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Chimeric antigen receptor T (CAR-T) cell immunotherapy for sarcomas: From mechanisms to potential clinical applications

  • Pichaya Thanindratarn
    Affiliations
    Department of Orthopedic Surgery, Sarcoma Biology Laboratory, David Geffen School of Medicine, University of California, Los Angeles, CA, USA

    Department of Orthopedic Surgery, Chulabhorn Hospital, HRH Princess Chulabhorn College of Medical Science, Chulabhorn Royal Academy, Bangkok, Thailand
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  • Dylan C. Dean
    Affiliations
    Department of Orthopedic Surgery, Sarcoma Biology Laboratory, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
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  • Scott D. Nelson
    Affiliations
    Department of Pathology, University of California, Los Angeles, CA, USA
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  • Francis J. Hornicek
    Affiliations
    Department of Orthopedic Surgery, Sarcoma Biology Laboratory, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
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  • Zhenfeng Duan
    Correspondence
    Corresponding author at: Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, 615 Charles E. Young. Dr. South, Los Angeles, CA 90095, USA.
    Affiliations
    Department of Orthopedic Surgery, Sarcoma Biology Laboratory, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
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Published:November 25, 2019DOI:https://doi.org/10.1016/j.ctrv.2019.101934

      Highlights

      • Survival rates for sarcoma patients have plateaued in the past few decades and remain especially grim for those with recurrent or metastatic disease.
      • Given the limitations of current medical therapies, novel treatment strategies are urgently needed.
      • The Chimeric Antigen Receptor (CAR) T Cell therapy, has shown promising results in treating B-cell lymphoma and acute lymphoblastic leukemia.
      • Several sarcoma-associated antigens (SAA) amenable to CAR-T cell treatment have recently emerged with promising results and garnered interest, especially as new clinical trials and treatment strategies.

      Abstract

      Survival rates for sarcoma patients have plateaued in the past few decades and remain especially grim for those with recurrent or metastatic disease. This has prompted investigation into novel immunotherapies for sarcomas, especially after their recent and well-recognized successes in other cancers. One such modality, the Chimeric Antigen Receptor (CAR) T Cell therapy, has shown promising results in treating B-cell lymphoma and acute lymphoblastic leukemia. This novel therapy functions by fusing a specific antibody derived single-chain variable fragment (scFv) with a T-cell which recognizes a specific tumor-associated antigen (TAA). Several sarcoma-associated antigens (SAA) amenable to CAR-T cell treatment have recently emerged with encouraging results. These include human epidermal growth factor receptor 2 (HER2), disialoganglioside (GD2), interleukin 11 Receptor Subunit Alpha (IL-11RA), fibroblast activation protein (FAP), B7-H3, CD44v6, insulin-like growth factor 1 receptor (IGF-1R), and tyrosine kinase orphan-like receptor 1 (ROR1). Given the limitations of current medical therapies, novel treatment strategies are urgently needed. As a sarcoma treatment modality, CAR-T cell therapy is highly promising and continues to draw interest especially as new clinical trials emerge. Here we review recent breakthrough CAR-T cell studies in sarcoma, the targets which define them, and approaches to minimizing host cytotoxicity.

      Keywords

      Introduction

      Sarcomas are a group of mesenchyme-derived solid tumors with more than 50 histologic classes. They are broadly divided into bone or soft tissue subtypes. Approximately 16,250 patients are expected to receive a sarcoma diagnosis in the United States for 2019, with 3500 having the bone subtype and 12,750 having the soft tissue subtype [
      • Siegel Rebecca L.
      • Miller Kimberly D.
      • Jemal Ahmedin
      Cancer statistics, 2019.
      ]. Total deaths are estimated at 6930 annually, at a rate of 0.4 and 1.3 deaths per 100,000 people in bone and soft tissue sarcomas, respectively [
      • Siegel Rebecca L.
      • Miller Kimberly D.
      • Jemal Ahmedin
      Cancer statistics, 2019.
      ,

      Noone AM HN, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2015. National Cancer Institute Bethesda, MD, https://seercancergov/csr/1975_2015/. 2018.

      ]. The conventional treatment strategy for localized sarcoma is complete surgical resection with adjuvant or neoadjuvant radiation [
      • Siegel Rebecca L.
      • Miller Kimberly D.
      • Jemal Ahmedin
      Cancer statistics, 2019.
      ]. Perioperative chemotherapy has been increasingly demonstrated to improve overall survival and disease-free survival in bone and high-risk soft tissue sarcoma treatment [
      • Pasquali S.
      • Pizzamiglio S.
      • Touati N.
      • Litiere S.
      • Marreaud S.
      • Kasper B.
      • et al.
      The impact of chemotherapy on survival of patients with extremity and trunk wall soft tissue sarcoma: revisiting the results of the EORTC-STBSG 62931 randomised trial.
      ,
      • Issels R.D.
      • Lindner L.H.
      • Verweij J.
      • Wessalowski R.
      • Reichardt P.
      • Wust P.
      • et al.
      Effect of neoadjuvant chemotherapy plus regional hyperthermia on long-term outcomes among patients with localized high-risk soft tissue sarcoma: the EORTC 62961-ESHO 95 randomized clinical trial.
      ,
      • Gronchi A.
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      • Quagliuolo V.
      • Broto J.M.
      • Pousa A.L.
      • Grignani G.
      • et al.
      Histotype-tailored neoadjuvant chemotherapy versus standard chemotherapy in patients with high-risk soft-tissue sarcomas (ISG-STS 1001): an international, open-label, randomised, controlled, phase 3, multicentre trial.
      ,
      • Casali P.G.
      • Bielack S.
      • Abecassis N.
      • Aro H.T.
      • Bauer S.
      • Biagini R.
      • et al.
      Bone sarcomas: ESMO-PaedCan-EURACAN clinical practice guidelines for diagnosis, treatment and follow-up.
      ]. However, the prognosis for sarcoma patients has not improved significantly over the past few decades. According to data from the Surveillance, Epidemiology, and End Results (SEER) Program from 2008 to 2014, the five-year overall survival for patients with bone or soft tissue sarcoma are 66.9% and 64.9%, respectively [

      Noone AM HN, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2015. National Cancer Institute Bethesda, MD, https://seercancergov/csr/1975_2015/. 2018.

      ]. Additionally, the sarcoma recurrence rate remains high at approximately 35%. Sarcoma patients with metastasis have even worse outcomes, with a 10 – 25% five-year overall survival rate and a median overall survival of 1.5 – 2 years [
      • Siegel Rebecca L.
      • Miller Kimberly D.
      • Jemal Ahmedin
      Cancer statistics, 2019.
      ,

      Noone AM HN, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2015. National Cancer Institute Bethesda, MD, https://seercancergov/csr/1975_2015/. 2018.

      ]. Despite increasing efforts on targeted therapies, including tyrosine kinase inhibitors (e.g., pazopanib, sorafenib, regorafenib) for soft tissue and bone sarcomas, clinical results have not significantly improved in these patients [
      • Miwa S.
      • Yamamoto N.
      • Hayashi K.
      • Takeuchi A.
      • Igarashi K.
      • Tsuchiya H.
      Therapeutic targets for bone and soft-tissue sarcomas.
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      • Sleijfer S.
      • Ray-Coquard I.
      • Papai Z.
      • Le Cesne A.
      • Scurr M.
      • Schoffski P.
      • et al.
      Pazopanib, a multikinase angiogenesis inhibitor, in patients with relapsed or refractory advanced soft tissue sarcoma: a phase II study from the European organisation for research and treatment of cancer-soft tissue and bone sarcoma group (EORTC study 62043).
      ,
      • van der Graaf W.T.
      • Blay J.Y.
      • Chawla S.P.
      • Kim D.W.
      • Bui-Nguyen B.
      • Casali P.G.
      • et al.
      Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a randomised, double-blind, placebo-controlled phase 3 trial.
      ,
      • Grignani G.
      • Palmerini E.
      • Dileo P.
      • Asaftei S.D.
      • D'Ambrosio L.
      • Pignochino Y.
      • et al.
      A phase II trial of sorafenib in relapsed and unresectable high-grade osteosarcoma after failure of standard multimodal therapy: an Italian Sarcoma Group study.
      ,
      • Santoro A.
      • Comandone A.
      • Basso U.
      • Soto Parra H.
      • De Sanctis R.
      • Stroppa E.
      • et al.
      Phase II prospective study with sorafenib in advanced soft tissue sarcomas after anthracycline-based therapy.
      ,
      • Duffaud F.
      • Mir O.
      • Boudou-Rouquette P.
      • Piperno-Neumann S.
      • Penel N.
      • Bompas E.
      • et al.
      Efficacy and safety of regorafenib in adult patients with metastatic osteosarcoma: a non-comparative, randomised, double-blind, placebo-controlled, phase 2 study.
      ]. Given the limitations of current medical therapies, novel treatment strategies are urgently needed to improve sarcoma patient outcomes.
      Cancer immunotherapy is rapidly expanding and divided into three main categories: tumor vaccinations, adoptive T cell transfer (ACT), and immune checkpoint blockade. Recently, advanced ex vivo cellular engineering has shown ACT to have durable clinical responses in various cancers. In brief, the ACT method modifies patient-specific T cells for enhanced targeting of tumor-specific antigen. The three ACT types used for cancer immunotherapy include tumor-infiltrating lymphocytes (TILs), T cell receptor (TCR) T cells, and CAR-T cells [
      • June C.H.
      • O'Connor R.S.
      • Kawalekar O.U.
      • Ghassemi S.
      • Milone M.C.
      CAR T cell immunotherapy for human cancer.
      ]. Of note, CAR-T cells have shown great success in treating CD19 + B-cell lymphoma and acute lymphoblastic leukemia, leading to their recent approval by the FDA as a new cancer treatment modality. Subsequent interest has been generated for CAR-T cell-based technology for sarcoma therapy [
      • June C.H.
      • O'Connor R.S.
      • Kawalekar O.U.
      • Ghassemi S.
      • Milone M.C.
      CAR T cell immunotherapy for human cancer.
      ]. Here we review the basis of CAR-T cell immunotherapy and evidence for clinical application in sarcoma treatment.

      Mechanism of CAR-T cell therapy

      CAR T cell therapy relies on the manipulation of autologous T cells ex vivo. In short, a specific antibody derived single-chain variable fragment (scFv), acting as the specific receptor, is fused with a T-cell signaling domain. When reintroduced in vivo, the T-cell can be activated after binding of the specifically engineered antigen binding site without the need of a major histocompatibility complex (MHC). The current CARs are categorized into three generations according to their signaling compartments and have three major components [
      • June C.H.
      • O'Connor R.S.
      • Kawalekar O.U.
      • Ghassemi S.
      • Milone M.C.
      CAR T cell immunotherapy for human cancer.
      ]. First, the extracellular antigen identification component (scFv) recognizes tumor-associated antigen (TAA). Second, the transmembrane component (CD3, CD8, CD28, or FcεRI) fixes scFv to the surface of T cells and transduces the signal. The final component is the intracellular signal transduction segment (CD8, CD28, or CD137 intracellular area and CD3ζ), which contains the immune-receptor tyrosine-based activation motif (ITAM) (Fig. 1). While the first-generation CARs only contain an intracellular CD3ζ domain, the second- and third-generation CARs have additional co-stimulatory domains [
      • June C.H.
      • O'Connor R.S.
      • Kawalekar O.U.
      • Ghassemi S.
      • Milone M.C.
      CAR T cell immunotherapy for human cancer.
      ] (Fig. 1). Various co-stimulatory molecules such as CD28, 4-1BB (CD137), and OX40 have been used to enhance CAR-T cell proliferation and survival. Recently, newly designed so-called “fourth-generation CAR” constructs have been equipped with nuclear factor of activated T cell-responsive expression elements, allowing for inducible transgenic products (Fig. 1). Currently available expressible components of these fourth-generation CARs include pro-inflammatory cytokines such as IL-12, IL-8, IL-9, IL-15, and IL-18. These T cells redirected for universal cytokine killing (TRUCK engineered T cells) are advantageous in that they can release tailored cytokines upon CAR-T cell activation. When a cytokine such as IL-12 is released, it increases CAR-T cell activation. Additionally, TRUCKs activate innate immune cells such as NK cells and macrophages, which in turn support the antitumor response by destroying the antigen-negative cancer cells which CAR-T cells are unable to recognize as they lack a CAR-recognized target [
      • Zhang L.
      • Kerkar S.P.
      • Yu Z.
      • Zheng Z.
      • Yang S.
      • Restifo N.P.
      • et al.
      Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment.
      ,
      • Morgan M.A.
      • Schambach A.
      Engineering CAR-T cells for improved function against solid tumors.
      ]. Another advantage of fourth-generation CARs is that the cellular and potentially toxic cytokine milieu is more precisely controlled by the CAR-T, as there is an additional level of control within their highly engineered transcription mechanism.
      Figure thumbnail gr1
      Fig. 1Structural features of chimeric antigen receptors. An antigen-binding domain typically consists of variable heavy (VH) and light (VL) chains from a monoclonal antibody assembled through a linker sequence to form a single chain variable fragment (scFv). (A) In first-generation CAR, the scFv is coupled via a hinge and transmembrane domain to an intracellular T cell signaling domain, typically the CD3ζ of the T cell receptor. In the second and third generation CARs, the structure contains an additional one or two co-stimulatory domains, respectively. (B) the novel fourth generation CAR is equipped with a nuclear factor of activated T cell responsive expression element, for an inducible transgenic product such as interleukin-12. (C) A dual-targeting CAR with two CARs expressed by the same T cell, each targeting an independent antigen. T cell signaling domains are split between the two CARs to enable optimal T cell activation only upon simultaneous engagement with both antigens.
      The differences between TCR-T cell therapy and CAR-T cell therapy are summarized in Table 1 [
      • June C.H.
      • O'Connor R.S.
      • Kawalekar O.U.
      • Ghassemi S.
      • Milone M.C.
      CAR T cell immunotherapy for human cancer.
      ]. Briefly, TCRs consist of α and β-subunits associated with a surface CD3 signaling complex, whereas CARs contain scFv derived from the variable domains of tumor targeting antibodies and a CD3ζ chain as well as other less specific costimulatory elements [
      • June C.H.
      • O'Connor R.S.
      • Kawalekar O.U.
      • Ghassemi S.
      • Milone M.C.
      CAR T cell immunotherapy for human cancer.
      ] (Fig. 1). Hence, TCR T cells require the TCR to interact with TAA bound to MHC on the tumor cells or antigen-presenting cells (APCs), whereas CAR-T cell activation is MHC independent. Although the TCR-MHC interaction is normally important for reducing host autoimmunity, it is also a barrier for designing precision immunotherapy. Moreover, in the case of relatively low affinity TCR binding, there is potential for cross-reactivity and off-target neurotoxicity and cardiotoxicity [
      • June C.H.
      • O'Connor R.S.
      • Kawalekar O.U.
      • Ghassemi S.
      • Milone M.C.
      CAR T cell immunotherapy for human cancer.
      ,
      • Garrido F.
      • Aptsiauri N.
      • Doorduijn E.M.
      • Garcia Lora A.M.
      • van Hall T.
      The urgent need to recover MHC class I in cancers for effective immunotherapy.
      ]. In contrast, the absence of MHC restriction in CAR-T cell therapy offers several advantages. It circumvents immune-evasion if HLA expression is modified while also maintaining its TCR binding affinity and antigenic intracellular processing [
      • June C.H.
      • O'Connor R.S.
      • Kawalekar O.U.
      • Ghassemi S.
      • Milone M.C.
      CAR T cell immunotherapy for human cancer.
      ,
      • Figueroa J.A.
      • Reidy A.
      • Mirandola L.
      • Trotter K.
      • Suvorava N.
      • Figueroa A.
      • et al.
      Chimeric antigen receptor engineering: a right step in the evolution of adoptive cellular immunotherapy.
      ].
      Table 1Comparison between CAR- and TCR-T cell therapies
      • June C.H.
      • O'Connor R.S.
      • Kawalekar O.U.
      • Ghassemi S.
      • Milone M.C.
      CAR T cell immunotherapy for human cancer.
      .
      CAR-T cells therapyTCR-T cells therapy
      T-cell signaling amplification from synthetic biologyT-cell signaling amplification derived by evolution of the TCR
      Avidity-controllableLow-avidity, unless engineered
      CAR targets tumor surface structure such as proteins, glycansTCR targets intracellular proteome of the tumor cell
      MHC-independent recognition of tumor targetsMHC class I expression and HLA matching requisition
      Cytokine release syndrome, which could be more severe than with TCR T cell therapyOff-tumor target toxicity, which is difficult to predict
      Abbreviations: chimeric antigen receptor (CAR), human lymphocyte antigen (HLA), major histocompatibility complex (MHC), T cell receptor (TCR).
      The general workflow for CAR-T cell therapy involves collecting T cells from the patient with subsequent ex vivo activation. The CAR molecules are then introduced to the T cells by retroviral or lentiviral transduction. Those CAR-T cells with preferred expression patterns are then selected and expanded, and eventually infused back into the patient (Fig. 2). Recent works have identified several novel candidate TAAs amenable to CAR-T cell therapy in various sarcomas with promising results at the pre-clinical and clinical level.
      Figure thumbnail gr2
      Fig. 2Workflow of chimeric antigen receptor (CAR) T cell production. T cells from peripheral blood are collected via leukapheresis, followed by apheresis. The T cells are then transduced by viral (retroviral or lentiviral) or non-viral vector loading genes of CAR inserted artificially. Afterward, the modified CAR-T cells are expanded and purified. Eventually, cell quality and sterility will be examined before the cell products are infused into patients.

      Application of CAR-T cells in sarcomas

      Human epidermal growth factor receptor 2 (HER2)

      HER2 (also known as Erb-b2 receptor tyrosine kinase) is a member of the human epidermal growth factor receptor (HER) family, which includes EGFR (HER1), ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4). A major HER ligand is epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), which promotes downstream signaling of pathways such as Ras/Raf/MEK/ERK1/2 as well as the phospholipase pathway [
      • Roskoski Jr., R.
      The ErbB/HER family of protein-tyrosine kinases and cancer.
      ]. HER2 can exhibit both ligand-dependent and ligand-independent activity [
      • Roskoski Jr., R.
      The ErbB/HER family of protein-tyrosine kinases and cancer.
      ]. In breast cancer, HER2 is routinely tested for as it informs prognosis, metastasis, and treatment [
      • Sergina N.V.
      • Moasser M.M.
      The HER family and cancer: emerging molecular mechanisms and therapeutic targets.
      ]. Aside from acting as a biomarker, HER2 induces tumor cell proliferation, angiogenesis, migration, and survival. In an effort to mitigate its role in oncogenesis, targeted HER2 monoclonal antibodies such as trastuzumab have been developed and significantly improved outcomes in HER2-positive breast cancer patients. HER2 overexpression has also been reported in other solid tumors such as medulloblastoma, gastric, endometrial, esophageal, and lung cancers [
      • Iqbal N.
      • Iqbal N.
      Human epidermal growth factor receptor 2 (HER2) in cancers: overexpression and therapeutic implications.
      ]. Importantly, overexpression or amplification of the HER2 gene has also been observed in sarcomas.
      HER2 expression correlates with clinical features in various sarcomas including osteosarcoma, synovial sarcoma, and Ewing’s sarcoma [
      • Morris C.D.
      • Gorlick R.
      • Huvos G.
      • Heller G.
      • Meyers P.A.
      • Healey J.H.
      Human epidermal growth factor receptor 2 as a prognostic indicator in osteogenic sarcoma.
      ,
      • Ahmed N.
      • Brawley V.S.
      • Hegde M.
      • Robertson C.
      • Ghazi A.
      • Gerken C.
      • et al.
      Human epidermal growth factor receptor 2 (HER2) -specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma.
      ,
      • Tabak S.A.
      • Khalifa S.E.
      • Fathy Y.
      HER-2 immunohistochemical expression in bone sarcomas: a new hope for osteosarcoma patients.
      ,
      • Mardanpour K.
      • Rahbar M.
      • Mardanpour S.
      Coexistence of HER2, Ki67, and p53 in osteosarcoma: a strong prognostic factor.
      ,
      • Olsen R.J.
      • Lydiatt W.M.
      • Koepsell S.A.
      • Lydiatt D.
      • Johansson S.L.
      • Naumann S.
      • et al.
      C-erb-B2 (HER2/neu) expression in synovial sarcoma of the head and neck.
      ,
      • Scotlandi K.
      • Manara M.C.
      • Hattinger C.M.
      • Benini S.
      • Perdichizzi S.
      • Pasello M.
      • et al.
      Prognostic and therapeutic relevance of HER2 expression in osteosarcoma and Ewing's sarcoma.
      ]. In one such example, expression of HER2 correlates with worse histologic response and survival for osteosarcoma patients [
      • Gorlick R.
      • Huvos A.G.
      • Heller G.
      • Aledo A.
      • Beardsley G.P.
      • Healey J.H.
      • et al.
      Expression of HER2/erbB-2 correlates with survival in osteosarcoma.
      ,
      • Abdou A.G.
      • Kandil M.
      • Asaad N.Y.
      • Dawoud M.M.
      • Shahin A.A.
      • Abd Eldayem A.F.
      The prognostic role of Ezrin and HER2/neu expression in osteosarcoma.
      ]. Although the anti-HER2 antibody (trastuzumab) was found to be ineffective in osteosarcoma treatment [
      • Ebb D.
      • Meyers P.
      • Grier H.
      • Bernstein M.
      • Gorlick R.
      • Lipshultz S.E.
      • et al.
      Phase II trial of trastuzumab in combination with cytotoxic chemotherapy for treatment of metastatic osteosarcoma with human epidermal growth factor receptor 2 overexpression: a report from the children's oncology group.
      ], there are opportunities within the realm of immunotherapy. For example, in trastuzumab-resistant medulloblastoma, HER2 CAR-T cells were able to recognize and kill low-HER2 cells [
      • Ahmed N.
      • Ratnayake M.
      • Savoldo B.
      • Perlaky L.
      • Dotti G.
      • Wels W.S.
      • et al.
      Regression of experimental medulloblastoma following transfer of HER2-specific T cells.
      ]. It is therefore an avenue that warrants further exploration in sarcoma therapy.
      HER2 CAR-T cells have induced in vitro therapeutic responses in osteosarcoma cell lines through IFN-γ and IL-2 [
      • Ahmed N.
      • Salsman V.S.
      • Yvon E.
      • Louis C.U.
      • Perlaky L.
      • Wels W.S.
      • et al.
      Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression.
      ]. In a xenograft model, HER2 CAR-T cell treatment caused tumor regression of both localized and metastatic lesions, and even prolonged survival in mice inoculated with the low-HER2 expression osteosarcoma cell line [
      • Ahmed N.
      • Salsman V.S.
      • Yvon E.
      • Louis C.U.
      • Perlaky L.
      • Wels W.S.
      • et al.
      Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression.
      ]. Another study showed HER2 CAR-T cells to reduce in vitro sarcosphere forming capacity of two osteosarcoma cell lines [
      • Rainusso N.
      • Brawley V.S.
      • Ghazi A.
      • Hicks M.J.
      • Gottschalk S.
      • Rosen J.M.
      • et al.
      Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma.
      ].
      At the clinical level, a phase I/II trial used HER2 CAR-T cells to treat refractory or metastatic HER2-positive osteosarcoma, Ewing’s sarcoma, primitive neuroectodermal tumor (PNET), and desmoplastic small round cell tumor (DSRCT) [
      • Ahmed N.
      • Brawley V.S.
      • Hegde M.
      • Robertson C.
      • Ghazi A.
      • Gerken C.
      • et al.
      Human epidermal growth factor receptor 2 (HER2) -specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma.
      ]. The T cells were transduced with retroviral particles encoding HER2-CD28-CD3ζ to create second-generation CAR-T cells. Aside from one patient experiencing fever after a high dose infusion which later resolved with ibuprofen, no serious adverse events were observed [
      • Ahmed N.
      • Brawley V.S.
      • Hegde M.
      • Robertson C.
      • Ghazi A.
      • Gerken C.
      • et al.
      Human epidermal growth factor receptor 2 (HER2) -specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma.
      ]. HER2 CAR-T cells were detectable in vivo three hours after infusion within the peripheral blood by quantitative polymerase chain reaction (qPCR) analysis [
      • Ahmed N.
      • Brawley V.S.
      • Hegde M.
      • Robertson C.
      • Ghazi A.
      • Gerken C.
      • et al.
      Human epidermal growth factor receptor 2 (HER2) -specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma.
      ]. In terms of clinical response, 4 of 17 patients achieved stable disease for 12 weeks to 14 months [
      • Ahmed N.
      • Brawley V.S.
      • Hegde M.
      • Robertson C.
      • Ghazi A.
      • Gerken C.
      • et al.
      Human epidermal growth factor receptor 2 (HER2) -specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma.
      ]. Three of these patients had their residual tumor removed without additional therapy, with one specimen achieving ≥ 90% tumor necrosis. Each of these three patients remained in remission at 16 months follow-up. The median overall survival was 10.3 months (5.1 to 29.1 months) with a median follow-up time of 10.1 months (1.1 to 37 months) [
      • Ahmed N.
      • Brawley V.S.
      • Hegde M.
      • Robertson C.
      • Ghazi A.
      • Gerken C.
      • et al.
      Human epidermal growth factor receptor 2 (HER2) -specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma.
      ]. In summary, HER2 is a promising CAR target in select sarcomas; however, future work is required to improve target specificity and to better predict patient response to therapy.

      Disialoganglioside (GD2)

      Disialoganglioside (GD2) is expressed on various tumors including neuroblastoma, melanoma, osteosarcoma, Ewing’s sarcoma, rhabdomyosarcoma, and DSRCT, with relatively restricted expression in normal tissue [
      • Dobrenkov K.
      • Ostrovnaya I.
      • Gu J.
      • Cheung I.Y.
      • Cheung N.K.
      Oncotargets GD2 and GD3 are highly expressed in sarcomas of children, adolescents, and young adults.
      ,
      • Navid F.
      • Santana V.M.
      • Barfield R.C.
      Anti-GD2 antibody therapy for GD2-expressing tumors.
      ]. This relative specificity makes GD2 an attractive target for cancer immunotherapy. The monoclonal anti-GD2 antibody 3F8 has been utilized in the treatment of neuroblastoma and melanoma. The other anti-GD2 antibody hu14.18K322A is currently in phase II clinical trials for the treatment of refractory or recurrent neuroblastoma in combination with chemotherapy. Of note, first generation GD2 CAR-T cells improved overall survival in patients with high-risk neuroblastoma when combined with standard treatment [
      • Yu A.L.
      • Gilman A.L.
      • Ozkaynak M.F.
      • London W.B.
      • Kreissman S.G.
      • Chen H.X.
      • et al.
      Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma.
      ].
      With regards to sarcomas, a recent study showed GD2 to be expressed in 18 of 18 (100%) patients with osteosarcoma, 7 of 35 (20%) patients with Ewing’s sarcoma, and 2 of 15 (13%) patients with rhabdomyosarcoma [
      • Long A.H.
      • Highfill S.L.
      • Cui Y.
      • Smith J.P.
      • Walker A.J.
      • Ramakrishna S.
      • et al.
      Reduction of MDSCs with all-trans retinoic acid improves CAR therapy efficacy for sarcomas.
      ]. A comparable lysis of tumor cells formed with third generation GD2 CAR-T cells incorporating 14g2a-scFv with CD28, OX40, and CD3ζ domains, was observed in both neuroblastoma and GD2 + sarcoma cell lines [
      • Long A.H.
      • Highfill S.L.
      • Cui Y.
      • Smith J.P.
      • Walker A.J.
      • Ramakrishna S.
      • et al.
      Reduction of MDSCs with all-trans retinoic acid improves CAR therapy efficacy for sarcomas.
      ]. Of note, a large number of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) were observed in pediatric sarcoma xenografts, and were suspected to cause CAR-T cell inhibition. In response, the researchers combined GD2 CAR-T cells plus all-trans retinoic acid capable of reducing MDSCs and observed significantly improve antitumor response in pediatric sarcoma xenografts [
      • Long A.H.
      • Highfill S.L.
      • Cui Y.
      • Smith J.P.
      • Walker A.J.
      • Ramakrishna S.
      • et al.
      Reduction of MDSCs with all-trans retinoic acid improves CAR therapy efficacy for sarcomas.
      ]. These results support GD2 CAR-T cells as a promising treatment for sarcoma when combined with backing immunotherapy. Currently there is a phase I clinical trial implementing third generation GD2 CAR-T cells combined with a suicide switch caspase dimerization domain (ICD9), which enables CAR-T apoptosis in the case of serious toxicity. Eligible patients include children and young adults with solid tumors including osteosarcoma and soft tissue sarcoma (NCT02107963).

      Interleukin 11 receptor, alpha subunit (IL-11RA)

      Interleukin 11 (IL-11) is a stromal cell-derived cytokine with variable activity in inflammation-associated cancers [
      • Xu D.H.
      • Zhu Z.
      • Wakefield M.R.
      • Xiao H.
      • Bai Q.
      • Fang Y.
      The role of IL-11 in immunity and cancer.
      ]. The expression of IL-11 and its receptor, interleukin 11 receptor alpha subunit (IL-11RA), have been proposed to promote tumorigenesis and tumor cell growth in sarcomas such as osteosarcoma and chondrosarcoma [
      • Lewis V.O.
      • Ozawa M.G.
      • Deavers M.T.
      • Wang G.
      • Shintani T.
      • Arap W.
      • et al.
      The interleukin-11 receptor alpha as a candidate ligand-directed target in osteosarcoma: consistent data from cell lines, orthotopic models, and human tumor samples.
      ,
      • Li T.M.
      • Wu C.M.
      • Huang H.C.
      • Chou P.C.
      • Fong Y.C.
      • Tang C.H.
      Interleukin-11 increases cell motility and up-regulates intercellular adhesion molecule-1 expression in human chondrosarcoma cells.
      ,
      • Liu T.
      • Ma Q.
      • Zhang Y.
      • Ke S.
      • Yan K.
      • Chen X.
      • et al.
      Interleukin-11 receptor alpha is overexpressed in human osteosarcoma, and near-infrared-labeled IL-11Ralpha imaging agent could detect osteosarcoma in mouse tumor xenografts.
      ]. Moreover, high IL-11RA expression has been demonstrated in osteosarcoma cell lines and pulmonary metastases tissues, with absent or low expression in the surrounding normal tissue [
      • Huang G.
      • Yu L.
      • Cooper L.J.
      • Hollomon M.
      • Huls H.
      • Kleinerman E.S.
      Genetically modified T cells targeting interleukin-11 receptor alpha-chain kill human osteosarcoma cells and induce the regression of established osteosarcoma lung metastases.
      ].
      Third-generation IL-11RA CAR-T cells have in vitro cytotoxicity to the osteosarcoma cell lines SAOS-2, LM7, CCH-OS-D, and KRIB [
      • Huang G.
      • Yu L.
      • Cooper L.J.
      • Hollomon M.
      • Huls H.
      • Kleinerman E.S.
      Genetically modified T cells targeting interleukin-11 receptor alpha-chain kill human osteosarcoma cells and induce the regression of established osteosarcoma lung metastases.
      ]. In a mouse model mimicking pulmonary metastatic osteosarcoma, intravenous IL-11RA CAR-T cells homed to the metastatic lung lesions while sparing the surrounding healthy lung tissue. The number of visible lung metastases were also decreased [
      • Huang G.
      • Yu L.
      • Cooper L.J.
      • Hollomon M.
      • Huls H.
      • Kleinerman E.S.
      Genetically modified T cells targeting interleukin-11 receptor alpha-chain kill human osteosarcoma cells and induce the regression of established osteosarcoma lung metastases.
      ]. Specifically, three of five mice treated with IL-11RA CAR-T cells had no visible metastases, while all five mice treated with controlled T cells had metastases. These results suggest IL-11RA CAR-T cells are promising for osteosarcoma with pulmonary metastases.

      Fibroblast activation protein, alpha (FAPα)

      The tumor microenvironment has emerged as a major contributor to solid tumor development. Non-malignant cells such as cancer-associated fibroblasts (CAFs) cultivate a pro-tumorigenic environment by secreting growth factors and providing physical support [
      • Tao L.
      • Huang G.
      • Song H.
      • Chen Y.
      • Chen L.
      Cancer associated fibroblasts: an essential role in the tumor microenvironment.
      ]. Therefore, the inhibition of CAFs has become increasingly recognized as a therapeutic strategy. The identification of fibroblast activation protein alpha (FAPα) on CAFs has led efforts of selective targeting. FAPα is a type II integral membrane serine protease of the prolyl oligopeptidase family [
      • Brennen W.N.
      • Isaacs J.T.
      • Denmeade S.R.
      Rationale behind targeting fibroblast activation protein-expressing carcinoma-associated fibroblasts as a novel chemotherapeutic strategy.
      ]. FAPα functions in fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis [
      • Brennen W.N.
      • Isaacs J.T.
      • Denmeade S.R.
      Rationale behind targeting fibroblast activation protein-expressing carcinoma-associated fibroblasts as a novel chemotherapeutic strategy.
      ]. Many studies have shown increased FAPα expression on stromal fibroblasts in malignancies such as breast, colorectal, skin, and pancreatic cancer, as well as in bone and soft tissue sarcomas [
      • Brennen W.N.
      • Isaacs J.T.
      • Denmeade S.R.
      Rationale behind targeting fibroblast activation protein-expressing carcinoma-associated fibroblasts as a novel chemotherapeutic strategy.
      ,
      • Garin-Chesa P.
      • Old L.J.
      • Rettig W.J.
      Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers.
      ].
      Various FAPα-targeting modalities have been studied, including FAPα-CAR-T cell therapy. In mouse models using FAPα-CAR-T cells for mesothelioma and lung cancer treatment, there was significant antigen-specific cytotoxicity and inhibition of FAPα positive stromal cells compared to untreated and vector-control-transduced CAR-T cell-treated tumors [
      • Wang L.C.
      • Lo A.
      • Scholler J.
      • Sun J.
      • Majumdar R.S.
      • Kapoor V.
      • et al.
      Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity.
      ]. Mechanistically, the antitumor effects of FAPα-CAR-T cell therapy can be increased with increased injection frequency, diacylglycerol kinase deficient CAR-T cells, or when combined with vaccination therapy [
      • Wang L.C.
      • Lo A.
      • Scholler J.
      • Sun J.
      • Majumdar R.S.
      • Kapoor V.
      • et al.
      Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity.
      ]. Given FAPα is selectively overexpressed in osteosarcoma and expression correlates with clinical stage, histological grade, metastasis, and poor survival, there is significant incentive for future study aimed at identifying the mechanism and function of FAPα CAR-T cells.

      Insulin-like growth factor 1 receptor (IGF-1R) and tyrosine kinase orphan-like receptor 1 (ROR1)

      Insulin-like growth factor 1 receptor (IGF-1R) is a transmembrane receptor tyrosine kinase and promoter of tumor cell survival with notable prognostic significance in sarcoma. Several multidrug-resistant osteosarcoma cell lines have shown to be particularly sensitive to IGF-1R inhibition [
      • Duan Z.
      • Choy E.
      • Harmon D.
      • Yang C.
      • Ryu K.
      • Schwab J.
      • et al.
      Insulin-like growth factor-I receptor tyrosine kinase inhibitor cyclolignan picropodophyllin inhibits proliferation and induces apoptosis in multidrug resistant osteosarcoma cell lines.
      ]. Recent clinical trials evaluating anti-IGF-1R monoclonal antibodies in recurrent or refractory Ewing’s sarcoma demonstrated a 10 – 14% overall response rate with modest median progression-free survivals of less than 2 years [
      • Pappo A.S.
      • Patel S.R.
      • Crowley J.
      • Reinke D.K.
      • Kuenkele K.P.
      • Chawla S.P.
      • et al.
      R1507, a monoclonal antibody to the insulin-like growth factor 1 receptor, in patients with recurrent or refractory Ewing sarcoma family of tumors: results of a phase II sarcoma alliance for research through collaboration study.
      ,
      • Malempati S.
      • Weigel B.
      • Ingle A.M.
      • Ahern C.H.
      • Carroll J.M.
      • Roberts C.T.
      • et al.
      Phase I/II trial and pharmacokinetic study of cixutumumab in pediatric patients with refractory solid tumors and Ewing sarcoma: a report from the children's oncology group.
      ]. There is an ongoing phase II randomized controlled trial investigating combination chemotherapy with anti-IGF-1R antibody in Ewing’s sarcoma with yet to be published results. At this time, therefore, IGF-1R CAR-T cell therapy is still within the early stages of clinical investigation.
      Tyrosine kinase orphan-like receptor 1 (ROR1), also known as neurotrophic tyrosine kinase receptor-related 1 (NTRKR1), is another transmembrane protein with vital roles in cancer cell migration, invasion, and metastasis. Overexpression of ROR1 has been observed in hematologic malignancies and solid tumors alike, including Ewing’s sarcoma, osteosarcoma, and rhabdomyosarcoma [
      • Yang J.
      • Baskar S.
      • Kwong K.Y.
      • Kennedy M.G.
      • Wiestner A.
      • Rader C.
      Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies.
      ,
      • Baskar S.
      • Wiestner A.
      • Wilson W.H.
      • Pastan I.
      • Rader C.
      Targeting malignant B cells with an immunotoxin against ROR1.
      ,
      • Zhang S.
      • Chen L.
      • Cui B.
      • Chuang H.Y.
      • Yu J.
      • Wang-Rodriguez J.
      • et al.
      ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth.
      ,
      • Yamaguchi T.
      • Yanagisawa K.
      • Sugiyama R.
      • Hosono Y.
      • Shimada Y.
      • Arima C.
      • et al.
      NKX2-1/TITF1/TTF-1-Induced ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma.
      ,
      • Zhang S.
      • Cui B.
      • Lai H.
      • Liu G.
      • Ghia E.M.
      • Widhopf 2nd, G.F.
      • et al.
      Ovarian cancer stem cells express ROR1, which can be targeted for anti-cancer-stem-cell therapy.
      ,
      • Huang X.
      • Park H.
      • Greene J.
      • Pao J.
      • Mulvey E.
      • Zhou S.X.
      • et al.
      IGF1R- and ROR1-specific CAR T cells as a potential therapy for high risk sarcomas.
      ]. A recent study evaluated CAR-T cells targeting IGF-1R or ROR1 in sarcomas [
      • Huang X.
      • Park H.
      • Greene J.
      • Pao J.
      • Mulvey E.
      • Zhou S.X.
      • et al.
      IGF1R- and ROR1-specific CAR T cells as a potential therapy for high risk sarcomas.
      ]. These IGF-1R and ROR1 CAR-T cells targeted sarcoma in mice xenograft models with high levels of IFN-γ, TNF-α, and IL-13 production. The IGF-1R and ROR1 CAR-T cells significantly reduced tumor growth in pre-established, localized, and systemically disseminated osteosarcoma mouse models. Moreover, IGF-1R and ROR1 CAR-T cells prolonged survival in a localized sarcoma mouse model. These data support IGF-1R and ROR1 CAR-T cells as potential treatments for sarcoma.

      B7-H3

      B7 homolog 3 (B7-H3, CD 276) is an inhibitory protein of the B7-CD28 superfamily with roles in inhibition of T cell activation, proliferation, and cytokine production [
      • Picarda E.
      • Ohaegbulam K.C.
      • Zang X.
      Molecular pathways: targeting B7–H3 (CD276) for human cancer immunotherapy.
      ]. Overexpression of B7-H3 is common in cancers such as melanoma, non-small-cell lung cancer, osteosarcoma, Ewing’s sarcoma and rhabdomyosarcoma [
      • Picarda E.
      • Ohaegbulam K.C.
      • Zang X.
      Molecular pathways: targeting B7–H3 (CD276) for human cancer immunotherapy.
      ,
      • Wang L.
      • Zhang Q.
      • Chen W.
      • Shan B.
      • Ding Y.
      • Zhang G.
      • et al.
      B7–H3 is overexpressed in patients suffering osteosarcoma and associated with tumor aggressiveness and metastasis.
      ,
      • Gregorio A.
      • Corrias M.V.
      • Castriconi R.
      • Dondero A.
      • Mosconi M.
      • Gambini C.
      • et al.
      Small round blue cell tumours: diagnostic and prognostic usefulness of the expression of B7–H3 surface molecule.
      ]. Notably, B7-H3 expression is very low in normal tissue. In osteosarcoma, B7-H3 expression negatively correlates with the number of TILs and promotes tumor cell invasion, indicating B7-H3 may impair T cell mediated antitumor immunity [
      • Wang L.
      • Zhang Q.
      • Chen W.
      • Shan B.
      • Ding Y.
      • Zhang G.
      • et al.
      B7–H3 is overexpressed in patients suffering osteosarcoma and associated with tumor aggressiveness and metastasis.
      ]. Moreover, osteosarcoma patients with high B7-H3 expression experience significantly shorter overall survival and progression-free survival times compared to those with low expression [
      • Wang L.
      • Zhang Q.
      • Chen W.
      • Shan B.
      • Ding Y.
      • Zhang G.
      • et al.
      B7–H3 is overexpressed in patients suffering osteosarcoma and associated with tumor aggressiveness and metastasis.
      ]. As a result of these findings, antibodies against B7-H3 have been explored for sarcoma treatment. Enoblituzumab, a humanized IgG1 monoclonal anti-B7-H3 antibody, was able to stabilize refractory B7-H3-expressing tumors such as melanoma and advanced solid tumors without dose-limiting toxicity (NCT01391143). Another ongoing study is investigating enoblituzumab in patients with B7-H3-expressing neoplasms including osteosarcoma and Ewing’s sarcoma (NCT02982941).
      B7-H3 CAR-T cells have recently been studied in vivo against xenograft osteosarcoma and Ewing’s sarcoma models [
      • Majzner R.G.
      • Theruvath J.L.
      • Nellan A.
      • Heitzeneder S.
      • Cui Y.
      • Mount C.W.
      • et al.
      CAR T cells targeting B7–H3, a pan-cancer antigen, demonstrate potent preclinical activity against pediatric solid tumors and brain tumors.
      ]. The B7-H3 CAR-T cells completely regressed osteosarcoma and Ewing’s sarcoma xenografts, leading to significant survival improvements compared to the control mice. In the metastatic osteosarcoma model, 9 of 10 mice who received B7-H3 CAR-T cell treatment survived at least 5 months longer than the untreated mice [
      • Majzner R.G.
      • Theruvath J.L.
      • Nellan A.
      • Heitzeneder S.
      • Cui Y.
      • Mount C.W.
      • et al.
      CAR T cells targeting B7–H3, a pan-cancer antigen, demonstrate potent preclinical activity against pediatric solid tumors and brain tumors.
      ]. Therefore, there is promising evidence B7-H3 CAR-T cells mediate antitumor immunity against established and metastatic osteosarcomas. In addition, an advantage of B7-H3 CAR-T cell therapy is the low B7-H3 expression found in normal tissue [
      • Majzner R.G.
      • Theruvath J.L.
      • Nellan A.
      • Heitzeneder S.
      • Cui Y.
      • Mount C.W.
      • et al.
      CAR T cells targeting B7–H3, a pan-cancer antigen, demonstrate potent preclinical activity against pediatric solid tumors and brain tumors.
      ].

      CD44v6

      CD44 is a cell-surface glycoprotein with roles in cell proliferation, differentiation, migration, angiogenesis, cytokine presentation, and signaling for cell survival [
      • Zoller M.
      CD44: can a cancer-initiating cell profit from an abundantly expressed molecule?.
      ]. However, CD44, particularly the isoform 6 of adhesive receptor CD44 (CD44v6), is also a prominent cancer-initiating cell marker of tumorigenesis and metastasis [
      • Wang Z.
      • Zhao K.
      • Hackert T.
      • Zoller M.
      CD44/CD44v6 a reliable companion in cancer-initiating cell maintenance and tumor progression.
      ]. CD44v6 expression has been reported in various malignancies and is associated with worse outcomes and metastasis [
      • Xu Y.Y.
      • Guo M.
      • Yang L.Q.
      • Zhou F.
      • Yu C.
      • Wang A.
      • et al.
      Regulation of CD44v6 expression in gastric carcinoma by the IL-6/STAT3 signaling pathway and its clinical significance.
      ,
      • Qiao G.L.
      • Song L.N.
      • Deng Z.F.
      • Chen Y.
      • Ma L.J.
      Prognostic value of CD44v6 expression in breast cancer: a meta-analysis.
      ,
      • Saito S.
      • Okabe H.
      • Watanabe M.
      • Ishimoto T.
      • Iwatsuki M.
      • Baba Y.
      • et al.
      CD44v6 expression is related to mesenchymal phenotype and poor prognosis in patients with colorectal cancer.
      ,
      • Wang J.L.
      • Su W.Y.
      • Lin Y.W.
      • Xiong H.
      • Chen Y.X.
      • Xu J.
      • et al.
      CD44v6 overexpression related to metastasis and poor prognosis of colorectal cancer: a meta-analysis.
      ]. A previous meta-analysis showed osteosarcoma patients with CD44v6 expression to have significantly worse survival and metastasis rates [
      • Zhang Y.
      • Ding C.
      • Wang J.
      • Sun G.
      • Cao Y.
      • Xu L.
      • et al.
      Prognostic significance of CD44V6 expression in osteosarcoma: a meta-analysis.
      ]. In soft tissue sarcoma, CD44v6 is associated with a higher risk for local recurrence [
      • Maula S.
      • Huuhtanen R.L.
      • Blomqvist C.P.
      • Wiklund T.A.
      • Laurila P.
      • Ristamaki R.
      The adhesion molecule CD44v6 is associated with a high risk for local recurrence in adult soft tissue sarcomas.
      ].
      Recently, CD44v6 was a therapeutic target for CAR-redirected cytokine-induced killer (CIK) T cells for soft tissue sarcoma patients [
      • Leuci V.
      • Casucci G.M.
      • Grignani G.
      • Rotolo R.
      • Rossotti U.
      • Vigna E.
      • et al.
      CD44v6 as innovative sarcoma target for CAR-redirected CIK cells.
      ]. CD44v6 had an expression average of 40% amongst soft tissue sarcomas such as liposarcoma, fibrosarcoma, leiomyosarcoma, and undifferentiated pleomorphic sarcoma. The CAR-CIK T cells killed soft tissue sarcomas in vitro with much greater activity than the unmodified CIK cells. They also had much less killing activity in low CD44v6-expressing sarcomas. Moreover, the addition of anti-NKG2D antibody did not interfere with CAR-CIK killing activity but did however affect unmodified CIK cells. The in vivo antitumor activity of CD44v6-CAR-CIK cells is also present in subcutaneous xenografts. Significant greater delay of tumor growth in CD44v6-CAR-CIK cell-treated mice was seen compared to untreated and control-treated mice [
      • Leuci V.
      • Casucci G.M.
      • Grignani G.
      • Rotolo R.
      • Rossotti U.
      • Vigna E.
      • et al.
      CD44v6 as innovative sarcoma target for CAR-redirected CIK cells.
      ]. For these reasons, CD44v6 may be a favorable target for CAR-T cells in future sarcoma clinical trials.

      Fetal acetylcholine receptor

      Nicotinic acetylcholine receptors are polypeptides essential to neurotransmission of the nervous and musculoskeletal systems. During the development of the neuromuscular junction, the fetal type of acetylcholine receptors (fAChRs, α2βγδ) change to the adult type (α2βεδ). fAChRs are nearly lost from mature muscle after birth; however, its expression is retained in some extraocular muscle and thymic myoid cells. They are also highly expressed in rhabdomyosarcoma [
      • Gattenloehner S.
      • Vincent A.
      • Leuschner I.
      • Tzartos S.
      • Muller-Hermelink H.K.
      • Kirchner T.
      • et al.
      The fetal form of the acetylcholine receptor distinguishes rhabdomyosarcomas from other childhood tumors.
      ], which has led to the advent of fAChR targeting CAR-T cell therapy in this cancer [
      • Simon-Keller K.
      • Barth S.
      • Vincent A.
      • Marx A.
      Targeting the fetal acetylcholine receptor in rhabdomyosarcoma.
      ].
      The first-generation of fAChR-directed CAR-T cells were marginally effective in killing rhabdomyosarcoma cells in vitro by IFN-γ secretion and >90% rhabdomyosarcoma cell lysis [
      • Gattenlohner S.
      • Marx A.
      • Markfort B.
      • Pscherer S.
      • Landmeier S.
      • Juergens H.
      • et al.
      Rhabdomyosarcoma lysis by T cells expressing a human autoantibody-based chimeric receptor targeting the fetal acetylcholine receptor.
      ]. Of note, an increase of fAChR expression after chemotherapy was observed in the residual tumors of patients with rhabdomyosarcoma in this same study. For these reasons, the fAChR-CAR-T cells have some potential as a treatment option for rhabdomyosarcoma, particularly post-chemotherapy. The second-generation fAChR-CAR-T cells with CD28 demonstrated activation improvement; however, rhabdomyosarcoma killing efficacy remained poor compared to CD20 + lymphoma or CEA-expressing adenocarcinoma cell lines [
      • Simon-Keller K.
      • Paschen A.
      • Eichmuller S.
      • Gattenlohner S.
      • Barth S.
      • Koscielniak E.
      • et al.
      Adoptive T-cell therapy of rhabdomyosarcoma.
      ]. It is likely that resolving the resistance mechanisms mitigating fAChR-CAR-T cells in rhabdomyosarcoma will improve this novel therapy.

      NKG2D/NKG2DL

      NK cell activating receptor group 2-member D (NKG2D) functions in antitumor immunity as a co-stimulatory transmembrane protein of the CD94/NKG2 family in NK cells, T cells, and activated macrophages [
      • Ding H.
      • Yang X.
      • Wei Y.
      Fusion Proteins of NKG2D/NKG2DL in cancer immunotherapy.
      ]. While NKG2D ligands (NKG2DLs) are rarely expressed in normal tissue, they are overexpressed in infected and malignant tissue. Binding of NKG2D and its ligand triggers NK cell activation, resulting in cytotoxic granule release and tumor cell apoptosis. Importantly, NKG2DL expression has been observed in osteosarcoma and Ewing’s sarcoma cell lines [
      • Fernandez L.
      • Valentin J.
      • Zalacain M.
      • Leung W.
      • Patino-Garcia A.
      • Perez-Martinez A.
      Activated and expanded natural killer cells target osteosarcoma tumor initiating cells in an NKG2D-NKG2DL dependent manner.
      ,
      • Lehner M.
      • Gotz G.
      • Proff J.
      • Schaft N.
      • Dorrie J.
      • Full F.
      • et al.
      Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor expressed by lentiviral transduction or mRNA transfection.
      ].
      Recently, the safety and antitumor capacity of second-generation NKG2D-directed CAR-T cells against osteosarcoma has been explored [
      • Fernandez L.
      • Metais J.Y.
      • Escudero A.
      • Vela M.
      • Valentin J.
      • Vallcorba I.
      • et al.
      Memory T cells expressing an NKG2D-CAR efficiently target osteosarcoma cells.
      ]. The lentiviral transduction of NKG2D-1-4BB-CD3ζ remarkably increased NKG2D expression on the CAR-T cell surface and showed strong genetic stability. These NKG2D-CAR-T cells demonstrated significantly increased in vitro cytotoxic activity against osteosarcoma cell lines compared to non-transduced T cells. No cytolytic activity against healthy cell lines was apparent. In an orthotopic osteosarcoma murine model, the NKG2D-CAR-T cell treated group had lower tumor burden and extended survival times compared to control groups [
      • Fernandez L.
      • Metais J.Y.
      • Escudero A.
      • Vela M.
      • Valentin J.
      • Vallcorba I.
      • et al.
      Memory T cells expressing an NKG2D-CAR efficiently target osteosarcoma cells.
      ]. Similarly, NKG2D-CD28- CD3ζ CAR-T cells, which were created using a lentivirus vector, resulted in Ewing’s sarcoma cell death in vitro [
      • Lehner M.
      • Gotz G.
      • Proff J.
      • Schaft N.
      • Dorrie J.
      • Full F.
      • et al.
      Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor expressed by lentiviral transduction or mRNA transfection.
      ]. These studies defined NKG2D/NKG2DLs as promising targets for CAR-T cells in sarcoma treatment.

      NY-EYO-1

      Cancer-testis antigens (CTA) are proteins with roles in immunological maturation normally restricted to human male germ cells. However, these antigens can be re-expressed in various malignancies. New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a well-known CTA with re-expression in numerous cancers including sarcomas [
      • Thomas R.
      • Al-Khadairi G.
      • Roelands J.
      • Hendrickx W.
      • Dermime S.
      • Bedognetti D.
      • et al.
      NY-ESO-1 based immunotherapy of cancer: current perspectives.
      ]. With regards to sarcomas, the most homogenous NY-ESO-1 expression has been observed in myxoid and round cell liposarcoma and synovial sarcoma [
      • Hemminger J.A.
      • Ewart Toland A.
      • Scharschmidt T.J.
      • Mayerson J.L.
      • Kraybill W.G.
      • Guttridge D.C.
      • et al.
      The cancer-testis antigen NY-ESO-1 is highly expressed in myxoid and round cell subset of liposarcomas.
      ,
      • Jungbluth A.A.
      • Antonescu C.R.
      • Busam K.J.
      • Iversen K.
      • Kolb D.
      • Coplan K.
      • et al.
      Monophasic and biphasic synovial sarcomas abundantly express cancer/testis antigen NY-ESO-1 but not MAGE-A1 or CT7.
      ]. Many immunotherapy clinical trials utilizing cancer vaccines, adoptive T cell therapy, and combination treatment with immune checkpoint inhibitors have targeted NY-ESO-1 [
      • Thomas R.
      • Al-Khadairi G.
      • Roelands J.
      • Hendrickx W.
      • Dermime S.
      • Bedognetti D.
      • et al.
      NY-ESO-1 based immunotherapy of cancer: current perspectives.
      ]. However, most of these adoptive T cell therapies have targeted the NY-ESO-1 signal TCR transduction pathway intracellularly and in an MHC dependent manner. For traditional CAR-T cells, the TAA must be expressed on the tumor surface in order to bind with the chimeric receptor. More recently, CAR on T cells mimicking TCR function which recognize NY-ESO-1 (NY-ESO-1 CAR/TCR) in the context of HLA-A2, and HLA-A2-T cells modified to serve as NY-ESO-1 antigen presenting cells (NY-ESO-1T-APC), have both been developed and studied in multiple myeloma [
      • Patel K.
      • Olivares S.
      • Singh H.
      • Hurton L.V.
      • Huls M.H.
      • Qazilbash M.H.
      • et al.
      Combination immunotherapy with NY-ESO-1-specific CAR+ T cells with T-cell vaccine improves anti-myeloma effect.
      ]. In vitro studies demonstrated NY-ESO-1 CAR/TCR T cells can be expanded when co-cultured with NY-ESO-1T-APC. In multiple myeloma mouse models, NY-ESO-1 CAR/TCR T cell treatment produced superior antitumor effects compared to control. Furthermore, combined NY-ESO-1 CAR/TCR T cell and NY-ESO-1T-APC treatment exhibited even greater anti-myeloma activity. Therefore, TCR-enhancing CAR-T cells can target intracellular NY-ESO-1 yielding antitumor effects, with enhanced efficacy seen if there is a co-infusion of NY-ESO-1T-APC tumor vaccine [
      • Patel K.
      • Olivares S.
      • Singh H.
      • Hurton L.V.
      • Huls M.H.
      • Qazilbash M.H.
      • et al.
      Combination immunotherapy with NY-ESO-1-specific CAR+ T cells with T-cell vaccine improves anti-myeloma effect.
      ].

      Cytokine release syndrome

      Although a degree of immune stimulation and inflammation can be expected after ACT, severe cytokine release syndrome (CRS) has been observed with CD19-, BCMA-, and CD22 CAR-T cells and can cause considerable adverse effects. CRS is a systemic inflammatory response induced by excessive cytokine release after CAR-T cell infusion, with potential for widespread organ dysfunction [
      • Brudno J.N.
      • Kochenderfer J.N.
      Toxicities of chimeric antigen receptor T cells: recognition and management.
      ]. Cytokines such as IL-6, IFN-γ, TNF, IL-2, IL-8, and IL-10 become elevated and cause symptoms such as fever, tachycardia, and hypotension [
      • Brudno J.N.
      • Kochenderfer J.N.
      Toxicities of chimeric antigen receptor T cells: recognition and management.
      ]. Of note, this syndrome can be much more severe than the influenza-like syndrome commonly observed with TCR T-cell treatment. First-line treatment of CRS is aggressive supportive care and possible application of IL-6 receptor blockade with tocilizumab [
      • Brudno J.N.
      • Kochenderfer J.N.
      Toxicities of chimeric antigen receptor T cells: recognition and management.
      ]. Corticosteroids should be reserved for cases of tocilizumab non-responsiveness and neurotoxicity [
      • Brudno J.N.
      • Kochenderfer J.N.
      Toxicities of chimeric antigen receptor T cells: recognition and management.
      ]. Minimizing immune toxicity remains an important goal of CAR-T cell therapy.

      Future directions and conclusions

      The powerful clinical results seen in CAR-T cell immunotherapy in hematologic malignancies have led to an expansion of studies utilizing this therapy for sarcomas at the preclinical and clinical level. While CAR-T cells targeting multiple SAAs have been quite efficacious in sarcoma cell lines and animal models, confirmation within clinical trials is largely ongoing. As described in Table 2, there are multiple CAR-T cell clinical trials seeking to address these issues.
      Table 2Selected CAR-T cell clinical trial in patients with various types of sarcomas.
      AntigenIdentifierPhaseCAR designDisease conditionStatusNoteReference
      HER2NCT00902044I/II2nd generationHER2-positive sarcoma; osteosarcoma, Ewing’s sarcoma, PNET, rhabdomyosarcomaongoingNo serious adverse events related to this regimen.

      14 of 16 patients were observed HER2 CAR-T cells in vivo by qPCR analysis in the peripheral blood from dose level higher than 1 × 105/m2, and persisted up to 18 months.

      2 of 5 patients who had tumors removed had shown CD3 + T cell positivity by IHC and HER2 CAR-T cells by qPCR analysis within the tumor sites.

      4 of 17 patients had SD for 12 weeks to 14 months, with one demonstrated ≥ 90% tumor necrosis.

      The median OS is 10.3 months (5.1–29.1 months)
      • Ahmed N.
      • Brawley V.S.
      • Hegde M.
      • Robertson C.
      • Ghazi A.
      • Gerken C.
      • et al.
      Human epidermal growth factor receptor 2 (HER2) -specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma.
      GD2NCT01953900I3rd generationOsteosarcoma, neuroblastomaongoingThe purpose is to find the largest safe dose, side effect, and response of GD2 CAR-T cells in combination with a varicella zoster vaccine and lymohodepleting chemotherapy.

      No result is currently available.
      NCT02107963I3rd generationSarcoma, Osteosarcoma, neuroblastoma, melanomacompleteThe investigator utilized the 3rd generation GD2 CAR-T cells combined with a suicide switch comprising of a caspase dimerization domain (ICD9) to induce these CAR-T cells death in case of serious toxicity.

      No result is currently available.
      EGFR806NCT03618381I2nd generationRecurrent/refractory malignancies in children and young adults including bone and soft tissue sarcomaongoingThe purpose of this study is to evaluate the safety, feasibility, and efficacy of administering EGFR-CAR-T cells in the patients with recurrent or refractory hematologic malignancies, cancers, and sarcomas.

      The participants are currently enrolled.
      Sarcoma-specificNCT03356782I/II4th generationSarcoma, osteosarcoma, Ewing’s sarcomaongoingThe 4th generation CAR-T cells targeting sarcoma-specific antigen, such as CD133, GD2, Muc1, CD117, etc. will be utilized.
      Abbreviations: chimeric antigen receptor (CAR), desmoplastic small round cell tumors (DSRCT), epidermal growth factor receptor (EGFR), disialoganglioside (GD2), human epidermal growth factor receptor 2 (HER2), immunohistochemistry (IHC), malignant peripheral nerve sheath tumor (MPNST), overall survival (OS), primitive neuroectodermal tumor (PNET), stable disease (SD), quantitative polymerase chain reaction (qPCR).
      Compared to leukemias and lymphomas, solid tumors present several unique challenges to optimized CAR-T cell immunotherapy. For sarcomas, there is a paucity of specific and potent tumor-specific targets and high affinity CAR-domains, the tumor microenvironment is a barrier to penetration, and both intrinsic and extrinsic inhibitory mechanisms diminish CAR-T cell longevity [
      • Martinez M.
      • Moon E.K.
      CAR T cells for solid tumors: new strategies for finding, infiltrating, and surviving in the tumor microenvironment.
      ]. To offset the trafficking barrier, several works have trialed direct CAR-T cell infusion within solid tumors [
      • van Schalkwyk M.C.
      • Papa S.E.
      • Jeannon J.P.
      • Guerrero Urbano T.
      • Spicer J.F.
      • Maher J.
      Design of a phase I clinical trial to evaluate intratumoral delivery of ErbB-targeted chimeric antigen receptor T-cells in locally advanced or recurrent head and neck cancer.
      ,
      • Brown C.E.
      • Alizadeh D.
      • Starr R.
      • Weng L.
      • Wagner J.R.
      • Naranjo A.
      • et al.
      Regression of glioblastoma after chimeric antigen receptor T-cell therapy.
      ,
      • Adusumilli P.S.
      • Cherkassky L.
      • Villena-Vargas J.
      • Colovos C.
      • Servais E.
      • Plotkin J.
      • et al.
      Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity.
      ,
      • Choi B.D.
      • Suryadevara C.M.
      • Gedeon P.C.
      • Herndon 2nd, J.E.
      • Sanchez-Perez L.
      • Bigner D.D.
      • et al.
      Intracerebral delivery of a third generation EGFRvIII-specific chimeric antigen receptor is efficacious against human glioma.
      ]. As for the intravenous infusion method, tumor to CAR-T cell chemokine receptor mismatch can cause subdued tumor infiltration; however, this can potentially be resolved with better engineered and matched CAR T cell chemokine receptors [
      • Craddock J.A.
      • Lu A.
      • Bear A.
      • Pule M.
      • Brenner M.K.
      • Rooney C.M.
      • et al.
      Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b.
      ,
      • Newick K.
      • O'Brien S.
      • Sun J.
      • Kapoor V.
      • Maceyko S.
      • Lo A.
      • et al.
      Augmentation of CAR T-cell trafficking and antitumor efficacy by blocking protein kinase a localization.
      ]. Of note, oncolytic viruses have been used to specifically infect and lyse tumor cells, causing tumor chemokine secretion for enhanced CAR-T cell infiltration and tumor control in neuroblastoma [
      • Nishio N.
      • Diaconu I.
      • Liu H.
      • Cerullo V.
      • Caruana I.
      • Hoyos V.
      • et al.
      Armed oncolytic virus enhances immune functions of chimeric antigen receptor-modified T cells in solid tumors.
      ]. As an alternative, engineered CAR-T cells expressing degradative enzymes toward the extracellular matrix can improve tumor penetration and overall anti-tumor effects of CAR-T cells [
      • Caruana I.
      • Savoldo B.
      • Hoyos V.
      • Weber G.
      • Liu H.
      • Kim E.S.
      • et al.
      Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T lymphocytes.
      ].
      Even in the event of adequate infiltration, the tumor microenvironment presents a formidable barrier to CAR-T cell therapy as tumor cells and stroma produce immunosuppressive factors which dampen T cell activity and survival [
      • Shiao S.L.
      • Ganesan A.P.
      • Rugo H.S.
      • Coussens L.M.
      Immune microenvironments in solid tumors: new targets for therapy.
      ]. Specifically, there are various immune suppressor cells housed within the tumor microenvironment, such as M2 tumor-associated macrophages (M2-TAM), MDSCs, and regulatory T and B cells [
      • Shiao S.L.
      • Ganesan A.P.
      • Rugo H.S.
      • Coussens L.M.
      Immune microenvironments in solid tumors: new targets for therapy.
      ]. In response, there have been many efforts aimed towards manipulating the tumor microenvironment for anti-tumor activity via chemotherapeutic pre-conditioning, immune checkpoint inhibitors or specific antibodies, and combining MDSC depletion with CAR T cell therapy [
      • Menger L.
      • Sledzinska A.
      • Bergerhoff K.
      • Vargas F.A.
      • Smith J.
      • Poirot L.
      • et al.
      TALEN-mediated inactivation of PD-1 in tumor-reactive lymphocytes promotes intratumoral T-cell persistence and rejection of established tumors.
      ,
      • Li S.
      • Siriwon N.
      • Zhang X.
      • Yang S.
      • Jin T.
      • He F.
      • et al.
      Enhanced cancer immunotherapy by chimeric antigen receptor-modified T cells engineered to secrete checkpoint inhibitors.
      ,
      • John L.B.
      • Devaud C.
      • Duong C.P.
      • Yong C.S.
      • Beavis P.A.
      • Haynes N.M.
      • et al.
      Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells.
      ,
      • Ren J.
      • Liu X.
      • Fang C.
      • Jiang S.
      • June C.H.
      • Zhao Y.
      Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition.
      ,
      • Burga R.A.
      • Thorn M.
      • Point G.R.
      • Guha P.
      • Nguyen C.T.
      • Licata L.A.
      • et al.
      Liver myeloid-derived suppressor cells expand in response to liver metastases in mice and inhibit the anti-tumor efficacy of anti-CEA CAR-T.
      ]. In one such example, armored CARs which secrete activating cytokines such as IL-2, IL-12, and IL-15 showed enhanced therapeutic efficacy in preclinical studies [
      • Yeku O.O.
      • Purdon T.J.
      • Koneru M.
      • Spriggs D.
      • Brentjens R.J.
      Armored CAR T cells enhance antitumor efficacy and overcome the tumor microenvironment.
      ,
      • Chmielewski M.
      • Kopecky C.
      • Hombach A.A.
      • Abken H.
      IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression.
      ,
      • Wang W.
      • Ma Y.
      • Li J.
      • Shi H.S.
      • Wang L.Q.
      • Guo F.C.
      • et al.
      Specificity redirection by CAR with human VEGFR-1 affinity endows T lymphocytes with tumor-killing ability and anti-angiogenic potency.
      ]. As an alternative method, CARs incorporating dominant-negative receptors (DNRs) with a nullified intracellular domain can disrupt the downstream signaling cascade ordinarily responsible for immunosuppression [
      • Foster A.E.
      • Dotti G.
      • Lu A.
      • Khalil M.
      • Brenner M.K.
      • Heslop H.E.
      • et al.
      Antitumor activity of EBV-specific T lymphocytes transduced with a dominant negative TGF-beta receptor.
      ,
      • Cherkassky L.
      • Morello A.
      • Villena-Vargas J.
      • Feng Y.
      • Dimitrov D.S.
      • Jones D.R.
      • et al.
      Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition.
      ]. Finally, CAR-T cells can be equipped with a switchable receptor, which avoids immunosuppression by connecting the previously inhibitory immunosuppressive extracellular domain to an intracellular activating component. This is exemplified by the PD-1-CD28 switch receptor, which improves CAR-T cell infiltration and anti-tumor effects [
      • Liu X.
      • Ranganathan R.
      • Jiang S.
      • Fang C.
      • Sun J.
      • Kim S.
      • et al.
      A chimeric switch-receptor targeting PD1 augments the efficacy of second-generation CAR T cells in advanced solid tumors.
      ]. While these modalities are partially effective in dampening immunosuppression, future studies focused on combination therapy and novel CAR designs are needed to approach the results seen in hematologic malignancies.
      Exhaustion of CAR-T cells occurs from chronic antigen exposure and results in loss of T cell function, proliferation, and overall poor anti-tumor effects [
      • Davoodzadeh Gholami M.
      • Kardar G.A.
      • Saeedi Y.
      • Heydari S.
      • Garssen J.
      • Falak R.
      Exhaustion of T lymphocytes in the tumor microenvironment: significance and effective mechanisms.
      ]. To counteract this, PD-1 inhibitor or transduction of PD-1 DNR with CAR can reverse CAR-T cell exhaustion [
      • Cherkassky L.
      • Morello A.
      • Villena-Vargas J.
      • Feng Y.
      • Dimitrov D.S.
      • Jones D.R.
      • et al.
      Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition.
      ]. The co-stimulatory molecules such as CD28 and 4-1BB are also involved in T cell exhaustion and are potential therapeutic targets [
      • Zhang C.
      • Liu J.
      • Zhong J.F.
      • Zhang X.
      Engineering CAR-T cells.
      ]. These pathways should, therefore, inform future CAR designs, as they can directly reduce exhaustion and improve T cell proliferation and persistence.
      Aside from efficacy, another obstacle to widespread CAR-T cell therapy is patient safety. Undesired on-target on-tumor/off-tumor response and toxicity related to systemic cytokine release have been well-described, and underline the need for safety mechanisms when designing CAR-T cell therapy. Modern designs such as “chemical switches” or dual CAR constructs can control the activation-deactivation of CAR-T cells (Fig. 2). Safety modifications include suicide gene or pro-apoptotic caspase [
      • Budde L.E.
      • Berger C.
      • Lin Y.
      • Wang J.
      • Lin X.
      • Frayo S.E.
      • et al.
      Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma.
      ,
      • Gill S.
      • Porter D.L.
      CAR-modified anti-CD19 T cells for the treatment of B-cell malignancies: rules of the road.
      ] inclusion and coupling of scFv to signal transduction domains [
      • Jensen M.C.
      • Riddell S.R.
      Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells.
      ]. In addition, the CAR system can be coupled to CD3ζ and the co-stimulatory tails of scFvs, so that CAR-T cell activation occurs only upon binding both antigenic targets [
      • Fedorov V.D.
      • Themeli M.
      • Sadelain M.
      PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses.
      ,
      • Kloss C.C.
      • Condomines M.
      • Cartellieri M.
      • Bachmann M.
      • Sadelain M.
      Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells.
      ]. These modifications exemplify the next generation of CAR-T cell designs, and will likely expedite their specificity, efficacy, and safety profile for sarcoma patients.
      CAR-T cells are an emerging, adoptive immunotherapeutic approach with the advantages of native immunity without the restrictions of MHC presentation. Until recently, clinical studies of various CAR-T cell treatments in sarcomas have produced mixed results. Ongoing efforts revolve around identifying optimal CAR constructs, tumor-associated antigens, infusion doses, lymphodepletion regimens, all while preventing and managing toxicities. Current and future efforts will likely reveal CAR-T cell therapy to have application to sarcomas with limited therapeutic options. In addition, as technologies such as whole genome sequencing and RNA sequencing reveal new SAAs amenable to targeting, it is likely that clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mediated genome editing will be able to engineer precise TCRs with potent affinity towards any newly discovered CAR-T antigens.

      Declaration of Competing Interest

      The authors declare that there is no conflict of interest.

      Acknowledgement

      This work was supported, in part, by the Department of Orthopaedic Surgery at UCLA. Dr. Duan is supported, in part, through a Grant from Sarcoma Foundation of America (SFA) , 222433 , a Grant from National Cancer Institute (NCI)/National Institutes of Health (NIH) , UO1, CA151452-01 .

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