TCRvß-CART therapy mediates high-precision targeting of malignant T-cell clones.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of lymphoid malignancies associated with poor prognosis due to ineffective treatment options and high rates of relapse. The success of chimeric antigen receptor T-cell (CART) therapy for certain hematologic malignancies makes it an attractive treatment option for PTCLs. However, shared expression of potential target antigens by both malignant and healthy T cells poses a challenge. Current prospective CART approaches cause a high degree of on-target, off-tumor activity, resulting in fratricide during CART expansion, depletion of healthy T cells in vivo, and immune compromise in the patient. To limit off-tumor targeting, we sought to develop a CART platform specific for a given T-cell receptor vß (TCRvß) family that would endow CAR-modified T cells with the ability to mediate lysis of the clonal malignant population while preserving the majority of healthy T cells. Here, CAR constructs specific for multiple TCRvß family members were designed and validated. Our results demonstrate that TCRvß-family-specific CARTs (TCRvß-CARTs) recognize and kill TCRvß-expressing target cells. This includes specific self-depletion of the targeted cell subpopulation in the CART product and lysis of cell lines engineered to express a target TCRvß family. Furthermore, TCRvß-CARTs eliminated the dominant malignant TCRvß clone in 2 patient samples. Finally, in immunodeficient mice, TCRvß-CARTs eradicated malignant cells in a TCRvß-dependent manner. Importantly, the nontargeted TCRvß families were spared in all cases. Thus, TCRvß-CART therapy provides a potential option for high-precision treatment of PTCL with limited healthy T-cell depletion.

Expression of inducible factors reprograms CAR-T cells for enhanced function and safety.

Despite the success of CAR-T cell cancer immunotherapy, challenges in efficacy and safety remain. Investigators have begun to enhance CAR-T cells with the expression of accessory molecules to address these challenges. Current systems rely on constitutive transgene expression or multiple viral vectors, resulting in unregulated response and product heterogeneity. Here, we develop a genetic platform that combines autonomous antigen-induced production of an accessory molecule with constitutive CAR expression in a single lentiviral vector called Uni-Vect. The broad therapeutic application of Uni-Vect is demonstrated in vivo by activation-dependent expression of (1) an immunostimulatory cytokine that improves efficacy, (2) an antibody that ameliorates cytokine-release syndrome, and (3) transcription factors that modulate T cell biology. Uni-Vect is also implemented as a platform to characterize immune receptors. Overall, we demonstrate that Uni-Vect provides a foundation for a more clinically actionable next-generation cellular immunotherapy.

Dichotomous impact of affinity on the function of T cell engaging bispecific antibodies.

BACKGROUND: Bispecific T cell engagers represent the majority of bispecific antibodies (BsAbs) entering the clinic to treat metastatic cancer. The ability to apply these agents safely and efficaciously in the clinic, particularly for solid tumors, has been challenging. Many preclinical studies have evaluated parameters related to the activity of T cell engaging BsAbs, but many questions remain. MAIN BODY: This study investigates the impact of affinity of T cell engaging BsAbs with regards to potency, efficacy, and induction of immunomodulatory receptors/ligands using HER-2/CD3 BsAbs as a model system. We show that an IgG BsAb can be as efficacious as a smaller BsAb format both in vitro and in vivo. We uncover a dichotomous relationship between tumor-associated antigen (TAA) affinity and CD3 affinity requirements for cells that express high versus low levels of TAA. HER-2 affinity directly correlated with the CD3 engager lysis potency of HER-2/CD3 BsAbs when HER-2 receptor numbers are high (~200 K/cell), while the CD3 affinity did not impact potency until its binding affinity was extremely low (<600 nM). When HER-2 receptor numbers were lower (~20 K/cell), both HER-2 and CD3 affinity impacted potency. The high affinity anti-HER-2/low CD3 affinity BsAb also demonstrated lower cytokine induction levels in vivo and a dosing paradigm atypical of extremely high potency T cell engaging BsAbs reaching peak efficacy at doses >3 mg/kg. This data confirms that low CD3 affinity provides an opportunity for improved safety and dosing for T cell engaging BsAbs. T cell redirection also led to upregulation of Programmed cell death 1 (PD-1) and 4-1BB, but not CTLA-4 on T cells, and to Programmed death-ligand 1 (PD-L1) upregulation on HER-2HI SKOV3 tumor cells, but not on HER-2LO OVCAR3 tumor cells. Using this information, we combined anti-PD-1 or anti-4-1BB monoclonal antibodies with the HER-2/CD3 BsAb in vivo and demonstrated significantly increased efficacy against HER-2HI SKOV3 tumors via both combinations. CONCLUSIONS: Overall, these studies provide an informational dive into the optimization process of CD3 engaging BsAbs for solid tumors indicating that a reduced affinity for CD3 may enable a better therapeutic index with a greater selectivity for the target tumor and a reduced cytokine release syndrome. These studies also provide an additional argument for combining T cell checkpoint inhibition and co-stimulation to achieve optimal efficacy.

Folate Receptor Beta as a Direct and Indirect Target for Antibody-Based Cancer Immunotherapy.

Folate receptor beta (FRß) is a folate binding receptor expressed on myeloid lineage hematopoietic cells. FRß is commonly expressed at high levels on malignant blasts in patients with acute myeloid leukemia (AML), as well as on M2 polarized tumor-associated macrophages (TAMs) in the tumor microenvironment of many solid tumors. Therefore, FRß is a potential target for both direct and indirect cancer therapy. We demonstrate that FRß is expressed in both AML cell lines and patient-derived AML samples and that a high-affinity monoclonal antibody against FRß (m909) has the ability to cause dose- and expression-dependent ADCC against these cells in vitro. Importantly, we find that administration of m909 has a significant impact on tumor growth in a humanized mouse model of AML. Surprisingly, m909 functions in vivo with and without the infusion of human NK cells as mediators of ADCC, suggesting potential involvement of mouse macrophages as effector cells. We also found that TAMs from primary ovarian ascites samples expressed appreciable levels of FRß and that m909 has the ability to cause ADCC in these samples. These results indicate that the targeting of FRß using m909 has the potential to limit the outgrowth of AML in vitro and in vivo. Additionally, m909 causes cytotoxicity to TAMs in the tumor microenvironment of ovarian cancer warranting further investigation of m909 and its derivatives as therapeutic agents in patients with FRß-expressing cancers.

CAR-T cell-mediated depletion of immunosuppressive tumor-associated macrophages promotes endogenous antitumor immunity and augments adoptive immunotherapy.

The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Here, we demonstrate that a subset of TAMs that express folate receptor ß (FRß) possess an immunosuppressive M2-like profile. In syngeneic tumor mouse models, chimeric antigen receptor (CAR)-T cell-mediated selective elimination of FRß+ TAMs in the TME results in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8+ T cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRß-specific CAR-T cells also improves the effectiveness of tumor-directed anti-mesothelin CAR-T cells, while simultaneous co-administration of both CAR products does not. These results highlight the pro-tumor role of FRß+ TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens.

Quantitative Control of Gene-Engineered T-Cell Activity through the Covalent Attachment of Targeting Ligands to a Universal Immune Receptor.

Universal immune receptors represent a rapidly emerging form of adoptive T-cell therapy with the potential to overcome safety and antigen escape challenges faced by conventional chimeric antigen receptor (CAR) T-cell therapy. By decoupling antigen recognition and T-cell signaling domains via bifunctional antigen-specific targeting ligands, universal immune receptors can regulate T-cell effector function and target multiple antigens with a single receptor. Here, we describe the development of the SpyCatcher immune receptor, the first universal immune receptor that allows for the post-translational covalent attachment of targeting ligands at the T-cell surface through the application of SpyCatcher-SpyTag chemistry. The SpyCatcher immune receptor redirected primary human T cells against a variety of tumor antigens via the addition of SpyTag-labeled targeting ligands, both in vitro and in vivo. SpyCatcher T-cell activity relied upon the presence of both target antigen and SpyTag-labeled targeting ligand, allowing for dose-dependent control of function. The mutational disruption of covalent bond formation between the receptor and the targeting ligand still permitted redirected T-cell function but significantly compromised antitumor function. Thus, the SpyCatcher immune receptor allows for rapid antigen-specific receptor assembly, multiantigen targeting, and controllable T-cell activity.

CAR T Cells Targeting MISIIR for the Treatment of Ovarian Cancer and Other Gynecologic Malignancies.

The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor, and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a transforming growth factor ß (TGF-ß) receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) T cell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR T cell approach. Herein, we describe the development and evaluation of a CAR that targets MISIIR. T cells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity in vitro and eliminated MISIIR-overexpressing tumors in vivo. MISIIR CAR T cells also recognized a panel of human ovarian and endometrial cancer cell lines, and they lysed a battery of patient-derived tumor specimens in vitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology.

Multiparameter comparative analysis reveals differential impacts of various cytokines on CART cell phenotype and function ex vivo and in vivo.

Exogenous cytokines are widely applied to enhance the anti-tumor ability of immune cells. However, systematic comparative studies of their effects on chimeric antigen receptor (CAR)-engineered T (CART) cells are lacking. In this study, CART cells targeting folate receptor-alpha were generated and expanded ex vivo in the presence of different cytokines (IL-2, IL-7, IL-15, IL-18, and IL-21), and their expansion, phenotype and cytotoxic capacity were evaluated, in vitro and in vivo. Moreover, the effect of the administration of these cytokines along with CART cells in vivo was also studied. IL-2, IL-7, and IL-15 favored the ex vivo expansion of CART cells compared to other cytokines or no cytokine treatment. IL-7 induced the highest proportion of memory stem cell-like CART cells in the final product, and IL-21 supported the expansion of CART cells with a younger phenotype, while IL-2 induced more differentiated CART cells. IL-2 and IL-15-exposed CART cells secreted more proinflammatory cytokines and presented stronger tumor-lysis ability in vitro. However, when tested in vivo, CART cells exposed to IL-2 ex vivo showed the least anti-tumor effect. In contrast, the administration of IL-15 and IL-21 in combination with CART cells in vivo increased their tumor killing capacity. According to our results, IL-7 and IL-15 show promise to promote ex vivo expansion of CART cells, while IL-15 and IL-21 seem better suited for in vivo administration after CART cell infusion. Collectively, these results may have a profound impact on the efficacy of CART cells in both hematologic and solid cancers.

Tumor Regression and Delayed Onset Toxicity Following B7-H4 CAR T Cell Therapy.

B7-H4 protein is frequently overexpressed in ovarian cancer. Here, we engineered T cells with novel B7-H4-specific chimeric antigen receptors (CARs) that recognized both human and murine B7-H4 to test the hypothesis that B7-H4 CAR T cell therapy can be applied safely in preclinical models. B7-H4 CAR T cells specifically secreted IFN-γ and lysed B7-H4(+) targets. In vivo, B7-H4 CAR T cells displayed antitumor reactivity against B7-H4(+) human ovarian tumor xenografts. Unexpectedly, B7-H4 CAR T cell treatment reproducibly showed delayed, lethal toxicity 6-8 weeks after therapy. Comprehensive assessment of murine B7-H4 protein distribution uncovered expression in ductal and mucosal epithelial cells in normal tissues. Postmortem analysis revealed the presence of widespread histologic lesions that correlated with B7-H4(+) expression, and were inconsistent with graft versus host disease. Lastly, expression patterns of B7-H4 protein in normal human tissue were comparable to distribution in mice, advancing our understanding of B7-H4. We conclude that B7-H4 CAR therapy mediates control of cancer outgrowth. However, long-term engraftment of B7-H4 CAR T cells mediates lethal, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. This model system provides a unique opportunity for preclinical evaluation of safety approaches that limit CAR-mediated toxicity after tumor destruction in vivo.

Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level.

BACKGROUND: The poor prognosis and the limited efficacy of targeted therapy in patients with triple-negative breast cancer (TNBC) have raised the need for alternative therapies. Recent studies have demonstrated that folate receptor-alpha (FRα) may represent an ideal tumor-associated marker for immunotherapy for TNBC. METHODS: The aim of the present study was to apply a chimeric antigen receptor (CAR) approach for the targeting of FRα expressed on TNBC cells and evaluate the antitumor activity of CAR-engineered T cells in vitro and in vivo. RESULTS: We found that human T cells expressing a FRα-specific CAR were potent and specific killers of TNBC cells that express moderate levels of FRα in vitro and significantly inhibited tumor outgrowth following infusion into immunodeficient mice bearing an MDA-MB-231 tumor xenograft. However, the antitumor activity of the FRα CAR T cells was modest when compared to the same CAR T cells applied in an ovarian tumor xenograft model where FRα expression is more abundant. Notably, FRα CAR T cells induced superior tumor regression in vivo against MDA-MB-231 that was engineered for overexpression of FRα. CONCLUSIONS: Taken together, our results show that FRα CAR T cells can mediate antitumor activity against established TNBC tumor, particularly when FRα is expressed at higher levels. These results have significant implications for the pre-selection of patients with high antigen expression levels when utilizing CAR-based adoptive T cell therapies of cancer in future clinical trials.

Rigorous optimization and validation of potent RNA CAR T cell therapy for the treatment of common epithelial cancers expressing folate receptor.

Using lentiviral technology, we recently demonstrated that incorporation of CD27 costimulation into CARs greatly improves antitumor activity and T cell persistence. Still, virus-mediated gene transfer is expensive, laborious and enables long-term persistence, creating therapies which cannot be easily discontinued if toxic. To address these concerns, we utilized a non-integrating RNA platform to engineer human T cells to express FRα-specific, CD27 CARs and tested their capacity to eliminate human FRα(+) cancer. Novel CARs comprised of human components were constructed, C4-27z and C4opt-27z, a codon-optimized variant created for efficient expression. Following RNA electroporation, C4-27z and C4opt-27z CAR expression is initially ubiquitous but progressively declines across T cell populations. In addition, C4-27z and C4opt-27z RNA CAR T cells secrete high levels of Th-1 cytokines and display strong cytolytic function against human FRα(+) cancers in a time- and antigen-dependent manner. Further, C4-27z and C4opt-27z CAR T cells exhibit significant proliferation in vivo, facilitate the complete regression of fully disseminated human ovarian cancer xenografts in mice and reduce the progression of solid ovarian cancer. These results advocate for rapid progression of C4opt-27z RNA CAR to the clinic and establish a new paradigm for preclinical optimization and validation of RNA CAR candidates destined for clinical translation.

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity.

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate "on-target" toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and "on-target off-tumor" toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs.

Follicle-Stimulating Hormone Receptor as a Target in the Redirected T-cell Therapy for Cancer.

Adoptive transfer of T cells engineered to express chimeric immunoreceptors is an effective strategy to treat hematologic cancers; however, the use of this type of therapy for solid cancers, such as ovarian cancer, remains challenging because a safe and effective immunotherapeutic target has not yet been identified. Here, we constructed and evaluated a novel redirected T-cell-based immunotherapy targeting human follicle-stimulating hormone receptor (FSHR), a highly conserved molecule in vertebrate animals with expression limited to gonadal tissues, ovarian cancer, and cancer-associated vasculature. Receptor ligand-based anti-FSHR immunoreceptors were constructed that contained small binding fragments from the ligand for FSHR, FSH, fused to T-cell transmembrane and T-cell signaling domains. Human T cells transduced to express anti-FSHR immunoreceptors were specifically immunoreactive against FSHR-expressing human and mouse ovarian cancer cell lines in an MHC-nonrestricted manner and mediated effective lysis of FHSR-expressing tumor cells, but not FSHR-deficient targets, in vitro. Similarly, the outgrowth of human ovarian cancer xenografts in immunodeficient mice was significantly inhibited by the adoptive transfer of FSHR-redirected T cells. Our experimental observations show that FSHR is a promising immunotherapeutic target for ovarian cancer and support further exploration of FSHR-targeted immune therapy approaches for patients with cancer.

Targeting of folate receptor ß on acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells.

T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor ß (FRß) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRß (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRß as well as endogenous FRß(+) AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRß(+) THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRß expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRß expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34(+) HSCs in vitro. Our results indicate that FRß is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA.

A human ErbB2-specific T-cell receptor confers potent antitumor effector functions in genetically engineered primary cytotoxic lymphocytes.

The ErbB2 protein is a member of the tyrosine kinase family of growth factor receptors that is overexpressed in cancers of the breast, ovary, stomach, kidney, colon, and lung, and therefore represents an attractive candidate antigen for targeted cancer immunotherapy. Cytotoxic T lymphocytes specific for various immunogenic ErbB2 peptides have been described, but they often exhibit both poor functional avidity and tumor reactivity. In order to generate potent CD8(+) T cells with specificity for the ErbB2(369-377) peptide, we performed one round of in vitro peptide stimulation of CD8(+) T cells isolated from an HLA-A2(+) patient who was previously vaccinated with autologous dendritic cells pulsed with HLA class I ErbB2 peptides. Using this approach, we enriched highly avid ErbB2-reactive T cells with strong ErbB2-specific, antitumor effector functions. We then stimulated these ErbB2-reactive T cells with ErbB2(+) HLA-A2(+) tumor cells in vitro and sorted tumor-activated ErbB2(369-377) peptide T cells, which allowed for the isolation of a novel T-cell receptor (TCR) with ErbB2(369-377) peptide specificity. Primary human CD8(+) T cells genetically modified to express this ErbB2-specific TCR specifically bound ErbB2(369-377) peptide containing HLA-A2 tetramers, and efficiently recognized target cells pulsed with low nanomolar concentrations of ErbB2(369-377) peptide as well as nonpulsed ErbB2(+) HLA-A2(+) tumor cell lines in vitro. In a novel xenograft model, ErbB2-redirected T cells also significantly delayed progression of ErbB2(+) HLA-A2(+) human tumor in vivo. Together, these results support the notion that redirection of normal T-cell specificity by TCR gene transfer can have potential applications in the adoptive immunotherapy of ErbB2-expressing malignancies.

CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor.

PURPOSE: Upregulation of CD137 (4-1BB) on recently activated CD8(+) T cells has been used to identify rare viral or tumor antigen-specific T cells from peripheral blood. Here, we evaluated the immunobiology of CD137 in human cancer and the utility of a CD137-positive separation methodology for the identification and enrichment of fresh tumor-reactive tumor-infiltrating lymphocytes (TIL) or tumor-associated lymphocytes (TAL) from ascites for use in adoptive immunotherapy. EXPERIMENTAL DESIGN: TILs from resected ovarian cancer or melanoma were measured for surface CD137 expression directly or after overnight incubation in the presence of tumor cells and homeostatic cytokines. CD137(pos) TILs were sorted and evaluated for antitumor activity in vitro and in vivo. RESULTS: Fresh ovarian TILs and TALs naturally expressed higher levels of CD137 than circulating T cells. An HLA-dependent increase in CD137 expression was observed following incubation of fresh enzyme-digested tumor or ascites in IL-7 and IL-15 cytokines, but not IL-2. Enriched CD137(pos) TILs, but not PD-1(pos) or PD-1(neg) CD137(neg) cells, possessed autologous tumor reactivity in vitro and in vivo. In melanoma studies, all MART-1-specific CD8(+) TILs upregulated CD137 expression after incubation with HLA-matched, MART-expressing cancer cells and antigen-specific effector function was restricted to the CD137(pos) subset in vitro. CD137(pos) TILs also mediated superior antitumor effects in vivo, compared with CD137(neg) TILs. CONCLUSIONS: Our findings reveal a role for the TNFR-family member CD137 in the immunobiology of human cancer where it is preferentially expressed on tumor-reactive subset of TILs, thus rationalizing its agonistic engagement in vivo and its use in TIL selection for adoptive immunotherapy trials.

Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo.

Adoptive immunotherapy using T lymphocytes genetically modified to express a chimeric antigen receptor (CAR-T) holds considerable promise for the treatment of cancer. However, CAR-based therapies may involve on-target toxicity against normal tissues expressing low amounts of the targeted tumor-associated antigen (TAA). To specify T cells for robust effector function that is selective for tumor but not normal tissue, we developed a trans-signaling CAR strategy, whereby T-cell activation signal 1 (CD3z) is physically dissociated from costimulatory signal 2 (CD28) in two CARs of differing antigen specificity: mesothelin and a-folate receptor (FRa). Human T cells were genetically modified to coexpress signal 1 (anti-Meso scFv-CD3z) and signal 2 (anti-FRa scFv-CD28) CARs in trans. Trans-signaling CAR-T cells showed weak cytokine secretion against target cells expressing only one TAA in vitro, similar to first-generation CAR-T cells bearing CD3z only, but showed enhanced cytokine secretion upon encountering natural or engineered tumor cells coexpressing both antigens, equivalent to that of second-generation CAR-T cells with dual signaling in cis. CAR-T cells with dual specificity also showed potent anticancer activity and persistence in vivo, which was superior to first-generation CAR-T cells and equivalent to second-generation CARs. Importantly, second-generation CAR-T cells exhibited potent activity against cells expressing mesothelin alone, recapitulating normal tissue, whereas trans-signaling CAR-T cells did not. Thus, a dual specificity, trans-signaling CAR approach can potentiate the therapeutic efficacy of CAR-T cells against cancer while minimizing parallel reactivity against normal tissues bearing single antigen.

A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor.

Adoptive immunotherapies composed of T cells engineered to express a chimeric antigen receptor (CAR) offer an attractive strategy for treatment of human cancer. However, CARs have a fixed antigen specificity such that only one tumor-associated antigen (TAA) can be targeted, limiting the efficacy that can be achieved because of heterogeneous TAA expression. For this reason, a more generalized and effective application of CAR therapy would benefit from the capability to produce large panels of CARs against many known TAAs. In this study, we show a novel strategy to extend the recognition specificity potential of a bioengineered lymphocyte population, allowing flexible approaches to redirect T cells against various TAAs. Our strategy employs a biotin-binding immune receptor (BBIR) composed of an extracellular-modified avidin linked to an intracellular T-cell signaling domain. BBIR T cells recognized and bound exclusively to cancer cells pretargeted with specific biotinylated molecules. The versatility afforded by BBIRs permitted sequential or simultaneous targeting of a combination of distinct antigens. Together, our findings show that a platform of universal T-cell specificity can significantly extend conventional CAR approaches, permitting the tailored generation of T cells of unlimited antigen specificity for improving the effectiveness of adoptive T-cell immunotherapies for cancer.

Redirected antitumor activity of primary human lymphocytes transduced with a fully human anti-mesothelin chimeric receptor.

Cancer regression by gene-modified T cells bearing a chimeric antigen receptor (CAR) exodomain of mouse origin can be limited by the induction of transgene immunogenicity resulting in poor persistence and function in vivo. The development of functionally-active CAR of human origin can address this issue. Here, we constructed and evaluated fully human anti-mesothelin CARs comprised of a human mesothelin-specific single-chain antibody variable fragment (P4 scFv) coupled to T cell signaling domains. Primary human T cells expressing P4 CAR specifically produced proinflammatory cytokines, degranulated and exerted potent cytolytic functions when cultured with mesothelin-expressing tumors in vitro. P4 CAR T cells also mediated bystander killing of mesothelin-negative cancer cells during coculture. CAR reactivity was not abrogated by soluble tumor-secreted or recombinant mesothelin protein even at supraphysiological levels. Importantly, adoptive transfer of P4 CAR-expressing T cells mediated the regression of large, established tumor in the presence of soluble mesothelin in a xenogenic model of human ovarian cancer. Thus, primary human T cells expressing fully human anti-mesothelin CAR efficiently kill mesothelin-expressing tumors in vitro and in vivo and have the potential to overcome the issue of transgene immunogenicity that may limit CAR T cell trials that utilize scFvs of mouse origin.