In vitro elimination of epidermal growth factor receptor-overexpressing cancer cells by CD32A-chimeric receptor T cells in combination with cetuximab or panitumumab.

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). Although the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects the poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on natural killer (NK) cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32A) to a similar extent. Our study compares the ability of T cells, engineered with a novel low-affinity CD32A131R -chimeric receptor (CR), and those engineered with the low-affinity CD16158F -CR T cells, in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. After T-cell transduction, the percentage of CD32A131R -CR T cells was 74 ± 10%, whereas the percentage of CD16158F -CR T cells was 46 ± 15%. Only CD32A131R -CR T cells bound panitumumab. CD32A131R -CR T cells combined with the mAb 8.26 (anti-CD32) and CD16158F -CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116FcγR+ cells, in a reverse ADCC assay in vitro. Crosslinking of CD32A131R -CR on T cells by cetuximab or panitumumab and CD16158F -CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and the secretion of interferon gamma and tumor necrosis factor alpha. Neither cetuximab nor panitumumab induced Fcγ-CR T antitumor activity against Kirsten rat sarcoma (KRAS)-mutated HCT116, nonsmall-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. The ADCC of Fcγ-CR T cells was associated with the overexpression of EGFR on ECCs. In conclusion, CD32A131R -CR T cells are efficiently redirected by cetuximab or panitumumab against breast cancer cells overexpressing EGFR.

NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease.

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and ß genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165-specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.

IL-12 reprograms CAR-expressing natural killer T cells to long-lived Th1-polarized cells with potent antitumor activity.

Human natural killer T cells (NKTs) are innate-like T lymphocytes increasingly used for cancer immunotherapy. Here we show that human NKTs expressing the pro-inflammatory cytokine interleukin-12 (IL-12) undergo extensive and sustained molecular and functional reprogramming. Specifically, IL-12 instructs and maintains a Th1-polarization program in NKTs in vivo without causing their functional exhaustion. Furthermore, using CD62L as a marker of memory cells in human NKTs, we observe that IL-12 maintains long-term CD62L-expressing memory NKTs in vivo. Notably, IL-12 initiates a de novo programming of memory NKTs in CD62L-negative NKTs indicating that human NKTs circulating in the peripheral blood possess an intrinsic differentiation hierarchy, and that IL-12 plays a role in promoting their differentiation to long-lived Th1-polarized memory cells. Human NKTs engineered to co-express a Chimeric Antigen Receptor (CAR) coupled with the expression of IL-12 show enhanced antitumor activity in leukemia and neuroblastoma tumor models, persist long-term in vivo and conserve the molecular signature driven by the IL-12 expression. Thus IL-12 reveals an intrinsic plasticity of peripheral human NKTs that may play a crucial role in the development of cell therapeutics.

4-1BB-encoding CAR causes cell death via sequestration of the ubiquitin-modifying enzyme A20.

CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell cluster formation and cell death in the forms of both apoptosis and necroptosis in the absence of CAR tonic signaling. Mechanistic studies illustrate that 4-1BB sequesters A20 to the cell membrane in a TRAF-dependent manner causing A20 functional deficiency that in turn leads to NF-κB hyperactivity, cell aggregation via ICAM-1 overexpression, and cell death including necroptosis via RIPK1/RIPK3/MLKL pathway. Genetic modulations obtained by either overexpressing A20 or releasing A20 from 4-1BB by deleting the TRAF-binding motifs of 4-1BB rescue cell cluster formation and cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells.

Utilizing induced neural stem cell-based delivery of a cytokine cocktail to enhance chimeric antigen receptor-modified T-cell therapy for brain cancer.

Chimeric antigen receptor (CAR)-modified T-cell therapy has shown enormous clinical promise against blood cancers, yet efficacy against solid tumors remains a challenge. Here, we investigated the potential of a new combination cell therapy, where tumor-homing induced neural stem cells (iNSCs) are used to enhance CAR-T-cell therapy and achieve efficacious suppression of brain tumors. Using in vitro and in vivo migration assays, we found iNSC-secreted RANTES/IL-15 increased CAR-T-cell migration sixfold and expansion threefold, resulting in greater antitumor activity in a glioblastoma (GBM) tumor model. Furthermore, multimodal imaging showed iNSC delivery of RANTES/IL-15 in combination with intravenous administration of CAR-T cells reduced established orthotopic GBM xenografts 2538-fold within the first week, followed by durable tumor remission through 60 days post-treatment. By contrast, CAR-T-cell therapy alone only partially controlled tumor growth, with a median survival of only 19 days. Together, these studies demonstrate the potential of combined cell therapy platforms to improve the efficacy of CAR-T-cell therapy for brain tumors.

ALK inhibitors increase ALK expression and sensitize neuroblastoma cells to ALK.CAR-T cells.

Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy.

CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma.

BACKGROUND: Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40-60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. METHODS: In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. RESULTS: We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. CONCLUSIONS: In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

Bispecific binder redirected lentiviral vector enables in vivo engineering of CAR-T cells.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown considerable promise as a personalized cellular immunotherapy against B cell malignancies. However, the complex and lengthy manufacturing processes involved in generating CAR T cell products ex vivo result in substantial production time delays and high costs. Furthermore, ex vivo expansion of T cells promotes cell differentiation that reduces their in vivo replicative capacity and longevity. METHODS: Here, to overcome these limitations, CAR-T cells are engineered directly in vivo by administering a lentivirus expressing a mutant Sindbis envelope, coupled with a bispecific antibody binder that redirects the virus to CD3+ human T cells. RESULTS: This redirected lentiviral system offers exceptional specificity and efficiency; a single dose of the virus delivered to immunodeficient mice engrafted with human peripheral blood mononuclear cells generates CD19-specific CAR-T cells that markedly control the growth of an aggressive pre-established xenograft B cell tumor. CONCLUSIONS: These findings underscore in vivo engineering of CAR-T cells as a promising approach for personalized cancer immunotherapy.

Dual Targeting CAR-T Cells with Optimal Costimulation and Metabolic Fitness enhance Antitumor Activity and Prevent Escape in Solid Tumors.

Chimeric antigen receptor (CAR) T cells showed great activity in hematologic malignancies. However, heterogeneous antigen expression in tumor cells and suboptimal CAR-T cell persistence remain critical aspects to achieve clinical responses in patients with solid tumors. Here we show that CAR-T cells targeting simultaneously two tumor-associated antigens and providing transacting CD28 and 4-1BB costimulation, while sharing the sane CD3ζ-chain cause rapid antitumor effects in in vivo stress conditions, protection from tumor re-challenge and prevention of tumor escape due to low antigen density. Molecular and signaling studies indicate that T cells engineered with the proposed CAR design demonstrate sustained phosphorylation of T cell receptor-associated (TCR) signaling molecules and a molecular signature supporting CAR-T cell proliferation and long-term survival. Furthermore, metabolic profiling of CAR-T cells displayed induction of glycolysis that sustains rapid effector T cell function, but also preservation of oxidative functions, which are critical for T cell long-term persistence.

Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

Enhancing Chimeric Antigen Receptor T-Cell Efficacy in Solid Tumors.

Chimeric antigen receptor (CAR) T-cell therapy has been acclaimed as a revolution in cancer treatment following the impressive results in hematologic malignancies. Unfortunately, in patients with solid tumors, objectives responses to CAR T cells are still anecdotal, and important issues are driven by on-target but off-tumor activity of CAR T cells and by the extremely complex biology of solid tumors. Here, we will review the recent attempts to challenge the therapeutic impediments to CAR T-cell therapy in solid tumors. We will focus on the most promising strategies of antigen targeting to improve tumor specificity and address the tumor heterogeneity, efforts to circumvent the physical barriers of the tumor architecture such as subverted tumor vasculature, impediments of CAR T-cell trafficking and immune suppressive microenvironment.

GD2-specific CAR T cells encapsulated in an injectable hydrogel control retinoblastoma and preserve vision.

Retinoblastoma (RB) is a pediatric retinal tumor that overexpresses the ganglioside GD2. Although it is treatable in patients with early diagnosis, patients may lose one or two eyes. We generated GD2-specific chimeric antigen receptor T lymphocytes (GD2.CAR-Ts) and locally delivered them to mice with an in-situ grafting RB. When used in combination with the local release of interleukin (IL)-15 and an injectable hydrogel, we showed that GD2.CAR-Ts successfully eliminate RB tumor cells without impairment of the mouse vision.

A High-Avidity T-cell Receptor Redirects Natural Killer T-cell Specificity and Outcompetes the Endogenous Invariant T-cell Receptor.

T-cell receptor (TCR) gene transfer redirects T cells to target intracellular antigens. However, the potential autoreactivity generated by TCR mispairing and occurrence of graft-versus-host disease in the allogenic setting due to the retention of native TCRs remain major concerns. Natural killer T cells (NKT) have shown promise as a platform for adoptive T-cell therapy in cancer patients. Here, we showed their utility for TCR gene transfer. We successfully engineered and expanded NKTs expressing a functional TCR (TCR NKTs), showing HLA-restricted antitumor activity in xenogeneic mouse models in the absence of graft-versus-mouse reactions. We found that TCR NKTs downregulated the invariant TCR (iTCR), leading to iTCR+TCR+ and iTCR-TCR+ populations. In-depth analyses of these subsets revealed that in iTCR-TCR+ NKTs, the iTCR, although expressed at the mRNA and protein levels, was retained in the cytoplasm. This effect resulted from a competition for binding to CD3 molecules for cell-surface expression by the transgenic TCR. Overall, our results highlight the feasibility and advantages of using NKTs for TCR expression for adoptive cell immunotherapies. NKT-low intrinsic alloreactivity that associated with the observed iTCR displacement by the engineered TCR represents ideal characteristics for "off-the-shelf" products without further TCR gene editing.

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes.

PURPOSE: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice. EXPERIMENTAL DESIGN: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro, in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models. RESULTS: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK. CONCLUSIONS: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.

Eradication of Neuroblastoma by T Cells Redirected with an Optimized GD2-Specific Chimeric Antigen Receptor and Interleukin-15.

PURPOSE: A delay in encountering the cognate antigen while in the circulation, and the suboptimal costimulation received at the tumor site are key reasons for the limited activity of chimeric antigen receptor-redirected T cells (CAR-T) in solid tumors. We have explored the benefits of incorporating the IL15 cytokine within the CAR cassette to provide both a survival signal before antigen encounter, and an additional cytokine signaling at the tumor site using a neuroblastoma tumor model. EXPERIMENTAL DESIGN: We optimized the construct for the CAR specific for the NB-antigen GD2 without (GD2.CAR) or with IL15 (GD2.CAR.15). We then compared the expansion, phenotype, and antitumor activity of T cells transduced with these constructs against an array of neuroblastoma cell lines in vitro and in vivo using a xenogeneic metastatic model of neuroblastoma. RESULTS: We observed that optimized GD2.CAR.15-Ts have reduced expression of the PD-1 receptor, are enriched in stem cell-like cells, and have superior antitumor activity upon repetitive tumor exposures in vitro and in vivo as compared with GD2.CAR-Ts. Tumor rechallenge experiments in vivo further highlighted the role of IL15 in promoting enhanced CAR-T antitumor activity and survival, both in the peripheral blood and tissues. Finally, the inclusion of the inducible caspase-9 gene (iC9) safety switch warranted effective on demand elimination of the engineered GD2.CAR.15-Ts. CONCLUSIONS: Our results guide new therapeutic options for GD2.CAR-Ts in patients with neuroblastoma, and CAR-T development for a broad range of solid tumors.

Machine-Learning Prediction of Tumor Antigen Immunogenicity in the Selection of Therapeutic Epitopes.

Current tumor neoantigen calling algorithms primarily rely on epitope/major histocompatibility complex (MHC) binding affinity predictions to rank and select for potential epitope targets. These algorithms do not predict for epitope immunogenicity using approaches modeled from tumor-specific antigen data. Here, we describe peptide-intrinsic biochemical features associated with neoantigen and minor histocompatibility mismatch antigen immunogenicity and present a gradient boosting algorithm for predicting tumor antigen immunogenicity. This algorithm was validated in two murine tumor models and demonstrated the capacity to select for therapeutically active antigens. Immune correlates of neoantigen immunogenicity were studied in a pan-cancer data set from The Cancer Genome Atlas and demonstrated an association between expression of immunogenic neoantigens and immunity in colon and lung adenocarcinomas. Lastly, we present evidence for expression of an out-of-frame neoantigen that was capable of driving antitumor cytotoxic T-cell responses. With the growing clinical importance of tumor vaccine therapies, our approach may allow for better selection of therapeutically relevant tumor-specific antigens, including nonclassic out-of-frame antigens capable of driving antitumor immunity.

Treating hematological malignancies with cell therapy: where are we now?

INTRODUCTION: Adoptive cell therapy (ACT) is becoming an increasingly successful and widespread form of treatment for different types of cancer. Compared to chemotherapy or monoclonal antibodies, ACT is an active biological strategy, with infused immune cells featuring dynamic migration, expansion and long-term persistence properties. ACT in hematological malignancies offered the initial proof of principle of the feasibility for this innovative 'live-drug'. AREAS COVERED: In this review, the authors summarize the clinical results achieved with two specific strategies in hematological malignancies: chimeric antigen receptor (CAR) and T cell receptor (transgenic TCR) redirected T cells. Moreover, they discuss the recent pre-clinical studies aimed at increasing the feasibility, safety and efficacy of ACT. EXPERT OPINION: ACT can promote cancer regression in patients with leukemia, lymphoma and multiple myeloma. Nevertheless, more precise targeting of tumor cells and containment of side effects are needed. Overcoming tumor-associated immunosuppressive mechanisms and preventing tumor escape are also emerging as critical barriers. Finally, simplification in the manufacturing procedures should promote wider application of these technologies outside academic centers. Although the enthusiasm for ACT-based therapies is high, comprehensive and systematic clinical studies are required to advance the field.