. Conversely, serum levels of active TGFβ in Plt-Tgfβ1KO mice were comparable to those in WT mice (Fig. 7E), indicating that platelets are capable of trans-activating TGFβ from non-platelet sources. Significantly, total serum latent TGFβ levels were decreased only in Plt-Tgfβ1KO mice, but not in Plt-gp96KO or Plt-GARPKO mice (Fig. 7F). In summary, these data indicate that platelet-inherent GARP is the most important mechanism for systemic activation of TGFβ. This experiment also clearly demonstrates that serum levels of active TGFβ, rather than plasma levels, reflect only platelet activation.

It is hypothesized that platelet-specific GARP plays a crucial negative role in anti-tumor T-cell immunity. This hypothesis was explored by comparing the efficacy of adoptive T-cell therapy in melanoma in WT, Plt-Tgfβ1KO, and Plt-GARPKO recipient mice (Figure 8). B16-F1 melanomas were established in WT or KO mice, followed by lymphocyte depletion with cyclophosphamide (Cy) on day 9 and infusion of ex vivo activated Pmel T cells on day 10 (Figure 8A). Compared with WT mice, tumors were more effectively controlled in Plt-GARPKO mice (Figure 8A). This was associated with enhanced persistence (Figure 8B) and functionality (Figure 8C) of Pmel cells in the peripheral blood of Plt-GARPKO mice. In stark contrast, Plt-Tgfβ1KO mice, whose platelets expressed GARP and were still able to activate TGFβ, did not show improved tumor control (Figure 8D). Given that the growth of this transplantable tumor in syngeneic mice is mediated by both CD4 and CD8 immune stress, the generalization of these findings was investigated in the MC38 colon cancer system. Compared to WT mice, MC38 growth was significantly reduced in Plt-GARPKO mice (Figs. 9A–9C). Serum levels of active TGFβ were decreased in Plt-GARPKO mice carrying MC38 (9D). More importantly, staining for p-Smad2/3 in MC38 tumor sections demonstrated significantly reduced TGFβ signaling in MC38 cells from Plt-GARPKO mice (Figs. 9E and 9F). This was associated with a reduction in systemic bone marrow-derived suppressor cells (Fig. 9G) and tumor-infiltrating regulatory T cells (Fig. 9H) in Plt-GARPKO mice. In summary, this indicates that platelets are the primary source of TGFβ activity in the tumor microenvironment and exert an effective immunosuppressive effect against tumor immunity via GARP-TGFβ.

To determine the clinical relevance of platelet-mediated immunosuppression of tumors, the effects of platelets on immunotherapy were investigated from a pharmacological perspective. B16-F1 melanomas were established in C57BL/6 mice after subcutaneous injection on day 0, followed by lymphocyte depletion with Cy on day 7, and infusion of ex vivo induced Pmel cells along with antiplatelet (AP) agents: aspirin and clopidogrel on day 8. Aspirin and clopidogrel inhibited platelet activation by blocking cyclooxygenase and ADP receptors, respectively. Cy alone failed to control the tumor, and additional AP had no antitumor effect in this model (Fig. 10A, left panel). T-cell plus Cy provided good control of melanoma for approximately one month, but most mice eventually relapsed. Conversely, antiplatelet agents plus adoptive T-cell transfer were highly effective for B16-F1, with most mice achieving relapse-free survival exceeding 3 months (Fig. 10A, right panel). As further evidence, mice receiving simultaneous antiplatelet therapy and ACT maintained higher numbers of antigen-specific T cells in the blood, inguinal lymph nodes (ILN), and spleen (Fig. 10B). Importantly, antiplatelet agents provided no benefit when the metastatic T cells lacked IFNγ (Fig. 10C) or when anti-IFNγ neutralizing antibodies were administered (Fig. 10D), demonstrating that the effect of antiplatelet agents is immune-mediated.

Example 5 - Materials and Methods

Cell lines and mice. The pre-B cell line (70Z/3) was a generous gift from Brian Seed (Harvard University) (Randow and Seed, 2001). 4T1 mouse mammary epithelial carcinoma cell line, wild-type (WT) normal mouse mammary epithelial cells (NMuMG), and hnRNP E1-silenced NMuMG* subline. B16-F1 and 293FT cell lines were purchased from ATCC.

Female BALB/c, C57BL/6J, NOD-Rag ^-/- , NSG breeder pairs (NOD Scid Gamma), and Pmel 1T cell receptor (TCR) transgenic (Tg) mice aged 6–8 weeks were purchased from The Jackson Laboratory (Bar Harbor, MEUSA). All animal experiments involving mice were approved by the Medical University of South Carolina Animal Care and Use Institutional Committee and followed established guidelines. Control and treatment mice were co-housed, and age-matched female mice aged 6–8 weeks were used in all experiments.

Tissue microarrays and human serum. All human tumor microarrays (TMAs) were prepared from formalin-fixed paraffin-embedded tissue. TMAs for colon cancer, lung cancer, and one of two breast cancers were developed from samples collected at the Medical University of South Carolina (MUSC; Charleston, SC). Each patient sample in these TMAs was represented as two cores on a slide, with each core measuring 1 mm in diameter. TMAs for breast and prostate cancer were commercially available from Imgenex, Inc. (San Diego, CA). These patient samples were available in a single core with a diameter of 2 mm. Clinical and demographic information was obtained from the Cancer Registry at MUSC Hollins Cancer Center or from commercial sources. This study was approved by the MUSC Institutional Review Board (IRB).

Immunohistochemistry (IHC). The mouse anti-human GARP antibody (ALX-804-867-C100, Enzo Life Sciences) used in this study was first tested by Western blotting in untransfected and hGARP-transfected human embryonic kidney (HEK)-293 cells, and by IHC in mouse pre-B leukemia cells 70Z/3 transfected with hGARP and control vector. Both analyses demonstrated the specificity of the antibody and the dilutions used, from 1:250 (colon cancer) to 1:60 (all other cancers).

TMA slides were baked at 62°C for 2 hours, then deparaffinized in xylene and rehydrated. Antigen retrieval was then performed by boiling in citrate buffer (pH 6.0) in an autoclave for 30 minutes. Slides were incubated in 3% H2O2 in dH2O for 7 minutes, and nonspecific binding was blocked for 2 hours at room temperature with 2% normal horse serum. Samples were incubated with anti-h-GARP antibody at 4°C for 16 hours, then incubated with secondary antibody (Vectastain ABC kit) and developed using DAB substrate (Vector Labs SK-4100). Staining was specific for cytoplasm and cell membrane, with negative nuclear staining.

For mouse IHC, primary tumors and lungs were isolated. Tumor tissues were placed in OCT medium to obtain fresh frozen sections, or fixed overnight in 4% paraformaldehyde to obtain fixed sections. For hematoxylin and eosin (H&E) analysis of tumors and lungs, fixed tissues were incubated overnight in 70% ethanol before paraffin embedding and then cut for H&E staining. For p-Smad-2/3 on fresh frozen tumor sections, 5 μm sections were fixed with 4% paraformaldehyde and then incubated with 3% _H₂O₂ . To minimize nonspecific staining, sections were incubated with appropriate animal serum at room temperature for 20 min, then incubated overnight at 4 _° C with primary antibody p-Smad-2/3 (EP823Y; Abcam). Standard protocol for anti-rat Vectastain ABC Kil (Vector Labs) was followed.

The staining intensity of GARP and pSmad-2/3 was graded by a board-certified pathologist (S.S.) without the knowledge of sample identity (0: negative; 1: weak; 2: moderate; 3: strong, but not as strong as 4; 4: strong). The percentage of positive cells in each patient sample in the TMA was also calculated; in TMAs where samples were spotted in duplicate, the mean of the two cores was used as a representative value. Student's t-tests were performed to compare categorical variables, such as normal versus cancer or different disease stages or categories. Kaplan-Meier analysis of the association between GARP and survival was performed using X-tile software (Camp et al., 2004). Chi-square tests were used to test for statistical significance of differences between low and high GARP expression.

Immunofluorescence analysis. Freshly frozen tumor sections (5 μm) were air-dried, fixed in acetone for 10 minutes, and then incubated with phycoerythrin-conjugated anti-CD31 antibody (1:50). After imaging on an Olympus fluorescence microscope, the vascular density was determined by calculating the CD31 staining area using ImageJ v1.34 software (NIH).

GARP was knocked down using lentiviral expression of short hairpin RNA. Lentiviral vectors expressing short hairpin RNA (shRNA) targeting mouse GARP transcripts were purchased from Sigma-Aldrich (St. Louis, MO). Isotropic GARP shRNA and control scrambled lentiviral shRNA particles were generated in HEK293FT cells. To knock down GARP in NMuMG* cells, cells were transduced with GARP-targeting lentiviral supernatant and scrambled control cells. Knockdown efficiency was assessed using an anti-mouse GARP antibody (eBioscience) via RT-PCR (Applied Biosystems Step-One Plus) and flow cytometry (BD Verse).

Generation of vectors expressing GARP. GARP was amplified by PCR and subcloned between the BglII and HpaI sites of a MigR1 retroviral vector. A cDNA construct for expressing the recombinant GARP-Fc fusion protein was generated by ligating the extracellular domain of the GARP sequence to the Fc portion encoding the mouse IgG2a constant region. The Fc sequence was amplified from the phCMV1 vector by PCR, and GARP was amplified from the MigR1 retroviral vector by PCR. The two fragments were ligated and cloned into a MigR1 retroviral expression vector. Homotropic GARP and GARP-Fc retroviral particles were packaged into Pheonix-homotropic cells. Viral propagation and transduction of pre-B cells, 4T1 cells, and NMuMG* cells were performed according to established protocols (Wu et al., 2012; Zhang et al., 2015). Cells were stabilized by culturing for at least 72 hours in the presence of blastomycin 48 hours post-transduction.

Purification of GARP-Fc. To purify the GARP-Fc protein, the MigR1 vector was transfected into Chinese hamster ovary (CHO) cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Stably transfected clones were selected using blastomycin (5 μg/ml), and protein expression was quantified under reducing conditions by SDS-PAGE and Western blotting using anti-mouse GARP and anti-mouse Fc antibodies. Recombinant GARP-Fc was purified from the cell culture supernatant using protein A affinity chromatography (GE Health).

Generation and characterization of anti-GARP antibodies. Four BALB/c mice were immunized with recombinant human GARP (R&D Systems, Minneapolis, MN) and Freund's complete adjuvant, followed by boosting with SP2/0 cells stably expressing human GARP 2 to 3 times. Splenic B cells from mice with high anti-GARP antibody titers were fused with SP2/0 cells in the presence of polyethylene glycol. Hybridomas were selected in HAT medium and cloned using limiting dilution assays. Antibody specificity was screened and determined by ELISA and flow cytometry using 70Z/3 cells stably transduced with an empty vector (70Z/3-EV) and overexpressing human GARP (70Z/3-GARP).

Protein extraction, immunoprecipitation, and Western blot analysis were performed. Cells were harvested via trypsin-EDTA if necessary, washed in PBS, and lysed on ice in radioimmunoprecipitation assay (RIPA) lysis buffer in the presence of a protease inhibitor mixture (Sigma-Aldrich). Nucleus-free protein lysates were quantified by the Bradford assay (Bio-Rad) and analyzed by SDS-PAGE and Western blot under reducing conditions using anti-mouse GARP (AF6229; R&Dsystem), anti-mouse vimentin (D21H3; Cell signaling), anti-mouse E-cadherin (24E10; Cell Signaling), and anti-mouse p-Smad-2/3 (EP823Y; Abcam).

Cell proliferation and in vitro wound healing assays. NMuMG cells (4 x ^10⁵ ) were starved overnight in serum-free DMEM (Corningcellgrom). At the indicated time, starved cells were cultured with GARP-Fc in 2% FBSDMEM. To measure cell proliferation, 2.5 x ^10⁵ cells were seeded into complete medium (DMEM, 10% FCS, 1% penicillin-streptomycin) in 96-well plates and incubated overnight. Proliferation was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazol bromide (MTT), which was added to the cells at the indicated time and incubated at 37°C for another 3 hours. The medium was then removed and mixed with 100 μl DMSO by shaking for 15 minutes. The absorbance at 570 nm was then measured using a plate reader. Cell migration was measured by a wound healing assay: two parallel wounds were made using a 1 ml pipette tip at 100% confluence. Migration was assessed at 24, 48, and 72 hours, and the quantification of wound closure was measured using ImageJ software (NIH).

4T1 Tumor Model and GARP Antibody Therapy. ⁵ x 10⁵ cells (4T1 EV, 4T1 GARP, or 4T1 GARP-Fc) were subcutaneously (sq) injected into the fourth mammary fat pad of 6- to 8-week-old female BALB/c mice. Tumor growth was monitored three times weekly using digital calipers, and tumor volume was calculated using the following formula: Tumor volume ( ^mm³ ) = [(width) ^² × length]/². In the GARP antibody therapy experiment, starting 3 days post-tumor inoculation, mice were administered anti-GARP antibody or a polyclonal isotype control antibody (0.1 mg/mouse in 0.1 mL PBS; three times weekly) intraperitoneally (ip). For the combination therapy of cyclophosphamide (CY) and antibody, mice were treated with a single injection of CY (4 mg/mouse) 3 days post-tumor inoculation, in addition to antibody treatment. At the endpoint, mice were sacrificed, and the primary tumor, draining lung, spleen, lung, and liver were isolated. Tumor-infiltrating lymphocytes were isolated by digestion with collagenase D (Sigma) followed by density separation mediated by Histopaque-1083 (Sigma).

B16-F1 Tumor Model and Adoptive T-Cell Therapy (ACT). In three groups (B16 EV and B16 GARP-Fc; n = 5–7 per group), 2.5 x ^10⁵ cells were seeded sq in the right flank of 6–8-week-old female C57BL/6J mice and treated with a single intraperitoneal injection of CY (4 mg/mouse) one day prior to adoptive T-cell therapy. To obtain gp100-specific T cells, spleen cells from Pmel TCR transgenic female mice were stimulated for 3 days with hgp100 (25–33 epitopes, 1 μg/ml, American Peptide Company) and mouse IL-12 (10 ng/ml, Shenandoa). One day after CY injection, ACT was performed via tail vein injection of 2 x ^10⁶ activated Pmel T cells per recipient mouse. Primary tumor growth was monitored three times weekly using calipers. Peripherally adopted Pmel cells were monitored at 2, 3, 4, and 5 weeks post-ACT. In vitro Pmel IFN-γ production was assessed by stimulating Pmel cells at 37°C for 3 hours in the presence of hgp100 and brevidin A (BFA), and analyzed by flow cytometry.

NMuMG tumor model. 5 x ^10⁵ cells (NMuMG*-EV, GARP knockdown NMuMG*) were subcutaneously seeded into the fourth and left mammary fat pads of female NOD-Rag-1 ^-/- (n = 5 per group; 6 to 8 weeks old). Animals were weighed and tumors were measured weekly. At the endpoint, primary tumors, lungs, and livers were harvested. In another experiment, ⁵ x 10⁵ cells (NMuMG-GARP-Luc, NMuMG-GARP-Fc-Luc, or NMuMG-Luc cells) were subcutaneously seeded into the fourth and left mammary fat pads of female NOD-Rag-1 ^-/ - mice (n = 4–5 per group; 6 to 8 weeks old). In vivo luciferase imaging was assessed weekly as follows: Mice were anesthetized by intraperitoneal injection of D-luciferin (Perkin Elmer) at a dose of 150 mg/kg per mouse. Bioluminescence images were then acquired using the Xenogen IVIS imaging system. The bioluminescent signal was quantized into photon flux (photons/s/ ^cm² ) in a defined target region using Living Image software (Xenogen).

TGF-β1, GARP, and GARP-TGF-β1 analysis. Active TGF-β1, total TGF-β1, and soluble GARP in human and mouse serum were measured using the TGF-β1 and GARP ELISA kit (BioLegend, San Diego, CA) according to the manufacturer's protocol. To measure the GARP-TGF-β1 complex by ELISA, 96-well plates were coated with TGF-β1 capture antibody according to the manufacturer's instructions (BioLegend, San Diego, CA). Samples were incubated at room temperature for 2 hours, followed by a further 2 hours incubation with our lab-developed anti-hGARP detection antibody.

For MFB-F11 functional assays, MFB-F11 cells (a generous gift from Tony Wyss-Coray, Stanford University) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin. 2 x ^10⁴ cells were seeded per well and incubated overnight. Cells were serum starved for 2–3 hours before the addition of diluted serum or tumor supernatant. The diluted serum or tumor supernatant samples were incubated for 24 hours and then analyzed using QUANTI-Blue medium (InvivoGen, San Diego, CA) (Tesseur et al., 2006).

Statistical analysis. In TMAs with duplicate sample sizes, the means of the two cores were used as representative values. Student's t-tests were performed to compare categorical variables, such as normal versus cancer or different disease stages or categories. Kaplan-Meier analysis of the association between GARP and survival was performed using X-tile software (Camp et al., 2004). Chi-square tests were used to test for statistical significance of differences between low and high GARP expression. Tumor curve analysis was performed using two-way ANOVA; all other experiments were analyzed using two-tailed Student's t-tests with GraphPad Prism. All data are presented as mean ± SEM. A p-value less than 0.05 was considered statistically significant.

Example 6 - Humanization report of antibody 4D3

Computational modeling. A homologous model of MAb 4D3 Fv was built using pdb 1KC5 as the model structure, and the humanization design was double-checked using another heterologous model built on pdb 1MCP and pdb 32C2.

Reverse mutation design rules. During the humanization process, mouse CDRs are transplanted into human frame receptors to study residues that differ between the human and mouse frame receptors. Reverse mutations from human residues to mouse residues are designed according to the following rules:

If a new contact (unusual interaction, hydrogen bond, hydrophobic interaction) is formed between the human residue and a mouse Fv CDR residue, canonical residue, interface residue, or vernier residue, the human residue needs to be reverted to a mouse residue. If the old contact (unusual interaction, hydrogen bond, hydrophobic interaction) between the mouse residue and the canonical residue, interface residue, or vernier residue is lost when a human residue replaces a mouse residue, the human residue needs to be reverted to a mouse residue. Substitution of mouse canonical residues, interface residues, or vernier residues with human residues should be carefully studied and generally avoided.

Schrödinger surface analysis data. Schrödinger surface analysis was performed on mouse MAb 4D3 Fv and huVHv1VLv1. Further investigation of the surface analysis data is necessary to address any polymerization issues observed in the humanized leaders in future benchtop laboratory settings.

Schrödinger post-translational modification data. Schrödinger post-translational modifications were performed on mouse MAb4D3 Fv and huPIIO-1VH1VL1 (data from the humanized version with the highest humanization percentage). Only typical PTM motifs with side chains and >50% 3D surface accessibility were classified as “high-risk” residues (e.g., “NG” is a typical deamidation site, “QG” is atypical). These types of PTMs were found in the huMAbPIIO-1VH1VL1 PTM analysis file.

Studies of T-cell epitopes, B-cell epitopes, and MHC II epitopes are presented. All potential T-cell epitopes, B-cell epitopes, MHC II epitopes, and antigenic epitopes predicted by Protean 3D within the highest humanized version PIIO-1VH1VL1 containing reversion mutations are listed. Those framework epitopes contain reversion mutations. Removal of these reversion mutations may result in loss of affinity and/or exploitability.

Humanization candidates were ranked. Four humanized VHs and three VLs were designed, resulting in 12 VH/VL combinations. IgG proteins from 12 humanized leaders and wild-type HCL64 clones were produced on a small scale, and the unpurified culture supernatant was used for the following ELISA assays.

Table A - FACS analysis using Ag(+) cells

Binding assays were performed on culture supernatants using FACS. FACS results showed that all humanized leaders maintained similar binding aptitude to the wild-type 4D3 chimeric clone. The plateau phase MFI was relatively low. To double-check binding, FACS binding assays were repeated. To rapidly assess the thermal stability of the humanized leaders, the inventors treated supernatant samples at 70°C for 5 minutes, and then repeated FACS assays under the same conditions using the same samples. The results showed that the 4D3 chimeric clone and all designed humanized clones were completely resistant to heat treatment.

Table B - Preliminary Thermal Stability Test

No clones showed any nonspecific binding to Ag(-) cells.

Table C - FACS analysis using Ag(-) cells

The concentration of all antibody samples was 5 ug/ml.

Conclusion. Since all the designed humanized clones were very similar in terms of specific binding and thermal stability, the inventors primarily selected clones VH1VL1, VH1VL2, and VH2VL1 for further testing based on the percentage of humanization (VH1>VH2>VH3>VH4, VL1>VL2>VL3).

Methods - Transient transfection. Wild-type 4D3 (chimeric) and humanized VH/VL DNA were synthesized. Expi293 cells were transiently transfected using different VH/VL combinations. Three days post-transfection, culture supernatants were collected, and IgG levels were measured using a protein A sensor on a Gator (similar to Octet, ProbeLife, located in Palo Alto, CA), with corrections performed using ELISA.

Methods - FACS Analysis. Cell Preparation : Harvest cells and wash once with PBS + 2% FBS. Adjust the cell density to 1.5E6/mL in PBS/2% FBS. Add 100 μL of cells to each well of a 96-well U-plate. Antibody Sample Preparation : Adjust the antibody concentration in the supernatant to 10 μg/mL using PBS + 2% FBS, and perform serial 5-fold dilutions to obtain three antibody solutions of different concentrations. The blank is prepared with PBS + 2% FBS. Add 100 μL of antibody solution to each well of a 96-well U-plate, arranged in the same manner as in cell preparation. Incubation : Mix the antibody sample (100 μL) with the cells (100 μL) and incubate at room temperature for 1 hour, then centrifuge at 1000 rpm (rotary plate) for 3 minutes. Aspirate the supernatant and wash the cells once with PBS + 2% FBS. Incubation with secondary antibody : Cy3-conjugated AffiniPure goat anti-human IgG was diluted 250-fold with PBS + 2% FBS and added at 100 μl/well to a 96-well U-plate, then incubated at room temperature for 30 min. The plate was then centrifuged at 1000 rpm for 3 min, and the supernatant was aspirated. Cells were washed twice with PBS + 2% FBS. Cells were resuspended in 200 μl PBS + 2% FBS and analyzed on FACS. The MFI of total viable cells was used as a binding signal.

Method - Heat treatment. The culture supernatant was serially diluted with cell culture medium to the indicated IgG concentration and heated at 70°C for 5 minutes on a PCR instrument, followed by rapid cooling to room temperature.

The selected humanized leaders were characterized using purified IgG. Based on the culture supernatant results, the inventors selected three humanized leaders, VH1VL1, VH1VL2, and VH2VL1. Expi 293 cells were co-transfected with the VH and VL plasmid DNA of each of the selected leaders, and IgG was purified for each candidate. FACS analysis was repeated using purified antibodies to compare the specific binding ability of the humanized leaders to wild-type chimeras. Preliminary assays were performed to compare their thermostability and non-specific binding. The results are shown in Figure 11.

Conclusion. Using purified IgG antibodies, the results confirmed that the selected candidates had very similar binding affinities. After treatment at 70°C for 5 minutes, all three leaders showed similar binding abilities compared to the chimeric antibody.

Methods - FACS Analysis. Cell Preparation : Harvest cells and wash once with PBS + 2% FBS. Adjust the cell density to 1.5E6/mL in PBS/2% FBS. Add cells at 100 μl/well to a 96-well U-plate. Antibody Sample Preparation : Adjust the antibody concentration to 20 μg/mL using PBS + 2% FBS, then perform serial dilutions to obtain antibody solutions of different concentrations. The blank is prepared with PBS + 2% FBS, and the antibody solution is added to a 96-well U-plate at 100 μl/well using the same method as cell preparation. Incubation : Mix antibody sample (100 μl) with cells (100 μl) and incubate at room temperature for 1 hour. Then centrifuge the plate at 1000 rpm (rotary drum) for 3 minutes. Aspirate the supernatant and wash cells once with PBS + 2% FBS. Incubation with secondary antibody : Cy3-conjugated AffiniPure goat anti-human IgG was diluted 250-fold with PBS + 2% FBS and added at 100 μl/well to a 96-well U-plate, then incubated at room temperature for 30 min. The plate was then centrifuged at 1000 rpm for 3 min, and the supernatant was aspirated. Cells were washed twice with PBS + 2% FBS. FACS detection : Cells were resuspended in 200 μL PBS + 2% FBS and then subjected to FACS analysis.

Method - Heat treatment. The antibody was heated at 70°C for 5 minutes on a PCR instrument, and then rapidly cooled to room temperature.

Affinity measurements. Label-free kinetic binding assays were performed on a Gator (similar to Octet). The results showed that the three humanized leaders (H1L1, H1L2, and H2L1, also referred to in this paper as VH1VL1, VH1VL2, and VH2VL1, respectively) had the same KD values as the chimera.

Table D - Dynamics combined with analysis data

Methods. Affinity was measured on a Gator (ProbeLife, Palo Alto) instrument. Briefly, purified IgG samples were diluted to 2 μg/ml in K buffer, and antigen was diluted to 5 μg/ml. Antibody-loaded anti-human Fc probes were immersed in antigen wells for 5 min, and then transferred to K buffer wells for 5 min. The sample plate was shaken at 1000 rpm throughout the process. Data analysis was performed using Gator software (ProbeLife).

The non-specific binding of the humanized lead antibody to "glycoproteins, lipids, and proteins" was assessed. A baculovirus (BV) ELISA was used as a preliminary assay to assess the potential risk of non-specific binding. Results are shown in Figure 12 and Table E below.

Table E - Original BV Combined Data

Conclusions and Summary. In the baculovirus (BV) ELISA, the inventors used rituximab Mab as a reference. If an antibody showed weaker BV binding than rituximab, this would be considered a nonspecific binding problem. With purified antibodies, results showed that chimeric, VH1VL1, VH2VL1, and rituximab had similar binding, with clone VH1VL2 showing a slightly higher signal. Humanized clones VH1VL1, VH1VL2, and VH2VL1 (also referred to herein as H1L1, H1L2, and H2L1, respectively) were very similar in terms of specific binding, preliminary thermostability, and nonspecific binding assays (BV ELISA). In the BV ELISA assay, clone VH1VL2 showed a slightly higher signal than the other clones, but none of the clones showed any nonspecific binding to Ag(-) cells (see Experiments 1.1.1 and 2.1.1). If a clone binds only to BV and not to 293 cells, it will be considered to have a low risk of nonspecific binding. Therefore, clones VH1VL1, VH1VL2, and VH2VL1 can be considered as candidates for further evaluation.

Method - Baculovirus ELISA. Each well of the plate was plated with 50 μl of baculovirus sample diluted 1:500 in PBS. The plate was incubated overnight at 4°C. The plate was washed three times with 300 μl of wash buffer, and 200 μl of blocking buffer (1% BSA) was added at room temperature for 60 minutes. The plate was washed three times with 300 μl of wash buffer, and 100 μl of diluted antibody was added to each well at different concentrations, followed by incubation at room temperature for 1 hour. The plate was washed six times with 300 μl of wash buffer, and 100 μl of 1:5000 HRP-conjugated secondary antibody was added to PBS, followed by incubation at room temperature for 1 hour. The plate was washed six times with 300 μl of wash buffer. Developing buffer was added, and the plate was read.

Developability analysis of the PIIO-1 humanization lead was conducted. Thermostability of the antibody was analyzed using DSF/SLS. Samples were submitted to the UNcle system (Unchained Labs) for analysis. For DSF and SLS, a temperature ramp of 1 °C/min was performed, monitored from 20 °C to 95 °C. SLS was measured at 266 nm and 473 nm using UNcle. Tm and Tagg were calculated and analyzed using UNcle analysis software. DSF: Differential Scanning Fluorescence Assay; SLS: Static Light Scattering; Tm: Melting Temperature; Tagg 266: Thermal accumulation during SLS at 266 nm; Tagg 473: Thermal accumulation during SLS at 473 nm. A summary is shown in Table F below:

Table F

IgG is a multi-domain structure, and each domain has its own melting temperature (Tm). The CH2 domain typically has a Tm of around 70°C in PBS, while the CH3 domain is more stable, exhibiting a Tm of around 80°C. Due to significant sequence variation, the Tm range of Fab is wide, typically ranging from around 50°C to 85°C. Therefore, the Tm values measured by various analytical techniques are usually "apparent" transition temperatures, rather than the true Tm of each domain. For the entire IgG, there are typically 2 to 3 Tm values in DSF measurements, which presents some challenges in determining which Tm represents which domain.

All measured huPIIO-1 clones had two or three Tm values, and it is likely that the higher one (Tm2) represents CH3, while Tm1 represents Fab+CH2. DSF results showed that the three huPIIO-1 candidates had similar thermal stability to the 4D3 wild-type clone.

Tagg is the temperature at which SLS begins to detect aggregated particles. Tagg266 measures SLS at 266 nm, which is more sensitive and suitable for detecting smaller aggregated particles. Tagg473 measures SLS at 473 nm and is more suitable for detecting larger particles.

Compared to the 4D3 wild-type clone, all huPIIO-1 candidates have slightly different Tagg values, which means that the candidates have similar aggregation potential to the wild-type clone.

Aggregation potential analyzed by DLS. DLS was performed on the Uncle system (Unchained Labs). DLS was measured at 25°C. Data were calculated and analyzed using Uncle analysis software. DLS: Dynamic Light Scattering, PDI: Polydispersity Index, PDI = (Standard Deviation / Mean Hydrodynamic Radius). Results are summarized in Table G below:

Summary of G-DLS Results

Dynamic light scattering (DLS) is used to detect aggregation in antibody samples. The “mode diameter” is the diameter of the protein particles, and the “mass percentage” is the percentage of the amount in each size fraction. The “PDI” is the polydispersity index; the higher the index, the greater the polydispersity of the sample. As shown in the table above, VH1VL2 and VH2VL1 have similar or better PDIs compared to WT, with VH1VL1 having a slightly worse PDI than WT. Peak 1 is the main peak and represents the IgG monomer. The inventors considered both the “peak 1 mass percentage” and the “PDI” value when selecting the leader. VH1VL2 and VH2VL1 have very similar peak 1 mass percentages and PDI values to the chimeric clones.

Heterogeneity was analyzed by capillary electrophoresis (CE). Samples were prepared in reducing and non-reducing labeled buffers prior to CE analysis. A summary of the results for reducing CE-SDS and non-reducing CE-SDS is shown in Figure 13A/Table H and Figure 13B/Table I, respectively.

Table H

Table I

sample 10KD moving time Main peak (%) Main peak movement time other(%) 4D3 Chimera 13.187 98.7 29.575 1.5 huPIIO-1VH1VL1 13.212 97.6 29.408 2.4 huPIIO-1VH1VL2 13.241 97.6 29.442 2.4 huPIIO-1VH2VL1 13.267 89.1 29.567 10.8

Compared to the chimeric 4D3 clone, the humanized clone VH1VL2 exhibited very similar binding affinity, thermal stability (heat treatment), CE purity, and aggregation potential in DLS assays. In DLS, VH1VL2 showed a slightly higher PDI than the chimeric clone, while in SLS assays it also demonstrated significantly better Tagg266 and Tagg473 (78.8 vs. 75.0, 79.3 vs. 75.4), indicating a very low risk of aggregation for VH1VL2.

Example 7 - Antibody against GARP (LRRC32)

Table J. GARP Monoclonal Antibody Clones

One of the goals of the Ohio State University (OSU) SPORE research in lung cancer is to generate novel cancer therapies targeting GARP. Anti-human GARP antibodies were generated by immunizing mice with recombinant human GARP (hGARP) and boosting them with irradiated SP2/0 myeloma cells stably expressing hGARP. The antigen specificity of the generated clones was confirmed by flow cytometry (Figure 17A). Of the seven clones reported here, all recognized hGARP on Tregs, while only five clones (excluding clones 1C12 and huPIIO-1) recognized hGARP on platelets. To further characterize antibody function, we tested whether the clones recognized free GARP or the GARP-TGFβ complex (GARP-LAP).

Using an overexpression system with 293 cells, we found that only the huPIIO-1 clone recognized free GARP, while other antibodies recognized both free GARP and the GARP-LAP complex (Fig. 17B). Given the low affinity of this antibody for mouse GARP (mGARP), we used a series of HA-labeled mGARP/hGARP chimeras to map the epitopes of these clones. We found that only huPIIO-1 recognized aa171-297 (Fig. 17C). Importantly, only huPIIO-1 blocked the binding of exogenous human LTGFβ-1 (huLTGFβ1) to surface GARP (Fig. 17D). Therefore, we generated and validated an anti-GARP antibody library to further test the ability of GARP as a true immuno-oncology target.

Example 8 - Generation of GARP humanized mice.

Most anti-GARP antibodies recognize human GARP but not mouse GARP. To facilitate translational work, we generated human GARP knock-in ( ^hLrrc32KI ) mice (Figs. 18A–18C). We confirmed that humanized 4D3 (huPIIO-1) recognizes GARP on Tregs but not on platelets (Fig. 18D). Furthermore, we found that intravenous administration of huPIIO-1 was well-tolerated, without causing significant thrombocytopenia or marked toxicity (Figs. 18E–18F). These findings validate huPIIO-1 as a strong candidate for clinical development and provide a systematic approach to investigating the potential mechanism of action of this drug.

Example 9-huPIIO-1 has immunomodulatory activity in Lrrc32 humanized mice in the following aspects.

Next, we used hGARP mice to determine whether the anti-GARP antibody huPIIO-1 could modulate the host immune response. Two experiments were performed. First, tumor-free hGARP mice were intravenously injected with either huPIIO-1 or IgG1 (200 μg, 3 doses, every 2 days), followed by immunophenotypic analysis. We observed higher cytosis in the peripheral lymph nodes (pLN) of huPIIO-1-treated mice, which was associated with an increased frequency of CD8 ⁺ T cells (Figs. 27A–27B). We also observed elevated Ki67 levels in CD8 ⁺ T cells, indicating that the enhanced cytosis was due to increased proliferation (Fig. 27E). Furthermore, we noted a moderate but significantly reduced frequency of Tregs in the pLN (Fig. 27D). Second, hGARP mice were subcutaneously injected with MB-49 bladder cancer cells expressing hGARP, followed by treatment with huPIIO-1. We then examined TGFβ activity by intracellular staining of pSMAD2/3 in tumor-infiltrating immune cells. We found that huPIIO-1 inhibited TGFβ activity in all examined subsets of immune cells, including T cells, B cells, M1 and M2 macrophages, and dendritic cells in the TME (Figure 25). In summary, we conclude that huPIIO-1 possesses immunomodulatory activity, likely through its ability to block GARP binding and activate LTGFβ.

Example 10 - huPIIO-1 monotherapy promotes CD8 ⁺ T cell recruitment to the TME and confers single-agent anticancer activity in Lrrc32 humanized mice.

Recent studies have shown that the TGFβ pathway not only attenuates T cell effector function but also blocks CD8 ⁺ T cell transport to the TME by specifically inhibiting CXCR3 expression. We then investigated whether huPIIO-1 could induce CXCR3 expression on CD8 ⁺ T cells and thus contribute to antitumor activity (Fig. 26H). We found that huPIIO-1 exhibited significant single-agent activity against MB49 (Figs. 26I–26J), which was associated with increased CD8 ⁺ T cell activity in the drained lymph node (LN) (Fig. 26K) and TME (Fig. 26L). To examine the contribution of CXCR3 in this process, we blocked CRCX3 with an antagonistic antibody during huPIIO-1 treatment. We observed that this antibody completely eliminated the antitumor efficacy of huPIIO-1 (Figs. 26I–26J). Mechanistically, anti-CXCR3 blocked CD8 ⁺ T cell recruitment in both the dLN and TME (Figs. 26K–26L). Overall, the data indicate that huPIIO-1 promotes T cell transport via CXCR3 by removing TGFβ from the TME.

Example 11 - The potential of the anti-GARP antibody huPIIO-1 to overcome resistance to PD-1 blockers in lung cancer.

Mechanistically, PD-1 blockers primarily exert their effects by targeting the progenitor-depleted CD8 ⁺ T cell population (expressing TCF-1, SlamF6, and moderate to low PD-1) in the tumor microenvironment (TME). These cells undergo a proliferative burst upon treatment, increasing differentiation into a terminally depleted population and leading to tumor clearance. As single-therapy data have shown, huPIIO-1 significantly modulates CD8 ⁺ T cells in both the TME and draining lung nuclei (LN). Therefore, we hypothesized that GARP expression may contribute to PD-1/L1 ICB resistance. To explore this hypothesis, we first mined the POPLAR database, which compares the PD-L1 blocker atezolizumab with chemotherapy (docetaxel) in platinum-resistant advanced NSCLC. In this multicenter international phase II trial, bulk RNAseq data from pre-treatment tumors were obtained from 86 patients recruited primarily at a US site. We divided these patients into high-GARP expression and low-GARP expression groups based on the median expression level of the LRRC32 transcript. We found that for patients with low GARP expression, atezolizumab treatment had a greater benefit in both overall survival (HR = 1.89, p = 0.086; atezolizumab group n = 22 vs. docetaxel group n = 21) and disease control rate (68.2% in the atezolizumab group vs. 9.5% in the docetaxel group, p = 0.047703). In patients with high GARP expression, atezolizumab (n = 21) and docetaxel (n = 23) showed no difference in OS (p = 0.9655) or disease control rate. Although exploratory in nature, these data suggest that high GARP expression may contribute to PD-L1 ICB resistance. To further explore this hypothesis, we tested combination therapy of the anti-GARP antibody huPIIO-1 and a PD-1 blocker in mouse Lewis lung cancer (LLC) and CMT-167 lung cancer models, both of which are considered to be immune-cold tumors resistant to single-agent PD-1 ICBs. To this end, human GARP KI mice were challenged with LCC and then treated with 200 μg/mouse of isotype huPIIO-1, anti-PD-1 antibody, or a combination of huPIIO-1 and PD-1, starting on day 8 post-tumor challenge, every 3 days for a total of 4 injections. We found that the combination of huPIIO-1 and PD-1 blockers had the greatest effect in slowing LLC growth compared to single-therapy treatment (Figure 36A). Although endpoint TIL analysis revealed a decrease in CD8 ⁺ TCF-1 ⁺ T cell frequency in all groups receiving PD-1 blockers, only the combination group was associated with an increase in subsequent TOX expression. These data are interesting for two reasons: 1) TOX is known to be associated with and required for terminal T cell depletion and the generation of memory T cells; and 2) TOX expression has been shown to be downstream of TCR stimulation. Importantly, recent work has demonstrated the direct role of TGFβ signaling in inhibiting anti-tumor CD8 ⁺ T cell responses by raising the TCR activation threshold. Therefore, these data indicate that huPIIO-1 can improve PD-1 blockade response by enhancing TCR stimulation through increased differentiation of progenitor-consuming cells. Finally, we also confirmed that huPIIO-1 can overcome anti-PD-1 resistance in CMT-167. This activity was significantly correlated with an increased CD8 ⁺ T cell population in the TME (Figs. 19A–19B). Furthermore, we analyzed CD8+ TIL dynamics using an established T cell cohort (CD45, CD3, CD8, CD4, Foxp3, CD69, CD25, PD-1, Tim3, Slamf6, TOX, Tcf-1, CD44, CD62L, CTLA4, Lag-3, Klrg1, T-bet, Ki-67, GARP, EOMES, Vista, TIGIT, CX3CR1, ICOS, CXCR3, OX40, CD28, GITR, CD101, CD95, and granzyme B) via spectroscopic flow cytometry ( ^Cytek Aurora). We found that the combination therapy caused a significant increase in two CD8 ⁺ T cell clusters (Fig. 19C to 19E), reflecting newly activated effector progenitor cells (cluster #10: CD44 ⁺ Tox ^- PD-1 ^- GZMB ^- Vista ⁺ Tigit ^low ) and Teff-like cells (cluster #3: CD44 ⁺ Tox ^- Tcf1 ^- PD-1 ^- GZMB ⁺⁺ Vista ⁺ Tigit ^low ).

All methods disclosed and claimed herein can be performed and carried out according to this disclosure without excessive experimentation. While the compositions and methods of this disclosure have been described in conjunction with preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the methods and steps or the order of steps in the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent to those skilled in the art that certain chemically and physiologically relevant agents can replace the agents described herein while achieving the same or similar results. It will be apparent to those skilled in the art that all such similar substitutions and modifications are considered to be within the spirit, scope, and concept of this disclosure as defined by the appended claims.

Example 12: High LRRC32-TGFB expression in human cancers is associated with unfavorable TME and poor clinical response to ICB.

To understand the immunological and clinical significance of GARP expression in cancer, we first mined the Cancer Immune Landscape Database, which developed a global immune profiling classification through bulk transcriptomic analysis of over 10,000 patients from TCGA. The wound healing classification (C1) reflects the induced expression of genes related to angiogenesis. The interferon-γ (IFNγ)-dominant classification (C2) contains the highest populations of type 1 macrophages (M1) and CD8+ T cells with high T cell receptor (TCR) density. Increased helper T(Th)17 and Th1-related genes and decreased tumor cell proliferation were included in the inflammatory classification (C3). A low Th1/high type 2 macrophage (M2) response phenotype is characteristic of the lymphocyte depletion classification (C4). The immune resting classification (C5) shows the lowest lymphocyte infiltration and the highest M2 response. The TGFβ-dominant classification (C6) represents tumors with the highest TGFB gene signature. In 10 common solid tumor types, including bladder and breast cancer, we found that GARP expression was positively correlated with tumors rich in stromal matrix, TGFβ, and macrophage features, and negatively correlated with tumors exhibiting follicular helper T (Tfh) features, memory B cells, plasma cells, and activated dendritic cells (DCs) (Fig. 20A). The negative correlation between GARP expression and immune cells such as Tfh, B cells, plasma cells, and activated DCs suggests that GARP-rich TMEs are detrimental to the formation of tertiary lymphoid structures (TLS) in tumors, although this conclusion requires further histological investigation. In the lung squamous cell carcinoma cohort, GARP-rich tumors had greater TGFβ dominant immune features and lower activated NK cells, CD8+ T cells, and IFNγ features compared to low-GARP tumors (Figs. 20B–20C). Next, we evaluated the importance of LRRC32 expression and LRRC32-TGFB-related features for the responsiveness to immunotherapy in patients with metastatic urothelial carcinoma (mUC). We defined LRRC32-TGFB-related features using genes involved in TGFβ activation, such as αV integrins. LRRC32 expression and LRRC32-TGFB-related features were elevated in patients unresponsive to anti-PD-L1 ICBs (atezilzumab) (Fig. 20D). Elevated LRRC32 gene signature expression was primarily observed in immune-excluded tumors, and we found that high LRRC32 expression (Fig. 20E) and high LRRC32-TGFB-related gene signature (Fig. 20F) were significantly associated with poor overall survival in these patients. Therefore, we conclude that high LRRC32-TGFB expression in human cancers is associated with unfavorable TME and poor clinical response to anti-PD-L1 ICBs, and that GARP is a biologically relevant target for cancer immunotherapy.

Example 13: Anti-GARP antibody PIIO-1 blocks the formation of the GARP-LTGFβ1 complex.

To generate anti-GARP monoclonal antibodies (mAbs), mice were immunized with recombinant hGARP and then boosted with irradiated hGARP-expressing whole myeloma SP2/0 cells. We used flow cytometry to characterize the binding of seven antibodies that recognize hGARP. While all clones recognized hGARP on Tregs, only one clone failed to recognize hGARP on platelets (PIIO-1; Fig. 17A). GARP is known to exist biochemically in three main forms: ligand-free membrane-bound GARP; membrane-bound GARP-LTGFβ complex; and soluble GARP (released upon proteolysis). Tregs express both ligand-free and complexed GARP on their cell surface, while platelets express only the complexed form. Since PIIO-1 only recognizes GARP on Tregs and not on platelets, we can infer that it binds the ligand-free form of GARP (Fig. 21A). To confirm this prediction, we used HEK293FT cells transfected with plasmids expressing hGARP (with or without TGFβ1) to generate cells expressing ligandless GARP (293-hGARP) or the GARP-LTGFβ complex (293-hGARP-TGFβ1) (Figs. 21B and 17B). We generated a series of HA-labeled mouse GARP (mGARP)/hGARP chimeras using standard PCR-based cloning techniques and identified PIIO-1 binding to epitopes corresponding to amino acids 171 to 207 on hGARP (Fig. 21C), which are known sites for LTGFβ binding (Fig. 21D). Using a competitive binding assay, we found that LTGFβ1 blocks the binding of PIIO-1 to GARP (Fig. 17D). Importantly, we found that the expression of cell surface latent-related peptide (LAP) decreased in a dose-dependent manner in the presence of PIIO-1, indicating that PIIO-1 prevents the formation of the complex between GARP and exogenous LTGFβ1 [half-maximum inhibitory concentration (IC50) = 653.4 ng/ml] (Figure 21E). In summary, we generated a unique monoclonal antibody that specifically binds to ligandless GARP at the LTGFβ1 binding site and blocks the formation of the GARP-LTGFβ1 complex. This antibody specifically targets GARP on Treg cells and other cells expressing ligandless GARP, but does not recognize the TGFβ-GARP complex on platelets.

Example 14: Targeting tumor cells with GARP enhances the efficacy of PD-1 blockers in TNBC.

Since PIIO-1 can bind GARP on Treg cells, we next explored whether combining PIIO-1 with an anti-PD-1 ICB could enhance efficacy by transforming an unfavorable TME into a phenotype sensitive to immunotherapy. We orally implanted 4T1 mouse triple-negative breast cancer cells stably expressing hGARP (4T1-hGARP) into BALB/c mice. Mice with established tumors were treated every three days with either PIIO-1 (200 μg/mouse) or anti-PD-1 (150 μg/mouse) as monotherapy or in combination (day 7) (experimental protocol in Figure 22A). Combination therapy slowed tumor growth and prolonged overall survival, resulting in a 46% complete response rate in mice treated with both PIIO-1 and anti-PD-1 (Figures 22B–22D). Furthermore, lung metastases were significantly reduced in mice treated with PIIO-1 (monotherapy or combination therapy) (Figures 22E–22F). To assess the effect of PIIO-1 on downstream TGFβ signaling in the tumor microenvironment (TME), we stained endpoint-collected tumors targeting phosphorylated SMAD3 (pSMAD3) and α-smooth muscle actin (α-SMA). SMAD3 is phosphorylated upon TGFβ activation; α-SMA, a marker of cancer-associated fibroblasts (CAFs), is induced upon TGFβ activation. CAFs contribute to major treatment resistance and are an emerging target for cancer immunotherapy. We found that both pSMAD3 and α-SMA in the TME were reduced after PIIO-1 treatment (Fig. 22G), indicating that local TGFβ signaling was effectively attenuated. Combined treatment also reduced the total circulating active TGFβ (Fig. 22H). Finally, mice experiencing a complete response after combined treatment (Fig. 22C) were completely protected from re-attack by wild-type 4T1 (4T1-WT) tumor cells, demonstrating that PIIO-1 promotes an anti-tumor memory response (Fig. 22I). In summary, these results indicate that combining PIIO-1 with an anti-PD-1 lead enhances antitumor efficacy and antitumor memory, and this may be mediated by downregulation of TGFβ activity in the TME.

Example 15: Targeting TGFβ-GARP signaling regulates immune homeostasis and promotes the differentiation of anti-tumor effector CD8+ T cells in the TME.

Next, we generated hLRRC32KI mice in which the extracellular domain of mouse GARP was replaced by the corresponding human GARP domain from the germline (Figs. 18A–18C). This model allowed us to assess the preclinical safety and efficacy of PIIO-1. In the platelets of hLRRC32KI mice, hGARP association with mouse LAP was as effective as that of mGARP (Fig. 18D). PIIO-1 was well tolerated by intraperitoneal injection and did not cause significant thrombocytopenia or marked toxicity (Figs. 18E–18F). All PIIO-1-treated mice maintained stable body weight for at least 20 days, and there was no clinical evidence of heart failure such as fluid overload and shortness of breath. To assess the effect of PIIO-1 on the immune compartment in non-tumor-bearing hLRRC32KI mice, we administered PIIO-1 or mIgG1 (200 μg/mouse each) intravenously every two days for a total of three treatments, followed by tissue harvesting, single-cell isolation, and immunophenotypic analysis (Fig. 27A). PIIO-1 treatment was associated with increased cellularity and CD8+ T cell frequency in peripheral lymph nodes (pLNs) (Figs. 27B–27C). Furthermore, we observed a decrease in Tregs in pLNs following PIIO-1 treatment (Fig. 27D), consistent with the known role of TGFβ in inducing and maintaining the Treg lineage. Corresponding to the attenuated Treg function and reduced active TGFβ, PIIO-1 increased Ki67 expression and tumor necrosis factor α (TNFα) production by CD8+ T cells in pLNs (Figs. 27E–27F). No differences in immune cell composition were observed in other organs, such as the spleen, thymus, mesenteric lymph nodes (mLNs), or peripheral blood.

Next, we implanted MB-49 mouse urothelial carcinoma (an immunologically "neutral" tumor that only partially responds to PD-1 therapy) s.c. into hLRRC32KI mice. Starting four days after tumor implantation, we administered PIIO-1 or mIgG1 i.p. every three days for a total of four treatments. Mice treated with PIIO-1 showed a significant delay in tumor growth (Fig. 23A). Since mouse MB-49 does not express human GARP, this observed antitumor activity must be attributed to an increased antitumor immune response. Therefore, in a separate experiment, we treated MB-49 tumors with PIIO-1 or mIgG1 on day 6 every three days for a total of two (short-term) or six (long-term) doses. We harvested tumors 24 hours after the final treatment and isolated tumor-infiltrating lymphocytes (TILs) for analysis by high-dimensional flow cytometry. Short-term PIIO-1 increased the frequency of CD8+ T cells in the TME (Fig. 23B, left), and this effect was enhanced after long-term treatment (Fig. 23B, right). Long-term exposure to PIIO-1 also reduced both Treg frequency (Fig. 23C, left) and inhibitory function, as shown by the downregulation of CTLA4 and VISTA (Fig. 23C, right).

To examine the effects of PIIO-1 on CD8+ T cells in the TME at the single-cell level, we performed high-dimensional spectral flow cytometry using a 33-marker T cell depletion cohort. Dimensionality reduction was performed using the Unified Manifold Approximation and Projection (UMAP) method, which allowed the data to be displayed in two dimensions (Figs. 23D–23E). We then used FlowSOM for unsupervised cluster analysis to partition the data and allow for differential expression analysis between groups. This analysis identified 17 distinct clusters, one of which (cluster 14) was significantly enriched in CD8+ T cells from PIIO-1-treated mice. Cluster 14 showed elevated expression of activating markers including LAG-3, CD44, GITR, TIM-3, and PD-1 (Figs. 23D–23E), but excluding TOX (a transcription factor associated with terminal depletion). Our data support the hypothesis that PIIO-1 induces CD8+ T cell effector differentiation (Fig. 23D, orange circle population in UMAP) and blocks T cell depletion. In fact, with prolonged PIIO-1 treatment (starting on day 5, for a total of 4 doses), the terminally consumed population (cluster 9) decreased, as indicated by its high TOX state, with little or no production of effector cytokines (including IL-2, IL-21, TNFα, IFNγ, etc.; Figs. 23F–23G). In summary, our data suggest that PIIO-1 has a multifaceted role, simultaneously transforming immunologically neutral tumors into a pro-inflammatory state, increasing CD8+ T cell infiltration, and promoting activation while preventing the terminal consumption of these TILs. To support these results, we found that forced expression of hGARP in MB-49 cells (MB-49-hGARP) resulted in a higher frequency of tumor-infiltrating CD8+ T cells with a consumption phenotype compared to empty vector (EV) transfected MB-49 (cluster 9; Figs. 28A–28B).

Next, we performed spatial analysis of MB-49 tumors using multiplex IF imaging. Tumors were stained with CD45, CD8, and α-SMA and divided into internal, intermediate I, intermediate II, and external regions. Increased CD8+ T cell density in intermediate II indicated enhanced intratumoral invasion after PIIO-1 treatment (Fig. 29A). In the internal regions of mIgG1-treated tumors, α-SMA+ cell density was negatively correlated with CD8+ T cell density. PIIO-1 treatment reduced the degree of this negative correlation (Fig. 29B), suggesting that blocking the GARP-TGFβ axis reduced matrix formation and increased T cell invasion. Using spatial two-point correlation analysis, we found that CD8+ T cells more frequently co-localized in both the internal and intermediate II regions of PIIO-1-treated tumors compared to controls (Figs. 29C–29D). In conclusion, PIIO-1 treatment of MB-49 alters the intratumoral invasion dynamics of CD8+ T cells and mediates functional and spatial changes in their phenotype.

Example 16: Anti-GARP antibody enhances anti-PD-1 ICB against GARP-negative tumors.

Mechanistically, PD-1 blocks the depletion of CD8+ T cells in the tumor microenvironment (TME) by progenitor cells. These cells persistently express TCF-1 and SlamF6, as well as low levels of PD-1 and TIM-3. These cells undergo robust proliferation following anti-PD-1 treatment, leading to differentiation into effector phenotypes and inducing tumor clearance. Since PIIO-1 monotherapy significantly reduced CD8+ T cell depletion in the TME, we evaluated whether it could enhance the antitumor activity of anti-PD-1 ICBs. We treated hLRRC32KI mice with subcutaneous MB-49 tumors on day 4 sequentially with PIIO-1 (200 μg/mouse; six doses) and anti-PD-1 antibody (100 μg/mouse; four doses starting on day 10) (Fig. 24A). While PIIO-1 monotherapy moderately prolonged overall survival compared to control (Fig. 24B), the combination therapy with anti-PD-1 resulted in a complete tumor response in 60% of the mice (Fig. 24C). Finally, when we re-attacked cured mice with MB-49 cells, those mice that had previously received combination therapy had better anti-tumor memory function (Fig. 24D), indicating that PIIO-1 favorablely influences the generation of anti-tumor immune memory.

We also tested the combination therapy of PIIO-1 and anti-PD-1 in mouse Lewis lung cancer (LLC) and CMT-167 lung cancer models, both of which are immunocold tumors and resistant to anti-PD-1 ICB. Day 8 LLC tumors in hLRRC32KI mice were treated with single or combination therapy (PIIO-1 200 μg ± anti-PD-1 100 μg; four doses in total) every three days. The combination of PIIO-1 and anti-PD-1 was most effective in slowing LLC growth compared to anti-PD-1 monotherapy (Fig. 30A). These results were restated in the CMT-167 model, where the addition of PIIO-1 overcame the anti-PD-1 resistance observed in CMT-167, which was associated with increased CD8+ T cells in the TME (Figs. 30B–30C).

Example 17: Humanized PIIO-1 attenuates typical TGFβ signaling in tumor-infiltrating immune cells and promotes pro-inflammatory TME.

Next, we humanized PIIO-1 by fusing the complementarity-determining region (CDR) of the variable domain with the remainder of the chain derived from human IgG4. Humanized PIIO-1 exhibited the same affinity (Kd, 1–3 nM) as the human GARP parent antibody and similar single-agent antitumor efficacy in the MB-49 tumor model. Furthermore, treatment of MB49-carrying tumors with PIIO-1 reduced pSMAD2/3 signaling in the major tumor-infiltrating immune cell subset, which included T cells, B cells, macrophages, and dendritic cells (DCs) (Figs. 25A–25B), as well as T and B cells in the dLN (Figs. 31A–31B). Interestingly, on a per-cell basis, tumor-infiltrating CD8+ T cells exhibited the highest TGFβ signaling activity indicated by pSMAD levels (Fig. 25B). To identify the immune cell targets of PIIO-1, we injected it into tumor-bearing hLRRC32KI mice. Twenty-four hours later, tumor cells, dLNs, and spleen were harvested, and single-cell suspensions were analyzed for cell surface binding to PIIO-1. We found that PIIO-1 recognized only cells in tumor cells and dLNs, but not cells in the spleen (Fig. 31C). Treg cells were the major cell population binding PIIO-1 in dLNs (Fig. 31C). PIIO-1 preferentially targets tumor cells and dLNs, rather than the spleen, highlighting the favorable biodistribution of this antibody.

The antitumor function of cytotoxic CD8+ T cells requires lysis and the production of pro-inflammatory cytokines (e.g., TNFα and IFNγ). Furthermore, TGFβ is known to inhibit CD8+ T cell function and migration into the tumor microenvironment (TME). To further understand the mechanism of action of PIIO-1, we performed a batch transcriptomic analysis on day 10 MB-49 tumors from hLRRC32KI mice treated with PBS or PIIO-1 on days 6 and 9. mRNA expression analysis revealed increased levels of pro-inflammatory cytokines (e.g., the TNF superfamily, Il6) and chemokines (e.g., Ccl3, Ccl9, Cxcl14, Cxcl15) in PIIO-1-treated tumors (Fig. 25C), consistent with PIIO-1's ability to induce a pro-inflammatory TME. GSEA showed a similar pattern, particularly increased TNF-NFκB signaling and lymphocyte chemotaxis in PIIO-1-treated tumors (Fig. 25D). Deconvolution analysis of bulk tumor mRNA sequencing data demonstrated the enrichment of CD8+ T cells, mast cells, and activated NK cells in the tumor microenvironment (TME) after PIIO-1 administration (Figure 25E). TGFβ blocked mast cell activation by inhibiting the expression of high-affinity IgE receptor (FcεRI). In conclusion, we find that single-agent PIIO-1 treatment remodels the immunosuppressive TME and transforms it into an improved immune-adaptive state through an environment rich in pro-inflammatory cytokines and a large number of effector lymphocytes.

Example 18: Humanized PIIO-1 enhances anti-tumor immunity by promoting the recruitment of CD8+ T cells to tumors through CXCR3.

Next, we explored the role of CD8+ T cells in PIIO-1-induced protective immunity and its potential mechanisms. Depletion of CD8+ T cells completely eliminated the antitumor effect of PIIO-1 against MB-49 tumors (Figs. 26A–26B), highlighting the importance of CD8+ T cells in PIIO-1-mediated tumor control. To determine whether antitumor immunity depends on the sustained migration of activated T cells from the dLN to the tumor, we blocked T cell efflux from the dLN using the S1P receptor agonist FTY20 (Fig. 26C). We found that FTY20 eliminated the antitumor efficacy of PIIO-1 and effectively blocked T cell infiltration (Figs. 26D–26F), indicating that the expansion of CD8+ T cells pre-existing in the TME alone is unlikely to be a contributing factor to the antitumor activity of PIIO-1. Consistent with chemokine-mediated CD8+ T cell migration, we observed an enrichment of the CXCR3+CD8+ T cell population in the dLN following PIIO-1 administration (Fig. 26G), likely due to attenuated TGFβ signaling. Blocking CXCR3 during PIIO-1 treatment (Fig. 26H) completely eliminated the antitumor activity of PIIO-1 (Figs. 26I–26J), which was associated with a reduction in CD8+ T cells (but not Tregs) recruited to the TME (Figs. 26K–26L and Fig. 32). In summary, by blocking TGFβ activation within the TME, PIIO-1 partially promotes antitumor CD8+ T cell immunity by increasing CXCR3-dependent T cell transport to the tumor.

discuss

A key challenge in immuno-oncology is the primary and adaptive immune resistance to ICBs observed in most patients with cancer, including those with pancreatic cancer, ovarian cancer, and most TNBC cases. One potential mechanism of primary and acquired ICB resistance in advanced malignancies is associated with the accumulation of active TGFβ in the tumor microenvironment (TME), which drives immune dysfunction through multiple mechanisms such as inducing Tregs, excluding and inhibiting the function of effector CD8+ T cells, and restricting effector T cell migration to the TME. However, targeting TGFβ has proven difficult to treat human disease due to its highly background-dependent pleiotropic function. By using GARP-specific monoclonal antibodies that block the binding of LTGFβ to Tregs, tumor cells, and other cell types in the TME without affecting circulating platelets, we have achieved tumor-selective targeting of the GARP-TGFβ pathway and antitumor activity in various preclinical tumor models.

PIIO-1 offers advantages over other techniques attempting to deliver drugs that inhibit the TGFβ pathway. It targets only GARP-expressing cells primarily located in the TME, unlike systemic TGFβ blockers such as anti-TGFβ antibodies and small-molecule inhibitors targeting TGFβ signaling receptors. It differs from existing anti-GARP antibodies (such as ABBV-151) in several ways. First, PIIO-1 binds to ligandless GARP and blocks its binding to all LTGFβ isoforms. Second, platelets express abundant GARP-LTGFβ1 complexes due to their high levels of autocrine LTGFβ1. Antibodies targeting the GARP-LTGFβ1 complex (such as ABBV-151) carry the potential risk of platelet-related side effects; the unique epitope targeted by PIIO-1 (free GARP) eliminates this risk. Third, PIIO-1's preferential targeting of tumors and dLNs underscores its more favorable biodistribution than ABBV-151, which may be non-selectively distributed in peripheral blood, bone marrow, and spleen.

PIIO-1 monotherapy successfully modulated the tumor microenvironment (TME) by reducing active TGFβ signaling and associated matrix formation, and enhanced the accumulation of intratumoral effector CD8+ T cells. Furthermore, the combination of PIIO-1 and anti-PD-1 therapy demonstrated robust antitumor activity against GARP-positive tumors in humanized GARP knock-in mice. Mechanistic studies revealed several interesting biological insights related to the role of GARP in the TME. The increased migration of CD8+ T cells to the TME in response to PIIO-1 was likely expected, given the evidence of reduced matrix formation and thus reduced immune rejection. Increased chemokine production in the TME and the ability of TGFβ1 to suppress CXCR3 expression on CD8+ T cells may also support this migration. We confirmed that PIIO-1 promotes CXCR3+CD8+ T cells in the tumor dLN. Interestingly, we found that Treg migration to the TME does not require CXCR3. Therefore, the increased migration of CD8+ T cells from Tregs to the TME should translate into a proportional reduction in Tregs, which appears to be the case. Importantly, PIIO-1 treatment reduced CD8+ T cell depletion. Gabriel et al. recently reported using a chronic viral infection model that TGFβ1 maintains progenitor-depleted T cells by inhibiting mTOR activity, ultimately leading to a more terminally depleted CD8+ T cell state. In our study, we used single-cell high-dimensional flow cytometry to demonstrate that PIIO-1 treatment significantly blocked the formation of terminally depleted CD8+ T cells in the TME, as indicated by high TOX expression and almost no effector cytokine expression. Therefore, by blocking the production of active TGFβ within the TME and dLN, PIIO-1 enhances CD8+ T cell biology in two ways: first, it promotes the initiation and migration of antigen-specific T cells in the dLN, and second, it attenuates CD8+ T cell depletion in the TME.

Platelets are the primary source of active TGFβ through GARP-mediated maturation of latent TGFβ. Since PIIO-1 does not block the platelet GARP-LTGFβ axis, we conclude that targeting GARP in non-platelet compartments is sufficient to induce antitumor activity. Furthermore, unlike circulating platelets, extravasated tumor-infiltrating platelets may also be targets of PIIO-1; this hypothesis is being actively investigated using tissue-based spatial techniques.

In summary, we generated, humanized, and characterized a unique anti-GARP antibody that blocks the activation of all LTGFβ isoforms in the TME. Using our human LRRC32 knock-in mice and multiple preclinical tumor models, we demonstrated the potential druggability of the GARP-LTGFβ pathway for cancer immunotherapy. In doing so, we revealed several novel biological aspects of GARP, including how it promotes immune rejection, ICB resistance, CD8+ T cell depletion, and CD8+ T cell migration to the TME. Therefore, PIIO-1, as a promising immunotherapeutic agent against advanced cancers with ICB resistance, warrants further clinical development, whether as a monotherapy or in combination with ICBs.

method

Analysis of the Cancer Genome Atlas (TCGA) database

LRRC32 expression values were obtained from TCGA using RNA-seq data available in the cBioPortal database and further integrated with cancer immune landscape data using patient IDs. Independent t-tests were used to compare each parameter in the cancer immune landscape between the top 1/3 (high LRRC32) and bottom 1/3 expression groups (low LRRC32).

Generation of anti-human GARP (hGARP) antibodies

The generation of anti-hGARP antibodies has been described. BALB/c mice were immunized with recombinant human GARP (R&D Systems) in Freund's complete adjuvant and boosted 2 to 3 times with SP2/0-hGARP cells. Splenic B cells with high anti-GARP antibody titers from immunized mice were fused with SP2/0 cells in the presence of polyethylene glycol. Hybridoma selection was performed in HAT medium, and cloning was performed by limiting dilution assay.

LAP competitive binding assay

One × 10⁵ Jurkat-hGARP cells were incubated at 37°C for 30 min with 400 ng of recombinant human LTGFβ1 (R&D) and an indicated concentration of mouse IgG1 isotype control (mIgG1) or anti-GARP antibody. Cells were washed twice with PBS and flow cytometry was performed using anti-LAP antibody (eBioscience) to determine cell surface expression.

in vivo model

hLRRC32KI mice were treated intravenously (i.v.) with either mIgG1 or PIIO-1 (200 μg) three times, every other day. Indicator organs were collected on day 5. Single-cell suspensions were prepared and then stained and analyzed by flow cytometry.

TNBC model. 4T1-hGARP (1 × 10⁵ cells) was injected into the fourth mammary fat pad of 6- to 8-week-old female BALB/c mice. Antibodies were administered intraperitoneally (i.p.) on day 7 post-injection and continued every three days for a total of five injections. Key parameters measured included tumor growth, body weight, survival time to necessary euthanasia, lung metastasis, and serum TGFβ levels. To investigate the antitumor memory response, mice that completely rejected the tumor were then challenged again with 4T1-WT (5 × 10⁵ cells), and tumor growth and overall survival were closely monitored.

Bladder cancer model. MB-49 (1 × 10⁵ cells) was subcutaneously (sc) injected into the right flank of male hLRRC32KI mice. mIgG1 or PIIO-1 was administered intraperitoneally (ip) every three days on the indicated date. Indicator tissue was collected 24 hours after the last treatment. To investigate the efficacy of the combination therapy, PIIO-1 (200 μg) and anti-PD-1 antibody (100 μg) were delivered intraperitoneally every three days following MB-49 injection. PIIO-1 was initiated on day 4 for a total of 6 doses, and anti-PD-1 antibody was initiated on day 10 for a total of 4 doses. Tumors were monitored daily. Mice that completely rejected the tumor in the indicator group were then re-challenged with MB-49 (1 x ^10⁵ cells) sc. Tumor growth and overall survival were monitored.

MB-49 (1 x ^10⁵ cells) was subcutaneously injected into the right flank of male hLRRC32KI mice. Anti-CD8a antibody (200 μg, ip) was administered on days 4, 6, 8, 11, and 14. PIIO-1 (200 μg, ip) was administered on days 5, 8, 11, and 14. Tumor growth was monitored. To investigate the role of T cell migration in antitumor activity, FTY720 (2 mg/kg) was administered on day 6, every two days for a total of 6 doses. PIIO-1 (200 μg, ip) was administered on days 6, 9, 12, and 15. The experiment ended on day 17, during which tumor and other organ analyses were performed. The role of CXCR3 was also evaluated in the MB-49 model, with blocking anti-CXCR3 antibody and PIIO-1 (200 μg each, ip) administered on day 5 after MB-49 injection, every three days for a total of 4 treatments. Tumor growth was then monitored, with the final analysis performed on day 16.

Tumor size was measured by the longest width and length (in mm) and reported as tumor area (width × length). For the 4T1, LLC1, and CMT167 tumor models, treatment was initiated when the tumor area was approximately 30 mm² (≈75 mm³ tumor volume), and for the MB-49 model, treatment was initiated when the tumor area was approximately 12 to 24 mm² (≈18 to 48 mm³).

High-dimensional flow cytometry analysis and multiplex immunofluorescence (IF) microscopy

Antibody staining and high-dimensional flow cytometry (Cytek) analysis were performed. Multiplex IF was performed using Vectra Polaris. Detailed methods, including spatial analysis, are provided in the supplementary documentation.

RNA-seq alignment, preprocessing, and analysis

Sequencing was outsourced to Macrogen and performed on an Illumina Hiseq 6000. Reads were aligned to a GRCm38 reference using Hisat2 (v.2.0.5), and read counts were determined using featureCounts (v1.5.0-p3) software. Raw read counts were used for DEG analysis based on the DESeq2 package. Enrichment analysis of GO terms was performed via the R package clusterProfiler (v.3.18.0). Gene set enrichment analysis (GSEA) (v.4.0.3) was performed for enrichment analysis and visualization. Deconvolution was performed using TIMER 2.0. Detailed methodologies are provided in the supplementary documentation.

Statistical analysis

Student's t-test was performed to compare continuous variables, such as control and treatment groups, between two groups. Kaplan-Meier curves were used to visualize survival between the groups, and the log-rank test was used to quantify significance. Repeated measures ANOVA was used for tumor curve analysis. All data are presented as mean ± SEM. A p-value less than 0.05 was considered statistically significant. Multiple test correction was performed using the Turkey or Sidak procedure.

mice

Wild-type C57BL/6 (strain #00064) and BALB/c (strain #000651) mice were purchased from Jackson Laboratory (Bar Harbor, ME). hLRRC32KI mice with a C57BL/6 background were bred by Ingenious Targeting Laboratory (Ronkonkoma, NY). All in vivo experiments used age- and sex-matched mice. All experimental animals were 6 to 11 weeks old.

cell lines and mice

Jurkat, 4T1, and MB-49 cells with hGARP overexpression were used. Cancer cells were validated by gene expression analysis, in vivo growth, and histology. The MB-49 urothelial carcinoma cell line was kindly provided by Dr. Xue Li (Cedars-Sinai Medical Center, Los Angeles, CA). The 293FT and LLC1 lines were purchased from ATCC (Manassas, VA). The CMT-167 cell line was obtained from Sigma (St. Louis, MO). All cell lines were free of mycoplasma as determined by PCR. For all in vivo tumor experiments, tumor cells were used in the first four passages of the culture.

Generation of human/mouse GARP expression vectors

Human and mouse GARP were amplified by PCR and subcloned between the BglII and HpaI sites of the MigR1 retroviral vector.

For chimeric construction, we used the following primers:

20-60 Forward: GCTCTCTACTTGTCCGGGAACCAACTGCGGAGTATCCTGGCCTCACCC(SEQ ID NO:38)

20-60 reverse:GGGTGAGGCCAGGATACTCCGCAGTTGGTTCCCGGACAAGTAGAGAGC(SEQ ID NO:39)

61-100 Forward: CAGGCCCTGCCCTACCTGGAGCACCTCAGCCTGGCTCACAACCGGCTG(SEQ IDNO:40)

61-100 (reverse):

CAGCCGGTTGTGAGCCAGGCTGAGGTGCTCCAGGTAGGGCAGGGCCTG(SEQ ID NO:41)

101-140 positive:

AACAGCCTGCATGGCAATCTGGTGGAGCGGCTGCTGGGGGAGGCACCC(SEQ ID NO:42)

101-140 (reverse):

GGGTGCCTCCCCCAGCAGCCGCTCCACCAGATTGCCATGCAGGCTGTT(SEQ ID NO:43)

141-170 positive:

CGCCTGGCACGCCACAACCTTCTGGGACATGCCTGCGCTGGAGCAGCTT(SEQ ID NO:44)

141-170 (reverse direction):

AAGCTGCTCCAGCGCAGGCATGTCCCAGAAGGTGTGGCGTGCCAGGCG(SEQ ID NO:45)

171-207 Forward: ACTCACCTCAATCTCTCCAGAAACTCCCTCACCTGCATCTCCGACTTC(SEQ IDNO:46)

171-207 (reverse):

GAAGTCGGAGATGCAGGTGAGGGAGTTTCTGGAGAGATTGAGGTGAGT(SEQ ID NO:47)

208-265 forward: TTCCCTGACCTGGCCGTGTTCCCGAGACTCATCTACCTGAACTTGTCC (SEQ IDNO: 48)

208-265 (reverse):

GGACAAGTTCAGGTAGATGAGTCTCGGGAACACGGCCAGGTCAGGGAA(SEQ ID NO:49)

266-322 Forward: AATGAGATCGAACTGGTCCCTGCTAGCTTTCTTGAGCACCTGACCTCC(SEQ IDNO:50)

266-322 (reverse):

GGAGGTCAGGTGCTCAAGAAAGCTAGCAGGGACCAGTTCGATCTCATT(SEQ ID NO:51)

All constructs were subcloned into a MigR1 retroviral vector for retroviral production. The efficiency of mutagenesis was assessed by DNA sequencing. Chimeric constructs were transfected into 293FT cells, and cells with the desired construct expression levels were selected by FACS sorting.

In vivo mouse tumor model

Female hLRRC32KI mice were injected subc. into the right flank with LLC1 tumor cells (5 × 10⁵) or CMT-167 cells (1 × 10⁵). On day 8, mice were treated with PIIO-1 (200 μg), anti-PD-1 (100 μg), or a combination of both, every three days for a total of four treatments. Tumor growth was monitored, and tissue was collected on day 18. Flow cytometry analysis was performed at the end of the experiment.

MB-49-hGARP or -EV tumor cells (1 × 10⁵) were injected subcorporeally into the right flank of male C56BL/6 mice. Tumors were harvested on day 18. Single-cell suspensions were prepared, stained with appropriate antibodies, and then analyzed by flow cytometry. Tissue digestion, cell separation, and flow cytometry were performed.

Thymus, spleen, mesenteric lymph nodes (mLN), and peripheral lymph nodes (pLN) were dissociated into single-cell suspensions, and erythrocytes were removed using RBC lysis buffer (Biolegend). For tumor isolation, tissue was dissected and incubated at 37°C for 20 minutes with collagenase D (1 mg/mL; Roche), dispersin (0.05 U/mL; Worthington), and DNase I (100 mg/mL; Sigma-Aldrich). The digested tissue was then filtered through a 40-μm nylon filter (VWR). Blood cells were removed using RBC lysis buffer (Biolegend). The cell suspension was washed with PBS.

For flow cytometry staining, cells were washed twice in FACS buffer and blocked with FcR for 10 min at 4°C. Live/dead staining was performed for 10 min at 4°C using a fixable viability dye (Affymetrix) or live/dead blue (Thermofisher), followed by staining with a surface antibody mixture (described below) in FACS buffer at 4°C for 30 min. For intracellular staining, Foxp3/transcription factor staining buffer (eBioscience) was used according to the manufacturer's protocol. Cells were then incubated with antibodies in permeation buffer for 1 to 3 hours. Cells for cytokine production assessment were stimulated for 5 h at 37°C in T cell culture medium containing anti-CD3 (1 μg/ml)/CD28 (5 μg/ml) followed by FACS staining. Samples were immediately analyzed on BDFACSDiva, Fortessa, or Cytek Aurora, and data were analyzed using FlowJo (Tree Star) or OMIQ software.

For pSMAD2/3 staining, tissues were sieved in fixation buffer (Invitrogen) for 30 minutes and filtered through a 40-μm nylon filter (VWR). Cell suspensions were permeabilized in permeation buffer at room temperature (RT) for 30 minutes. Cell surface markers were stained in FACS buffer at room temperature for 1 hour. pSMAD2/3 and Foxp3 were stained in FACS buffer overnight at 4°C. Flow cytometry was performed immediately using a Cytek Aurora.

Immunophenotyping analysis group:

Anti-CD45 (clone 30-F11, Brilliant Violet 510, BioLegend), anti-CD3 (clone 17A2, BUV737, BD Biosciences), anti-CD8a (clone 53-6.7, BUV496, BD Biosciences), anti-CD4 (clone RM4-5, APC/Fire ^™ 810, BioLegend), anti-Foxp3 (clone FJK-16s, eFluor450, Invitrogen), anti-CD25 (clone PC61.5, Super Bright 600, Invitrogen), anti-CD11b (clone M1/70, AlexaFluor532, Invitrogen), anti-F4-80 (clone T45-2342, BUV395, BD Horizon), anti-CD11c (clone N418, Brilliant Violet) 750 (BioLegend), anti-MHC-II (clone M1/42, BUV615, BDBiosciences), anti-NK-1.1 (clone PK136, Brilliant Violet 570, BioLegend), anti-Ly-6C (clone HK1.4, Brilliant Violet 605, BioLegend), anti-Ly-6G (clone 1A8-Ly6g, Super Bright 436, Invitrogen), anti-CD103 (clone 2E7, Brilliant Violet 711, BioLegend), anti-PD-1 (clone J43, FITC, Invitrogen), anti-PD-L1 (clone B7-H1, Brilliant Violet) 421, BioLegend), anti-CD206 (clone MR6F3, APC-eflour780, Invitrogen), anti-CD38 (clone 90/CD38, PE/Cyanine7, BioLegend), anti-arginase 1 (clone A1exF5, Alexa Fluor 700, Invitrogen), anti-CD64 (clone X54-5/7.1, APC, BioLegend), XCR1 (clone ZET, PerCP/Cyanine5.5, BioLegend), anti-CD172 (clone P84, PE/Dazzle ^™ 594, BioLegend), anti-CD19 (clone 6D5, Spark NIR ^™ 685, BioLegend), anti-CD24 (clone M1/69, BV480, BD Biosciences);

T-cell consumption group:

Anti-CD45 (clone 30-F11, Brilliant Violet 510, BioLegend), anti-CD3 (clone 17A2, BUV737, BD Biosciences), anti-CD8a (clone 53-6.7, BUV496, BD Biosciences), anti-CD4 (clone RM4-5, APC/Fire ^™ 810, BioLegend), anti-Foxp3 (clone FJK-16s, eFluor450, Invitrogen), anti-CD25 (clone PC61.5, Super Bright 600, Invitrogen), anti-TOX (clone REA473, PE, Miltenyi Biotec), anti-CD44 (clone IM7, BUV611, Invitrogen), anti-CD62L (clone MEL-14, Brilliant Violet). Violet421 (BioLegend), anti-Slamf6 (clone 13G3-19D, APC, Invitrogen), anti-PD-1 (clone J43, APC-eflour780, Invitrogen), anti-Tim3 (clone RMT3-23, Brilliant Violet711, BioLegend), anti-Lag3 (clone C9B7W, BUV 805, BD Biosciences), anti-Klrg1 (clone 2F1, Pacific Orange, Invitrogen), anti-CD27 (clone LG.3A10, BUV563, BD Biosciences), anti-CD38 (clone 90/CD38, Brilliant Violet 750, BD Biosciences), anti-ICOS (clone 7E.17G9, Super Bright 436 (Invitrogen), anti-CD69 (clone H1.2F3, PE/Cyanine7, BioLegend), anti-TIGIT (clone 1G9, Brilliant Violet 650, BD Optibuild), anti-GITR (clone MIH44, BUV615, BD Biosciences), anti-CTLA4 (clone UC10-4B9, PE/Dazzle ^™ 594, BioLegend), anti-CD95 (clone Jo2, BV480, BD Biosciences), anti-Ki67 (clone B56, BUV395, BD Biosciences), anti-Tcf1 (clone C63D9, PE/Cyanine5, Cell Signaling Technology), anti-Bcl-2 (clone BCL/10C4, Alexa Fluor 647, BioLegend), anti-granzyme B (clone QA16A02, Alexa Fluor) 700, BioLegend), anti-T-bet (clone O4-46, Brilliant Violet 786, BD Biosciences);

Cytokine group:

Anti-CD45 (clone 30-F11, Brilliant Violet 510, BioLegend), anti-CD3 (clone 17A2, BUV737, BD Biosciences), anti-CD8a (clone 53-6.7, BUV496, BD Biosciences), anti-CD4 (clone RM4-5, APC/Fire ^™ 810, BioLegend), anti-Foxp3 (clone FJK-16s, eFluor450, Invitrogen), anti-CD11b (clone M1/70, Alexa Fluor 532, Invitrogen), anti-TOX (clone REA473, PE, Miltenyi Biotec), anti-Tcf1 (clone C63D9, PE/Cyanine7, Cell Signaling Technology), anti-TNFα (clone MP6-XT22, Percp-eflour 710, Invitrogen), anti-IFNγ (clone XMG1.2, Brilliant Violet). Violet 786 (BD Biosciences), anti-granzyme B (clone QA16A02, Alexa Fluor 700, BioLegend), anti-perforin (clone eBioOMAK-D, FITC, Invitrogen), anti-IL-2 (clone JES6-5H34, PE-eflu610, Invitrogen), anti-IL-4 (clone 11B11, BV605, BD Horizon), anti-IL-10 (clone JES5-16E3, APC, Invitrogen), anti-IL-17A (clone TC11-18H10, APC-Cy7, BD Pharmingen), anti-IL-21 (clone FFA21, eFluor660, Invitrogen);

Phosphorylation flow cytometry:

Anti-CD45 (clone 30-F11, Brilliant Violet 510, BioLegend), anti-CD3 (clone 17A2, BUV737, BD Biosciences), anti-CD8a (clone 53-6.7, BUV496, BD Biosciences), anti-CD4 (clone RM4-5, APC/Fire ^™ 810, BioLegend), anti-Foxp3 (clone FJK-16s, eFluor450, Invitrogen), anti-CD25 (clone PC61.5, Super Bright 600, Invitrogen), anti-CD11b (clone M1/70, AlexaFluor532, Invitrogen), anti-F4-80 (clone T45-2342, BUV395, BD Horizon), anti-CD11c (clone N418, Brilliant Violet) 750 (BioLegend), anti-MHC-II (clone M1/42, BUV615, BD Biosciences), anti-NK-1.1 (clone PK136, Brilliant Violet 570, BioLegend), anti-Ly-6C (clone HK1.4, Brilliant Violet 605, BioLegend), anti-Ly-6G (clone 1A8-Ly6g, Super Bright 436, Invitrogen), anti-CD206 (clone MR6F3, APC-eflour780, Invitrogen), anti-CD19 (clone 6D5, SparkNIR ^TM 685, BioLegend), anti-pSMAD2/3 (clone O72670, PE, BD Pharmaceuticals)

Multiplex immunofluorescence analysis

Samples were outsourced to Fred Hutch for IF staining, and the method was provided by Fred Hutch. Formalin-fixed paraffin-embedded tissues were cut into 4-micron sections and baked at 60°C for 1 hour. Slides were dewaxed using a dewaxing solution (Leica). Antigen retrieval (Bond Wash Solution) was applied at 100°C for 20 minutes. Endogenous peroxidase was _blocked with 3% _H₂O₂ for 5 minutes, followed by incubation of 10% normal mouse serum in TCT buffer (0.05M Tris, 0.15M NaCl, 0.25% casein, 0.1% Tween 20, pH 7.6) for 10 minutes. CD45 ICA antibody was applied for 1 hour, followed by secondary antibody staining for 10 minutes. Then, tertiary TSA amplification reagent (PerkinElmer OPAL fluor) was applied for 10 minutes. After the second and third applications, a high-roughness wash was performed using a high-salt TBST solution (0.05M Tris, 0.3M NaCl, and 0.1% Tween-20, pH 7.2–7.6). The table indicates that polymer HRP was used as the secondary antibody (Leica). See Table K below.

Table K

SMA staining was performed after a 20-minute peeling process in retrieval solution at 100°C. Prior to SMA staining, endogenous peroxidase blocking was performed using 3% H₂O₂. CD8a staining was repeated as with SMA. Finally, the slides were stained with DAPI for 5 minutes, rinsed, and covered with coverslips in Prolong Gold Antifade reagent (Invitrogen). Images were acquired using the filters and exposure times listed in Table L below on a Perkin Elmer Vectra 3.0 automated imaging system (Akoya Biosciences, Marlborough, MA).

Table L

Filter <![CDATA[Scan Exposure Time²：]]> <![CDATA[On-site exposure time²：]]> DAPI 25 200 FITC 150 250 CY3 30 70 Texas Red 25 40 CY5 150 200

In short, the slides were first scanned at 10x magnification using a long-pass filter to capture the entire tissue section. These images were annotated for regions of interest (ROIs) covering the entire tissue. Next, these ROIs were imaged using multispectral imaging settings for each biomarker. The generated .im3 multispectral images were quantized for CD45, CD8a, SMA, and DAPI. These ROIs were then imported into the inForm software for further analysis. First, the images were annotated for biomarkers and fluorophores. Autofluorescence signals and multiple fluorescence signals were separated. The images were normalized to exposure time. The inForm software allows for the development of machine learning-based tissue classification and cell segmentation. A subset of ROIs was sampled to create a training set for algorithms used in image processing, tissue segmentation, cell segmentation, and phenotypic analysis. These algorithms were applied to all ROIs in all images of the dataset for batch analysis. The resulting comprehensive data was further analyzed using the phenoptr package and R programming to identify and quantify cells for each tissue compartment (defined as tumor and stroma) and for each biomarker in the entire tissue section.

Region definition

Imaging cells were classified into stromal cells or tumor cells using a machine learning algorithm (from Akoya's notification software). Next, the largest tumor cell cluster was determined using a flood-fill algorithm. The region was discretized into a square lattice with a lattice constant of 30 μm, where a pixel was considered occupied if at least one tumor cell was present. Occupied pixels were connected to form clusters by linking the front faces of their shared nearest neighbors. The convex hull of the largest tumor cell cluster was calculated to define the boundary between the tumor mass and the outer stromal region. The centroid of the tumor was calculated by taking the average position of all tumor cells in the largest tumor cell cluster. Then, the tumor region was divided into three regions (middle I, middle II, and interior) based on their proximity to the centroid, using the boundary between the tumor mass and the stromal region and the centroid of the largest tumor cell cluster (Fig. 30A). If {xi(b),yi(b)} represents the position of a tumor cell within the boundary with the centroid as the origin, the boundary of the region within the tumor mass is given by {αxi(b),αyi(b)}, where α < 1. The α values corresponding to the boundaries are shown in Fig. 30A. The spatial distribution of CD8+ T cells and other cells in these regions was analyzed to assess changes in these cellular tissues based on their proximity to the center of the tumor region.

density

The density (σ) of a specific cell type (e.g., CD8+ T cells) in a region is calculated as the ratio of the total number of cells ( _NTot ) to the area (A) of that region. This is achieved by dividing the region (e.g., intermediate II) into a square lattice with a lattice constant of a = 30 μm and then calculating the area of the filled portion of the lattice.

Two-point correlation

We calculate the spatial two-point correlation of CD8+ T cells in a region (e.g., intermediate II) as follows (see page 34 for more information on two-point correlation). For any CD8+ T cell in the region (indexed by i), we plot an annular region of radius r and thickness δ (＝3 μm), with the CD8+ T cell at the center, and calculate the density of other CD8+ T cells in this annular region (Fig. 30B). Let _{ni, (r-δ/2, r+δ/2)} be defined as the number of CD8+ T cells in the ring, and the ring be defined as the area of the annular region. The density σi(r) of CD8+ T cells in the annular region surrounding the i-th CD8+ T cell is given by the following formula:

Where _{A_ring} = πrδ. The total number of CD8 ⁺ T cells in the region (N_CD8 ₊ ) and the density of CD8 ⁺ T cells (σ_CD8 ₊ = _{N_CD8+} / (region area)) are also calculated. Then, the pairwise correlation function is given by the following equation:

The calculation is performed over multiple radii r, and the resulting function is plotted as a function of r.

Batch RNA-seq analysis

Public data access and analysis.

Batch RNA-seq data for bladder cancer were downloaded to support survival analysis and LRRC32 gene expression analysis. 167 bladder tumor samples were selected based on the "Best Confirmed Overall Response" annotation, including 15 CR (complete response), PR (partial response), SD (stable disease), and PD (progressive disease). LRRC32-TGFB-related features included: LRRC32, ITGB6, ITGB8, ITGAV, ITGA2B, SELP, F2, and TGFB1 genes. DESeq 2 (v.1.30) normalization was applied prior to survival analysis and GARP gene expression analysis. Survival analysis was performed using the survival package (v 3.1).

Sample and library preparation

¹ x 10⁵ MB-49 cells were subcutaneously injected into the right flank of male hLRRC32KI mice. PIIO-1 (200 μg/mouse, ip) was administered twice, on days 6 and 9. Tumors were collected on day 10. Single-cell suspensions were prepared and RNA was isolated. Total RNA was isolated using the RNeasy kit (Qiagen) and then subjected to batch RNA sequencing. RNA quality was validated using an Agilent Bioanalyzer. Libraries were prepared using the NEBNext Ultra TM RNA Library Preparation Kit from Illumina (NEB, USA) as recommended by the manufacturer.

Comparison and quantification

Sequencing was outsourced to Macrogen and performed on an Illumina Hiseq 6000 with the following requirements: 150 pb reads, paired-end reads, and 300 M reads per sample. Reads containing adaptors were removed if N was greater than 10% (N represents unidentified bases), or identified as low-quality reads with a Q score (quality value) less than 5. The filtered reads were then aligned to the GRCm38 mouse genome using Hisat2 (v.2.0.5) with default settings, and read counts were determined using featureCounts (v1.5.0-p3) software. The raw read counts were normalized using the DESeq2 package with default settings.

Path enrichment analysis and deconvolution analysis

DEG is selected if the p-value is less than 0.001 and the absolute value of the logarithmic fold change is greater than 0.5. Based on the identified DEG, enrichment analysis of GO terms (biological processes, cellular components, and molecular functions) is performed via the R package clusterProfiler (v.3.18.0). GSEA (v.4.0.3) is also used for enrichment analysis and visualization.⁷ Deconvolution is performed using TIMER 2.0 following its tutorial.⁸

Immunohistochemistry (IHC)

Mouse tumor slides were processed and antigens were repaired. For mouse IHC, tissues were collected and fixed overnight in 4% paraformaldehyde, then incubated overnight in 70% ethanol, paraffin-embedded, and then cut for hematoxylin and eosin (H&E) staining. For pSMAD2/3 or α-SMA on paraffin-embedded tumor sections, 4 μm sections were incubated with 3% H2O2. To minimize nonspecific staining, sections were incubated with appropriate animal serum at room temperature for 20 minutes, then incubated overnight at 4°C with primary antibody against pSMAD2/3 (Abcam) or α-SMA (Abcam). Staining was then performed with secondary antibody (Vectastain ABC kit), followed by development with DAB substrate (Vector Labs SK-4100). The staining intensity grading of pSMAD2/3 or α-SMA is as follows, where the sample identity is unknown (0: negative; 1: weak; 2: moderate; 3: strong, but not as strong as 4; 4: strong).

Soluble TGFβ1 ELISA

Mouse blood was collected in Eppendorf tubes. Serum was collected after coagulation at room temperature for 1 hour and centrifugation at 5,000 rpm for 15 minutes. A capture ELISA for TGFβ1 was performed according to the manufacturer's instructions (BioLegend). Active TGFβ1 was measured without additional steps. Total TGFβ1 was measured after activation with 1M HCl for 10 minutes at room temperature and neutralization with 1.2N NaOH. Active and total TGFβ1 levels were measured using the TGFβ1 ELISA kit according to the manufacturer's protocol.

Combined measurement

Collect ¹ x 10⁵ Jurkat-hGARP cells and wash twice with PBS. Stain cells with live dead blue (1:1000, catalog L23105, Invitrogen) at 4°C for 15 min. Wash cells twice with FACS buffer and incubate with an indicated concentration (20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0 μg/ml) of isotype control or PIIO-1 in FACS buffer at 4°C for 30 min. Then wash twice with FACS buffer and further stain with anti-mouse Ig-PE or anti-human Fc-PE in FACS buffer at 4°C for 30 min. Surface GARP staining will be performed for flow cytometry.

Group

1. Mouse IgG1 isotype control (BioXcell)

2. Mouse PIIO-1 (hybridoma, BioXcell)

3. Mouse antibodies

4. PBS control of humanized antibodies

5. Humanized PIIO-1 (IgG4, Thermofisher)

6. Humanized PIIO-1 (IgG1, Ab studio)

7. Mouse anti-GARP antibody (Plato-1, Enzo)

Readings: Genomic mean fluorescence intensity of GARP at different antibody concentrations.

Competition Measurement

^Five Jurkat-hGARP cells (1 x 10⁵) were incubated at 37°C for 30 min with 400 ng of recombinant human LTGFβ1 (R&D) and isotype controls or PIIO-1 at indicated concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0 μg/ml). Cells were washed twice with PBS and then further analyzed by flow cytometry to determine LAP (eBioscience) expression on the cell surface.

Group:

1. Mouse IgG1 isotype control (BioXcell)

2. Mouse PIIO-1 (hybridoma, BioXcell)

3. Mouse antibodies

4. PBS control of humanized antibodies

5. Humanized PIIO-1 (IgG4, Thermofisher)

6. Humanized PIIO-1 (IgG1, Ab studio)

Readings: Genomic mean fluorescence intensity of LAP at different antibody concentrations.

Example 19: Combined data showing that PIIO-1 is superior to 4D3

We generated several antibodies against human GARP, including 4D3, humanized PIIO-1 IgG1, and humanized PIIO-1 IgG4. Recombinant humanized PIIO-1 IgG4 was prepared in CHO cells by Thermo Fisher, and humanized PIIO-1 IgG1 was generated by Ab Studio. We also performed several experiments using Plato-1 (a commercially available anti-GARP antibody (Enzo)). We conducted experiments to examine their ability to bind to GARP and their properties in inhibiting the interaction between GARP and latent extracellular TGFβ.

To determine whether they could recognize GARP on the cell surface, we utilized Jurkat cells overexpressing human GARP. Briefly, 1 × ^10⁵ Jurkat-hGARP cells (GARP-overexpressing Jurkat cells) were incubated at 4°C for 30 min with indicated concentrations (312.5, 156.25, 78, 39, 20, 9.7, 0 ng/ml) of anti-GARP antibody. They were then incubated with anti-mouse Ig-PE or anti-human Fc-PE secondary antibodies. GARP expression levels were assessed by flow cytometry, with the results quantified by the geometric mean of fluorescence intensity (gMFI). We found that all anti-GARP antibodies, except for the allotype control antibody (ISO), recognized GARP in a dose-dependent manner. However, 4D3 showed lower binding efficiency to GARP than PIIO-1 (Figure 33A). To determine whether these antibodies could block the binding between GARP and latent TGFβ1 (LTGFβ1), ¹ x 10⁵ Jurkat-hGARP cells were incubated with 400 ng of recombinant human LTGFβ1 (R&D) at 37°C for 30 min in the presence of indicated concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0 μg/ml) of either an isotype control or anti-GARP antibody. The cells were then thoroughly washed twice with PBS to remove free, unbound LTGFβ1. Cell surface LTGFβ1 was then detected by anti-LTGFβ1 antibody (eBioscience), followed by flow cytometry analysis and quantification. Using this competitive binding assay, we found that PIIO-1 blocked all LTGFβ1 binding to GARP; however, 4D3 or Plato-1 failed to block the binding between GARP and LTGFβ1. Importantly, we found that the competition between PIIO-1 and LTGFβ1 for GARP binding was dose-dependent (Figure 33B).

In summary, these experiments demonstrate that the original PIIO-1, humanized PIIO-1 IgG1, and humanized PIIO-1 IgG1 can effectively interact with GARP, thereby robustly blocking the binding between GARP and LTGFβ1. 4D3 has the ability to recognize GARP, but it cannot inhibit the interaction between GARP and LTGFβ1 as effectively as PIIO-1.

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