Tumour ischaemia by interferon-γ resembles physiological blood vessel regression.

The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models. Although IFNγ can impede tumour growth by acting directly on cancer cells, it must also act on the tumour stroma for effective rejection of large, established tumours. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ-GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.

Development of T-cell immunotherapy for hematopoietic stem cell transplantation recipients at risk of leukemia relapse.

Leukemia relapse remains the major cause of allogeneic hematopoietic stem cell transplantation (HCT) failure, and the prognosis for patients with post-HCT relapse is poor. There is compelling evidence that potent selective antileukemic effects can be delivered by donor T cells specific for particular minor histocompatibility (H) antigens. Thus, T-cell receptors (TCRs) isolated from minor H antigen-specific T cells represent an untapped resource for developing targeted T-cell immunotherapy to manage post-HCT leukemic relapse. Recognizing that several elements may be crucial to the efficacy and safety of engineered T-cell immunotherapy, we developed a therapeutic transgene with 4 components: (1) a TCR specific for the hematopoietic-restricted, leukemia-associated minor H antigen, HA-1; (2) a CD8 coreceptor to promote function of the class I-restricted TCR in CD4+ T cells; (3) an inducible caspase 9 safety switch to enable elimination of the HA-1 TCR T cells in case of toxicity; and (4) a CD34-CD20 epitope to facilitate selection of the engineered cell product and tracking of transferred HA-1 TCR T cells. The T-cell product includes HA-1 TCR CD4+ T cells to augment the persistence and function of the HA-1 TCR CD8+ T cells and includes only memory T cells; naive T cells are excluded to limit the potential for alloreactivity mediated by native TCR coexpressed by HA-1 TCR T cells. We describe the development of this unique immunotherapy and demonstrate functional responses to primary leukemia by CD4+ and CD8+ T cells transduced with a lentiviral vector incorporating the HA-1 TCR transgene construct.

Role of memory T cell subsets for adoptive immunotherapy.

Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. Besides the definition of optimal targets and antigen receptors, the differentiation status of transferred T cells is emerging as a crucial parameter for generating cell products with optimal efficacy and safety profiles. Long-living memory T cells subdivide into phenotypically as well as functionally different subsets (e.g. central memory, effector memory, tissue-resident memory T cells). This diversification process is crucial for effective immune protection, with probably distinct dependencies on the presence of individual subsets dependent on the disease to which the immune response is directed as well as its organ location. Adoptive T cell therapy intends to therapeutically transfer defined T cell immunity into patients. Efficacy of this approach often requires long-term maintenance of transferred cells, which depends on the presence and persistence of memory T cells. However, engraftment and survival of highly differentiated memory T cell subsets upon adoptive transfer is still difficult to achieve. Therefore, the recent observation that a distinct subset of weakly differentiated memory T cells shows all characteristics of adult tissue stem cells and can reconstitute all types of effector and memory T cell subsets, became highly relevant. We here review our current understanding of memory subset formation and T cell subset purification, and its implications for adoptive immunotherapy.

Misinitiation of intrathymic MART-1 transcription and biased TCR usage explain the high frequency of MART-1-specific T cells.

Immunity to tumor differentiation antigens, such as melanoma antigen recognized by T cells 1 (MART-1), has been comprehensively studied. Intriguingly, CD8(+) T cells specific for the MART-1(26(27)-35) epitope in the context of HLA-A0201 are about 100 times more abundant compared with T cells specific for other tumor-associated antigens. Moreover, MART-1-specific CD8(+) T cells show a highly biased usage of the Vα-region gene TRAV12-2. Here, we provide independent support for this notion, by showing that the combinatorial pairing of different TCRα- and TCRß- chains derived from HLA-A2-MART-1(26-35) -specific CD8(+) T-cell clones is unusually permissive in conferring MART-1 specificity, provided the CDR1α TRAV12-2 region is used. Whether TCR bias alone accounts for the unusual abundance of HLA-A2-MART-1(26-35) -specific CD8(+) T cells has remained conjectural. Here, we provide an alternative explanation: misinitiated transcription of the MART-1 gene resulting in truncated mRNA isoforms leads to lack of promiscuous transcription of the MART-1(26-35) epitope in human medullary thymic epithelial cells and, consequently, evasion of central self-tolerance toward this epitope. Thus, biased TCR usage and leaky central tolerance might act in an independent and additive manner to confer high frequency of MART-1(26-35) -specific CD8(+) T cells.

Human antitumor CD8+ T cells producing Th1 polycytokines show superior antigen sensitivity and tumor recognition.

Adoptive transfer of T cells expressing transgenic TCR with antitumor specificity provides a hopeful new therapy for patients with advanced cancer. To fulfill a large need for TCR with high affinity and specificity for various tumor entities, we sought to identify parameters for rapid selection of CTL clones with suitable characteristics. Twelve CTL clones displaying different Ag sensitivities for the same peptide-MHC epitope of the melanoma-associated Ag tyrosinase were analyzed in detail. Better MHC-multimer binding and slower multimer release are thought to reflect stronger TCR-peptide-MHC interactions; thus, these parameters would seem well suited to identify higher avidity CTL. However, large disparities were found comparing CTL multimer binding with peptide sensitivity. In contrast, CD8(+) CTL with superior Ag sensitivity mediated good tumor cytotoxicity and also secreted the triple combination of IFN-γ, IL-2, and TNF-α, representing a Th1 pattern often missing in lower avidity CTL. Furthermore, recipient lymphocytes were imbued with high Ag sensitivity, superior tumor recognition, as well as capacity for Th1 polycytokine secretion after transduction with the TCR of a high-avidity CTL. Thus, Th1 polycytokine secretion served as a suitable parameter to rapidly demark cytotoxic CD8(+) T cell clones for further TCR evaluation.

NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability.

The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy.

Minimal amino acid exchange in human TCR constant regions fosters improved function of TCR gene-modified T cells.

TCR gene therapy using adoptive transfer of TCR gene-modified T cells is a new strategy for treatment of cancer. One critical prerequisite for TCR gene therapy is sufficient expression of transferred TCRs. Several strategies to achieve optimal expression were developed, including "murinization," which replaces the human TCRalpha and TCRbeta constant regions by their murine counterparts. Using a series of mouse-human hybrid constructs, we have identified nine amino acids responsible for the improved expression of murinized TCRs. Five essential amino acid exchanges were identified in the TCRbeta C region, with exchange of a glutamic acid (human) for a basic lysine (mouse) at position 18 of the C region, being most important. For the TCRalpha C region, an area of four amino acids was sufficient for improved expression. The minimally murinized TCR variants (harboring only nine residues of the mouse sequence) enhanced expression of human TCRs by supporting preferential pairing of transferred TCR chains and a more stable association with the CD3 proteins. Most important, usage of minimally murinized TCR chains improved the function of transduced primary human T cells in comparison with cells transduced with wild-type TCRs. For TCR gene therapy, the utilization of minimally instead of completely murinized constant regions dramatically reduces the number of foreign residues and thereby the risk for immunogenicity of therapeutic TCRs.

A single TCR alpha-chain with dominant peptide recognition in the allorestricted HER2/neu-specific T cell repertoire.

T cells can recognize tumor cells specifically by their TCR and the transfer of TCR-engineered T cells is a promising novel tool in anticancer therapies. We isolated and characterized four allorestricted TCRs with specificity for the HER2/neu-derived peptide 369 (HER2(369)) demonstrating high peptide specificity. PBMCs transduced with especially one TCR, HER2-1, mediated specific tumor reactivity after TCR optimization suggesting that this TCR represents a potential candidate for targeting HER2 by TCR-transduced effector cells. Another TCR showed high-peptide specificity without tumor reactivity. However, the TCR alpha-chain of this TCR specifically recognized HER2(369) not only in combination with the original beta-chain but also with four other beta-chains of the same variable family deriving from TCRs with diverse specificities. Pairing with one beta-chain derived from another HER2(369)-specific TCR potentiated the chimeric TCRs in regard to functional avidity, CD8 independency, and tumor reactivity. Although the frequency of such TCR single chains with dominant peptide recognition is currently unknown, they may represent interesting tools for TCR optimization resulting in enhanced functionality when paired to novel partner chains. However, undirected mispairing with novel partner chains may also result in enhanced cross-reactivity and self-reactivity. These results may have an important impact on the further design of strategies for adoptive transfer using TCR-transduced T cells.

T-Cells Expressing a Highly Potent PRAME-Specific T-Cell Receptor in Combination with a Chimeric PD1-41BB Co-Stimulatory Receptor Show a Favorable Preclinical Safety Profile and Strong Anti-Tumor Reactivity.

The hostile tumor microenvironment (TME) is a major challenge for the treatment of solid tumors with T-cell receptor (TCR)-modified T-cells (TCR-Ts), as it negatively influences T-cell efficacy, fitness, and persistence. These negative influences are caused, among others, by the inhibitory checkpoint PD-1/PD-L1 axis. The Preferentially Expressed Antigen in Melanoma (PRAME) is a highly relevant cancer/testis antigen for TCR-T immunotherapy due to broad expression in multiple solid cancer indications. A TCR with high specificity and sensitivity for PRAME was isolated from non-tolerized T-cell repertoires and introduced into T-cells alongside a chimeric PD1-41BB receptor, consisting of the natural extracellular domain of PD-1 and the intracellular signaling domain of 4-1BB, turning an inhibitory pathway into a T-cell co-stimulatory pathway. The addition of PD1-41BB to CD8+ T-cells expressing the transgenic PRAME-TCR enhanced IFN-γ secretion, improved cytotoxic capacity, and prevented exhaustion upon repetitive re-challenge with tumor cells in vitro without altering the in vitro safety profile. Furthermore, a single dose of TCR-Ts co-expressing PD1-41BB was sufficient to clear a hard-to-treat melanoma xenograft in a mouse model, whereas TCR-Ts without PD1-41BB could not eradicate the PD-L1-positive tumors. This cutting-edge strategy supports development efforts to provide more effective TCR-T immunotherapies for the treatment of solid tumors.

Tumor rejection by local interferon gamma induction in established tumors is associated with blood vessel destruction and necrosis.

It has been shown that injecting a suspension of IFN-γ-secreting tumor cells results in their rejection. This effect has been attributed to IFN-γ preventing tumor stroma formation but not to a direct effect on the cancer cells. However, it is not known, which influence IFN-γ has on tumors with an established stroma. To address this question, the plasmacytoma cell line J558L was transduced with a vector allowing doxycycline-inducible IFN-γ gene expression. After the injection of the tumor cells into mice, IFN-γ was induced at different time points. Tumors did not grow when inducing IFN-γ immediately after tumor cell inoculation, while approximately half of the tumors were rejected when IFN-γ was induced in early established tumors within 2 weeks. Induction of IFN-γ 2-3 weeks after tumor cell inoculation was less efficient (0-17% rejection). IFN-γ induction in established tumors led to a reduction of CD146(+) endothelial cells and massive necrosis. Together, we show that vascularized tumors can be rejected by local IFN-γ expression, but that rejection of established tumors was less efficient over time. This suggests that transplanted tumors became less susceptible to local IFN-γ treatment the better they are established.

Dendritic cells pulsed with RNA encoding allogeneic MHC and antigen induce T cells with superior antitumor activity and higher TCR functional avidity.

Adoptive transfer of T cells expressing transgenic T-cell receptors (TCRs) with antitumor function is a hopeful new therapy for patients with advanced tumors; however, there is a critical bottleneck in identifying high-affinity TCR specificities needed to treat different malignancies. We have developed a strategy using autologous dendritic cells cotransfected with RNA encoding an allogeneic major histocompatibility complex molecule and a tumor-associated antigen to obtain allo-restricted peptide-specific T cells having superior capacity to recognize tumor cells and higher functional avidity. This approach provides maximum flexibility because any major histocompatibility complex molecule and any tumor-associated antigen can be combined in the dendritic cells used for priming of autologous T cells. TCRs of allo-restricted T cells, when expressed as transgenes in activated peripheral blood lymphocytes, transferred superior function compared with self-restricted TCR. This approach allows high-avidity T cells and TCR specific for tumor-associated self-peptides to be easily obtained for direct adoptive T-cell therapy or for isolation of therapeutic transgenic TCR sequences.

A safeguard eliminates T cell receptor gene-modified autoreactive T cells after adoptive transfer.

By transfer of T cell receptor (TCR) genes, antigen specificity of T cells can be redirected to target any antigen. Adoptive transfer of TCR-redirected T cells into patients has shown promising results. However, this immunotherapy bears the risk of autoreactive side effects if the TCR recognizes antigens on self-tissue. Here, we introduce a safeguard based on a TCR-intrinsic depletion mechanism to eliminate autoreactive TCR-redirected T cells in vivo. By the introduction of a 10-aa tag of the human c-myc protein into murine (OT-I, P14) and human (gp100) TCR sequences, we were able to deplete T cells that were transduced with these myc-tagged TCRs with a tag-specific antibody in vitro. T cells transduced with the modified TCR maintained equal properties compared with cells transduced with the wild-type receptor concerning antigen binding and effector function. More importantly, therapeutic in vivo depletion of adoptively transferred T cells rescued mice showing severe signs of autoimmune insulitis from lethal diabetes. This safeguard allows termination of adoptive therapy in case of severe side effects.

Development of a CD8 co-receptor independent T-cell receptor specific for tumor-associated antigen MAGE-A4 for next generation T-cell-based immunotherapy.

BACKGROUND: The cancer-testis antigen MAGE-A4 is an attractive target for T-cell-based immunotherapy, especially for indications with unmet clinical need like non-small cell lung or triple-negative breast cancer. METHODS: An unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived epitope (GVYDGREHTV) that was properly processed and presented on human leukocyte antigen (HLA)-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high-avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies. RESULTS: A MAGE-A4-reactive, HLA-A2-restricted T-cell receptor (TCR) was isolated from primed T cells of an HLA-A2-negative donor. The respective TCR-T-cell (TCR-T) product bbT485 was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared with TCR-Ts expressing a TCR derived from a tolerized T-cell repertoire to self-antigens. This natural high-avidity TCR was found to be CD8 co-receptor independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-Ts supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells and upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines on tumor-specific stimulation. CONCLUSION: The extensive pre-clinical assessment of safety and in vivo potency of bbT485 provide the basis for its use in TCR-T immunotherapy studies. The ability of this non-mutated high-avidity, co-receptor-independent TCR to activate CD8+ and CD4+ T cells could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T-cell subsets that goes beyond what is currently tested in the clinic.

Comparative analysis of TCR and CAR signaling informs CAR designs with superior antigen sensitivity and in vivo function.

Chimeric antigen receptor (CAR)-modified T cell therapy is effective in treating lymphomas, leukemias, and multiple myeloma in which the tumor cells express high amounts of target antigen. However, achieving durable remission for these hematological malignancies and extending CAR T cell therapy to patients with solid tumors will require receptors that can recognize and eliminate tumor cells with a low density of target antigen. Although CARs were designed to mimic T cell receptor (TCR) signaling, TCRs are at least 100-fold more sensitive to antigen. To design a CAR with improved antigen sensitivity, we directly compared TCR and CAR signaling in primary human T cells. Global phosphoproteomic analysis revealed that key T cell signaling proteins-such as CD3δ, CD3ε, and CD3γ, which comprise a portion of the T cell co-receptor, as well as the TCR adaptor protein LAT-were either not phosphorylated or were only weakly phosphorylated by CAR stimulation. Modifying a commonplace 4-1BB/CD3ζ CAR sequence to better engage CD3ε and LAT using embedded CD3ε or GRB2 domains resulted in enhanced T cell activation in vitro in settings of a low density of antigen, and improved efficacy in in vivo models of lymphoma, leukemia, and breast cancer. These CARs represent examples of alterations in receptor design that were guided by in-depth interrogation of T cell signaling.

Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette.

The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRalpha- and beta-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-gamma secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRalpha- and beta-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.

Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function.

Chimeric antigen receptors (CARs) link an antigen recognition domain to intracellular signaling domains to redirect T cell specificity and function. T cells expressing CARs with CD28/CD3ζ or 4-1BB/CD3ζ signaling domains are effective at treating refractory B cell malignancies but exhibit differences in effector function, clinical efficacy, and toxicity that are assumed to result from the activation of divergent signaling cascades. We analyzed stimulation-induced phosphorylation events in primary human CD8+ CD28/CD3ζ and 4-1BB/CD3ζ CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3ζ CARs activated faster and larger-magnitude changes in protein phosphorylation, which correlated with an effector T cell-like phenotype and function. In contrast, 4-1BB/CD3ζ CAR T cells preferentially expressed T cell memory-associated genes and exhibited sustained antitumor activity against established tumors in vivo. Mutagenesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3ζ CAR signal intensity was partly related to constitutive association of Lck with this domain in CAR complexes. Our data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor and that signal strength is a key determinant of T cell fate. Thus, tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity.

Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues.

Purpose: This study examines cell surface ROR1 expression in human tumors and normal tissues. ROR1 is considered a promising target for cancer therapy due to putative tumor-specific expression, and multiple groups are developing antibodies and/or chimeric antigen receptor-modified T cells to target ROR1. On-target, off-tumor toxicity is a challenge for most nonmutated tumor antigens; however, prior studies suggest that ROR1 is absent on most normal tissues.Experimental Design: Our studies show that published antibodies lack sensitivity to detect endogenous levels of cell surface ROR1 by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded tissues. We developed a ROR1-specific monoclonal antibody (mAb) targeting the carboxy-terminus of ROR1 and evaluated its specificity and sensitivity in IHC.Results: The 6D4 mAb is a sensitive and specific reagent to detect cell surface ROR1 by IHC. The data show that ROR1 is homogenously expressed on a subset of ovarian cancer, triple-negative breast cancer, and lung adenocarcinomas. Contrary to previous findings, we found ROR1 is expressed on several normal tissues, including parathyroid; pancreatic islets; and regions of the esophagus, stomach, and duodenum. The 6D4 mAb recognizes rhesus ROR1, and ROR1 expression was similar in human and macaque tissues, suggesting that the macaque is a suitable model to evaluate safety of ROR1-targeted therapies.Conclusions: ROR1 is a promising immunotherapeutic target in many epithelial tumors; however, high cell surface ROR1 expression in multiple normal tissues raises concerns for on-target off-tumor toxicities. Clinical translation of ROR1-targeted therapies warrants careful monitoring of toxicities to normal organs and may require strategies to ensure patient safety. Clin Cancer Res; 23(12); 3061-71. ©2016 AACR.

Inclusion of Strep-tag II in design of antigen receptors for T-cell immunotherapy.

Adoptive immunotherapy with genetically engineered T cells has the potential to treat cancer and other diseases. The introduction of Strep-tag II sequences into specific sites in synthetic chimeric antigen receptors or natural T-cell receptors of diverse specificities provides engineered T cells with a marker for identification and rapid purification, a method for tailoring spacer length of chimeric receptors for optimal function, and a functional element for selective antibody-coated, microbead-driven, large-scale expansion. These receptor designs facilitate cGMP manufacturing of pure populations of engineered T cells for adoptive T-cell therapies and enable in vivo tracking and retrieval of transferred cells for downstream research applications.

Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia.

The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies.

CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients.

BACKGROUND: T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. METHODS: We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS: The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. CONCLUSION: Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION: ClinicalTrials.gov NCT01865617. FUNDING: R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.