Pediatric cancers in need of new and effective interventions include relapsed or refractory brain tumors like glioblastoma and DIPG; sarcomas like osteosarcoma and rhabdomyosarcoma; and neuroblastoma.  Adoptive Immunotherapy with engineered T cells for pediatric cancers has been a major focus of laboratory research and is just now being clinically evaluated. The most effective lymphocyte effector populations are being defined with increasing specificity in order to meet the challenges that immunotherapy of solid tumors present, in contrast to the current success seen with pre-B ALL.  This Research Topic will focus of novel chimeric antigen receptor (CAR) formats, new genetic payloads that enhance T cell anti-tumor activity, and the creation or selection of lymphocyte populations that more effectively penetrate and resist the immune-inhibitory tumor microenvironment.

Caring Cross, Inc.

Baylor College of Medicine

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Frontiers in Oncology

Pediatric Oncology

Frontiers in Pediatrics

Advances in Engineered T Cells

Chimeric antigen receptor (CAR) T cells have shown great success in the treatment of CD19+ hematological malignancies, leading to their recent approval by the FDA as a new cancer treatment modality. However, their broad use is limited since a CAR targets a single tumor associated antigen (TAA), which is not effective against tumors with heterogeneous TAA expression or emerging antigen loss variants. Further, stably engineered CAR T cells can continually and uncontrollably proliferate and activate in response to antigen, potentially causing fatal on-target off-tumor toxicity, cytokine release syndrome, or neurotoxicity without a method of control or elimination. To address these issues, our lab and others have developed various universal immune receptors (UIRs) that allow for targeting of multiple TAAs by T cells expressing a single receptor. UIRs function through the binding of an extracellular adapter domain which acts as a bridge between intracellular T cell signaling domains and a soluble tumor antigen targeting ligand (TL). The dissociation of TAA targeting and T cell signaling confers many advantages over standard CAR therapy, such as dose control of T cell effector function, the ability to simultaneously or sequentially target multiple TAAs, and control of immunologic synapse geometry. There are currently four unique UIR platform types: ADCC-mediating Fc-binding immune receptors, bispecific protein engaging immune receptors, natural binding partner immune receptors, and anti-tag CARs. These UIRs all allow for potential benefits over standard CARs, but also bring unique engineering challenges that will have to be addressed to achieve maximal efficacy and safety in the clinic. Still, UIRs present an exciting new avenue for adoptive T cell transfer therapies and could lead to their expanded use in areas which current CAR therapies have failed. Here we review the development of each UIR platform and their unique functional benefits, and detail the potential hurdles that may need to be overcome for continued clinical translation.

Chimeric antigen receptor (CAR) T cells have shown great success in the treatment of CD19+ hematological malignancies, leading to their recent approval by the FDA as a new cancer treatment modality. However, their broad use is limited since a CAR targets a single tumor associated antigen (TAA), which is not effective against tumors with heterogeneous TAA expression or emerging antigen loss variants. Further, stably engineered CAR T cells can continually and uncontrollably proliferate and activate in response to antigen, potentially causing fatal on-target off-tumor toxicity, cytokine release syndrome, or neurotoxicity without a method of control or elimination. To address these issues, our lab and others have developed various universal immune receptors (UIRs) that allow for targeting of multiple TAAs by T cells expressing a single receptor. UIRs function through the binding of an extracellular adapter domain which acts as a bridge between intracellular T cell signaling domains and a soluble tumor antigen targeting ligand (TL). The dissociation of TAA targeting and T cell signaling confers many advantages over standard CAR therapy, such as dose control of T cell effector function, the ability to simultaneously or sequentially target multiple TAAs, and control of immunologic synapse geometry. There are currently four unique UIR platform types: ADCC-mediating Fc-binding immune receptors, bispecific protein engaging immune receptors, natural binding partner immune receptors, and anti-tag CARs. These UIRs all allow for potential benefits over standard CARs, but also bring unique engineering challenges that will have to be addressed to achieve maximal efficacy and safety in the clinic. Still, UIRs present an exciting new avenue for adoptive T cell transfer therapies and could lead to their expanded use in areas which current CAR therapies have failed. Here we review the development of each UIR platform and their unique functional benefits, and detail the potential hurdles that may need to be overcome for continued clinical translation.

The Emergence of Universal Immune Receptor T Cell Therapy for Cancer

Chimeric antigen receptor (CAR)-T cell therapy has transformed pediatric oncology by producing high remission rates and potent effects in CD19+ B-cell malignancies. This scenario is ideal as CD19 expression is homogeneous and human blood provides a favorable environment for CAR-T cells to thrive and destroy cancer cells (along with normal B cells). Yet, CAR-T cell therapies for solid tumors remain challenged by fewer tumor targets and poor CAR-T cell performances in a hostile tumor microenvironment. For acute myeloid leukemia and childhood solid tumors such as osteosarcoma, the primary treatment is systemic chemotherapy that often falls short of expectation especially for relapsed and refractory conditions. We aim to develop a CAR-T adaptor molecule (CAM)-based therapy that uses a bispecific small-molecule ligand EC17, fluorescein isothiocyanate (FITC) conjugated with folic acid, to redirect FITC-specific CAR-T cells against folate receptor (FR)-positive tumors. As previously confirmed in rodents as well as in human clinical studies, EC17 penetrates solid tumors within minutes and is retained due to high affinity for the FR, whereas unbound EC17 rapidly clears from the blood and from receptor-negative tissues. When combined with a rationally designed CAR construct, EC17 CAM was shown to trigger CAR-modified T cell activation and cytolytic activity with a low FR threshold against tumor targets. However, maximal cytolytic potential correlated with (i) functional FR levels (in a semi-log fashion), (ii) the amount of effector cells present, and (iii) tumors' natural sensitivity to T cell mediated killing. In tumor-bearing mice, administration of EC17 CAM was the key to drive CAR-T cell activation, proliferation, and persistence against FR+ pediatric hematologic and solid tumors. In our modeling systems, cytokine release syndrome (CRS) was induced under specific conditions, but the risk of severe CRS could be easily mitigated or prevented by applying intermittent dosing and/or dose-titration strategies for the EC17 CAM. Our approach offers the flexibility of antigen control, prevents T cell exhaustion, and provides additional safety mechanisms including rapid reversal of severe CRS with intravenous sodium fluorescein. In this paper, we summarize the translational aspects of our technology in support of clinical development.

Chimeric antigen receptor (CAR)-T cell therapy has transformed pediatric oncology by producing high remission rates and potent effects in CD19+ B-cell malignancies. This scenario is ideal as CD19 expression is homogeneous and human blood provides a favorable environment for CAR-T cells to thrive and destroy cancer cells (along with normal B cells). Yet, CAR-T cell therapies for solid tumors remain challenged by fewer tumor targets and poor CAR-T cell performances in a hostile tumor microenvironment. For acute myeloid leukemia and childhood solid tumors such as osteosarcoma, the primary treatment is systemic chemotherapy that often falls short of expectation especially for relapsed and refractory conditions. We aim to develop a CAR-T adaptor molecule (CAM)-based therapy that uses a bispecific small-molecule ligand EC17, fluorescein isothiocyanate (FITC) conjugated with folic acid, to redirect FITC-specific CAR-T cells against folate receptor (FR)-positive tumors. As previously confirmed in rodents as well as in human clinical studies, EC17 penetrates solid tumors within minutes and is retained due to high affinity for the FR, whereas unbound EC17 rapidly clears from the blood and from receptor-negative tissues. When combined with a rationally designed CAR construct, EC17 CAM was shown to trigger CAR-modified T cell activation and cytolytic activity with a low FR threshold against tumor targets. However, maximal cytolytic potential correlated with (i) functional FR levels (in a semi-log fashion), (ii) the amount of effector cells present, and (iii) tumors&apos; natural sensitivity to T cell mediated killing. In tumor-bearing mice, administration of EC17 CAM was the key to drive CAR-T cell activation, proliferation, and persistence against FR+ pediatric hematologic and solid tumors. In our modeling systems, cytokine release syndrome (CRS) was induced under specific conditions, but the risk of severe CRS could be easily mitigated or prevented by applying intermittent dosing and/or dose-titration strategies for the EC17 CAM. Our approach offers the flexibility of antigen control, prevents T cell exhaustion, and provides additional safety mechanisms including rapid reversal of severe CRS with intravenous sodium fluorescein. In this paper, we summarize the translational aspects of our technology in support of clinical development.

Preclinical Evaluation of Bispecific Adaptor Molecule Controlled Folate Receptor CAR-T Cell Therapy With Special Focus on Pediatric Malignancies

Development of chimeric antigen receptor (CAR)-modified T cells for the treatment of T-lineage leukemia and lymphoma has encountered several unique challenges. The most widely expressed tumor antigen targets for malignant T cells are often also expressed on non-malignant T cells. Transducing T cells with CARs targeted to these shared antigens can therefore promote over-activation or fratricide of CAR T cells, reducing their therapeutic potency. If fratricide is resolved, clinical CAR T cell activity may eliminate normal T-cell subsets and cause temporary immunosuppression. In this review, we summarize the preclinical development of CAR-based therapies for T-cell malignancies and discuss strategies to minimize toxicities associated with on-target fratricide and off-tumor activity.

Development of chimeric antigen receptor (CAR)-modified T cells for the treatment of T-lineage leukemia and lymphoma has encountered several unique challenges. The most widely expressed tumor antigen targets for malignant T cells are often also expressed on non-malignant T cells. Transducing T cells with CARs targeted to these shared antigens can therefore promote over-activation or fratricide of CAR T cells, reducing their therapeutic potency. If fratricide is resolved, clinical CAR T cell activity may eliminate normal T-cell subsets and cause temporary immunosuppression. In this review, we summarize the preclinical development of CAR-based therapies for T-cell malignancies and discuss strategies to minimize toxicities associated with on-target fratricide and off-tumor activity.

Chimeric Antigen Receptors for T-Cell Malignancies

High grade gliomas (HGG) comprise a heterogeneous group of brain malignancies with dismal prognosis. Current standard-of-care includes radiation, chemotherapy, and surgical resection when possible. Despite advances in each of these treatment modalities, survival rates for pediatric and adult HGG patients has remained largely unchanged over the course of several years. This is in stark contrast to the significant survival increases seen recently for a variety of hematological and other solid malignancies. The introduction and widespread use of immunotherapies have contributed significantly to these survival increases, and as such these therapies have been explored for use in the treatment of HGG. In particular, chimeric antigen receptor (CAR) T cell therapy has shown promise in clinical trials in HGG patients. However, unlike the tremendous success CAR T cell therapy has seen in B cell leukemia and lymphoma treatment, the success in HGG patients has been modest at best. This is largely due to the unique tumor microenvironment in the central nervous system, difficulty in accessing the tumor site, and heterogeneity in target antigen expression. The results of these features are poor CAR T cell proliferation, poor persistence, suboptimal cytokine secretion, and the emergence of antigen-loss tumor variants. These issues have called for the development of “next generation” CAR T cells designed to circumvent the barriers that have limited the success of current CAR T cell technologies in HGG treatment. Rapid advancements in gene editing technologies have provided several avenues for CAR T cell modification to enhance their efficacy. Among these are cytokine overexpression, gene knock-out and knock-in, targeting of multiple antigens simultaneously, and precise control of CAR expression and signaling. These “next generation” CAR T cells have shown promising results in pre-clinical models and may be the key to harnessing the full potential of CAR T cells in the treatment of HGG.

High grade gliomas (HGG) comprise a heterogeneous group of brain malignancies with dismal prognosis. Current standard-of-care includes radiation, chemotherapy, and surgical resection when possible. Despite advances in each of these treatment modalities, survival rates for pediatric and adult HGG patients has remained largely unchanged over the course of several years. This is in stark contrast to the significant survival increases seen recently for a variety of hematological and other solid malignancies. The introduction and widespread use of immunotherapies have contributed significantly to these survival increases, and as such these therapies have been explored for use in the treatment of HGG. In particular, chimeric antigen receptor (CAR) T cell therapy has shown promise in clinical trials in HGG patients. However, unlike the tremendous success CAR T cell therapy has seen in B cell leukemia and lymphoma treatment, the success in HGG patients has been modest at best. This is largely due to the unique tumor microenvironment in the central nervous system, difficulty in accessing the tumor site, and heterogeneity in target antigen expression. The results of these features are poor CAR T cell proliferation, poor persistence, suboptimal cytokine secretion, and the emergence of antigen-loss tumor variants. These issues have called for the development of &#x201c;next generation&#x201d; CAR T cells designed to circumvent the barriers that have limited the success of current CAR T cell technologies in HGG treatment. Rapid advancements in gene editing technologies have provided several avenues for CAR T cell modification to enhance their efficacy. Among these are cytokine overexpression, gene knock-out and knock-in, targeting of multiple antigens simultaneously, and precise control of CAR expression and signaling. These &#x201c;next generation&#x201d; CAR T cells have shown promising results in pre-clinical models and may be the key to harnessing the full potential of CAR T cells in the treatment of HGG.

Next Generation CAR T Cells for the Immunotherapy of High-Grade Glioma

Acute myeloid leukemia (AML) remains a challenging pediatric and adult disease. Given the elevated expression of the CD33 antigen on leukemic blasts, therapeutic approaches to AML now feature the approved antibody drug conjugate (Mylotarg, GO) and investigational CART cell approaches incorporating CD33-binding domains derived from humanized scFvs. We designed a functional chimeric antigen receptor utilizing a human targeting sequence, derived from a heavy chain variable domain, termed CAR33VH. Lentiviral-based expression vectors which encoded CAR constructs incorporating the novel binding domain (CAR33VH), or the My96 scFv control binder (My96CAR) in frame with a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3 zeta activation domain, were transduced into primary human CD4+ and CD8+ T cells, and CAR expression was confirmed by flow cytometry. CAR33VH, similar to My96CAR, demonstrated robust and specific cytotoxicity in short-term and long-term co-incubation killing assays against CD33+ AML lines. In overnight cytokine release assays in which CAR T cells were challenged with the CD33+ tumor cells HL-60, MOLM-14 and KG-1a, CAR33VH elicited IFN-gamma, TNF-alpha and IL-2. This was seen with CD33+ cell lines, but not when CAR T were cultured alone. Studies with a CD33− cell line engineered to stably express the full length CD33 variant 1, or the naturally occurring CD33 splice variant 2, revealed that both CAR33VH and My96CAR, target the V domain of CD33, suggesting a similar therapeutic profile. Colony-formation assays utilizing peripheral blood CD34+ hematopoietic stem cells treated with CAR33VH, My96CAR, or with an untransduced T cell control, yielded similar numbers of BFU-E erythroid and CFU-GM myeloid colonies, suggesting a lack of CAR-related overt toxicity. In an in vivo AML model, NSG mice engrafted with MOLM-14 cells stably expressing firefly luciferase, both CAR33VH and CARMy96 efficiently eliminated tumors. In conclusion, we demonstrate for the first time the feasibility and efficacy of employing human variable domain-only binder derived from a phage display library in an anti-AML CAR design. CAR33VH, comprised of a human heavy-chain variable fragment-only antigen binding domain, was efficient in tumor killing in vitro and in vivo, and showed comparable functionality to the scFv-based My96CAR.

Acute myeloid leukemia (AML) remains a challenging pediatric and adult disease. Given the elevated expression of the CD33 antigen on leukemic blasts, therapeutic approaches to AML now feature the approved antibody drug conjugate (Mylotarg, GO) and investigational CART cell approaches incorporating CD33-binding domains derived from humanized scFvs. We designed a functional chimeric antigen receptor utilizing a human targeting sequence, derived from a heavy chain variable domain, termed CAR33VH. Lentiviral-based expression vectors which encoded CAR constructs incorporating the novel binding domain (CAR33VH), or the My96 scFv control binder (My96CAR) in frame with a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3 zeta activation domain, were transduced into primary human CD4+ and CD8+ T cells, and CAR expression was confirmed by flow cytometry. CAR33VH, similar to My96CAR, demonstrated robust and specific cytotoxicity in short-term and long-term co-incubation killing assays against CD33+ AML lines. In overnight cytokine release assays in which CAR T cells were challenged with the CD33+ tumor cells HL-60, MOLM-14 and KG-1a, CAR33VH elicited IFN-gamma, TNF-alpha and IL-2. This was seen with CD33+ cell lines, but not when CAR T were cultured alone. Studies with a CD33− cell line engineered to stably express the full length CD33 variant 1, or the naturally occurring CD33 splice variant 2, revealed that both CAR33VH and My96CAR, target the V domain of CD33, suggesting a similar therapeutic profile. Colony-formation assays utilizing peripheral blood CD34+ hematopoietic stem cells treated with CAR33VH, My96CAR, or with an untransduced T cell control, yielded similar numbers of BFU-E erythroid and CFU-GM myeloid colonies, suggesting a lack of CAR-related overt toxicity. In an in vivo AML model, NSG mice engrafted with MOLM-14 cells stably expressing firefly luciferase, both CAR33VH and CARMy96 efficiently eliminated tumors. In conclusion, we demonstrate for the first time the feasibility and efficacy of employing human variable domain-only binder derived from a phage display library in an anti-AML CAR design. CAR33VH, comprised of a human heavy-chain variable fragment-only antigen binding domain, was efficient in tumor killing in vitro and in vivo, and showed comparable functionality to the scFv-based My96CAR.

A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

Application of the CAR targeting strategy in solid tumors is challenged by the need for adequate target antigens. As a consequence of their tissue origin, embryonal cancers can aberrantly express membrane-anchored gangliosides. These are carbohydrate molecules consisting of a glycosphingolipid linked to sialic acids residues. The best-known example is the abundant expression of ganglioside GD2 on the cell surface of neuroblastomas which derive from GD2-positive neuroectoderm. Gangliosides are involved in various cellular functions, including signal transduction, cell proliferation, differentiation, adhesion and cell death. In addition, transformation of human cells to cancer cells can be associated with distinct glycosylation profiles which provide advantages for tumor growth and dissemination and can serve as immune targets. Both gangliosides and aberrant glycosylation of proteins escape the direct molecular and proteomic screening strategies currently applied to identify further immune targets in cancers. Due to their highly restricted expression and their functional roles in the malignant behavior, they are attractive targets for immune engineering strategies. GD2-redirected CAR T cells have shown activity in clinical phase I/II trials in neuroblastoma and next-generation studies are ongoing. Further carbohydrate targets for CAR T cells in preclinical development are O-acetyl-GD2, NeuGc-GM3 (N-glycolyl GM3), GD3, SSEA-4, and oncofetal glycosylation variants. This review summarizes knowledge on the role and function of some membrane-expressed non-protein antigens, including gangliosides and abnormal protein glycosylation patterns, and discusses their potential to serve as a CAR targets in pediatric solid cancers.

Application of the CAR targeting strategy in solid tumors is challenged by the need for adequate target antigens. As a consequence of their tissue origin, embryonal cancers can aberrantly express membrane-anchored gangliosides. These are carbohydrate molecules consisting of a glycosphingolipid linked to sialic acids residues. The best-known example is the abundant expression of ganglioside GD2 on the cell surface of neuroblastomas which derive from GD2-positive neuroectoderm. Gangliosides are involved in various cellular functions, including signal transduction, cell proliferation, differentiation, adhesion and cell death. In addition, transformation of human cells to cancer cells can be associated with distinct glycosylation profiles which provide advantages for tumor growth and dissemination and can serve as immune targets. Both gangliosides and aberrant glycosylation of proteins escape the direct molecular and proteomic screening strategies currently applied to identify further immune targets in cancers. Due to their highly restricted expression and their functional roles in the malignant behavior, they are attractive targets for immune engineering strategies. GD2-redirected CAR T cells have shown activity in clinical phase I/II trials in neuroblastoma and next-generation studies are ongoing. Further carbohydrate targets for CAR T cells in preclinical development are O-acetyl-GD2, NeuGc-GM3 (N-glycolyl GM3), GD3, SSEA-4, and oncofetal glycosylation variants. This review summarizes knowledge on the role and function of some membrane-expressed non-protein antigens, including gangliosides and abnormal protein glycosylation patterns, and discusses their potential to serve as a CAR targets in pediatric solid cancers.