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Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell–like gene profile, including the canonical markers CD3ε, CD5, and CD8β, as well as the T cell lineage–commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56bright to CD56dim, we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rβ, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

CD8(+) T cells are critical mediators of adaptive immunity but may also exhibit innate-like properties such as surface expression of NKG2C, an activating receptor typically associated with natural killer (NK) cells. We demonstrate that, similar to NK cells, NKG2C(+)TCRαβ(+)CD8(+) T cells are associated with prior human cytomegalovirus (HCMV) exposure. In addition to expressing several NK cell markers such as CD56 and KIR, NKG2C(+)CD8(+) T cells are oligoclonal and do not upregulate PD-1 even in response to persistent activation. Furthermore, we found that NKG2C(+)CD8(+) T cells from some individuals exhibited strong effector function against leukemia cells and HCMV-infected fibroblasts, which was dictated by both NKG2C and TCR specificity. Transcriptomic analysis revealed that the transcription factor BCL11B, a regulator of T cell developmental fate, is significantly downregulated in NKG2C(+)CD8(+) T cells when compared to conventional NKG2C(−)CD8(+) T cells. BCL11B deletion in conventional CD8(+) T cells resulted in the emergence of a similar innate-like CD56(+)CD94(+)DAP12(+)NKG2C(+)CD45RA(+)CCR7(−)PD-1(−/low) T cell population with activity against HLA-E(+) targets. Based on their intrinsic capacity to recognize diseased cells coupled with lack of PD-1 induction, NKG2C(+)CD8(+) T cells represent a lymphocyte population that resides at the boundary between innate and adaptive immunity, presenting an attractive alternative for cellular therapy, including CAR T-based therapies.

CD8+ T cells not only are critical mediators of adaptive immunity but also may exhibit innate-like properties such as surface expression of NKG2C, an activating receptor typically associated with n...

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell–like gene profile, including the canonical markers CD3ε, CD5, and CD8β, as well as the T cell lineage–commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56(bright) to CD56(dim), we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rβ, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

Multiple rodent and primate preclinical studies have advanced CAR T cells into the clinic. However, no single model accurately reflects the challenges of effective CAR T therapy in human cancer patients. To evaluate the effectiveness of next-generation CAR T cells that aim to overcome barriers to durable tumor elimination, we developed a system to evaluate CAR T cells in pet dogs with spontaneous cancer. Here we report on this system and the results of a pilot trial using CAR T cells to treat canine diffuse large B cell lymphoma (DLBCL). We designed and manufactured CD20-targeting, second-generation canine CAR T cells for functional evaluation in vitro and in vivo using lentivectors to parallel human CAR T cell manufacturing. A first-in-species trial of five dogs with DLBCL treated with CAR T was undertaken. Canine CAR T cells functioned in an antigen-specific manner and killed CD20+ targets. Circulating CAR T cells were detectable post-infusion, however, induction of canine anti-mouse antibodies (CAMA) was associated with CAR T cell loss. Specific selection pressure on CD20+ tumors was observed following CAR T cell therapy, culminating in antigen escape and emergence of CD20-disease. Patient survival times correlated with ex vivo product expansion. Altering product manufacturing improved transduction efficiency and skewed toward a memory-like phenotype of canine CAR T cells. Manufacturing of functional canine CAR T cells using a lentivector is feasible. Comparable challenges to effective CAR T cell therapy exist, indicating their relevance in informing future human clinical trial design.

Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans.

CD19 chimeric antigen receptor (CAR) T cell therapy has shown great promise in treating human hematological malignancies. However, the full potential of CAR technology has not been reached in solid malignancies and limitations exist in utilizing genetically identical murine models to interrogate the safety and efficacy of anti-tumor immune therapies. Canines are an outbred population that naturally develop spontaneous disease. Here we have established a treatment model for CAR T cell therapy in outbred dogs with spontaneous B cell lymphoma. We developed a protocol to isolate, activate, and express CAR in canine T cells. We characterized a methodology utilizing anti-cCD3 antibody with a K562 antigen-presenting cell (APC) line that expresses human CD32 and canine CD86 to activate canine lymphocytes. This method yields robust (50-120x) expansion of cCD4+ or cCD8+ T cells from normal or lymphoma-diseased dogs. Canine T cells were then engineered utilizing mRNA electroporation to express a CAR targeting the B cell antigen CD20. An anti-canine CD20 (cCD20) CAR was selected for proof of principle studies to establish feasibility of efficient CAR expression and redirected effector function in canine T cells from a healthy and lymphoma-diseased dogs. The cCD20-Z CAR construct was efficiently, but transiently, expressed in canine T cells after mRNA electroporation. cCD20 CAR T cells exhibited specific lysis and IFN-γ secretion in response to cCD20+ target cells. These data indicate successful establishment of a method to isolate, activate, and express a fully functional CAR in canine T cells for use in adoptive immunotherapy. These results rationalized testing of this CAR approach in canines with advanced CD20+ malignancies. In an IACUC approved protocol, we administered three doses of autologous cCD20-Z RNA CAR+ T cells to a companion canine with advanced B cell lymphoma. Treatment was well-tolerated and no evidence of serious adverse events was observed. The anti-tumor effect, however, was transient, suggesting a need for product optimization. Future study will establish a lenti- or retroviral-based method to modify canine T cells to promote T cell persistence and durable anti-tumor effects in vivo. In conclusion, our study establishes the methodologies and feasibility of anti-cCD20 CAR therapy in canines with spontaneous B cell lymphoma that may represent a future clinical option for such companion pets. This work also lays the foundation for study of a large, outbred animal model that better resembles spontaneous, human malignancies to allow for improved assessment for potential toxicity risks prior to translation into human studies.

Chimeric antigen receptor (CAR) T cell therapy has shown great promise in treating human leukemias. However, for CAR T technology to achieve its full potential against solid malignancies, better pre-clinical models are needed that recapitulate the immune suppressive tumor microenvironment and the distribution of target antigens. Pet dogs are an outbred population that have a close phylogenetic relationship with man and frequently develop naturally occurring, spontaneous malignancies. We explored the feasibility of evaluating CAR T cell technologies in dogs with spontaneous cancers by assessing CD20-targeting CAR T cells in dogs with relapsed CD20+ B cell lymphoma.We have developed standard procedures for optimal ex vivo canine T cell activation, expansion, and lentiviral transduction. Canine PBLs were activated in vitro using artificial antigen presenting cells (aAPCs) loaded with anti-canine CD3, in the presence of rhIL-2 and rhIL-21, and the time of peak T cell activation and disappearance of aAPCs from the culture were determined. Peak CD25 expression was observed 3-4 days post-stimulation, when less than 5% of aAPCs remained in culture, eliminating potential competitor cells for viral infection. Transduction of canine T cells, 4 days post-stimulation using VSVg-pseudotyped lentivirus encoding a second-generation CD20-targeting CAR (CD20-28-ζ) at an MOI of 20 resulted in up to 26% surface expression of the CAR on T cells grown from a healthy dog. In vitro co-culture of CD20-specific canine CAR T cells derived from a lymphoma patient with CD20+ target cells resulted in antigen-specific CAR T cell expansion and effective killing.To determine whether canine CD20 CAR T cells could survive, expand and exhibit cytotoxic activity in vivo, three pet dogs with relapsed CD20+B cell lymphoma were treated with autologous CD20-28-ζ CAR T cells in an IACUC-approved protocol. PBLs were transduced (1.5-6.5%), expanded (22-153 fold) and infused into the patients either IV or IV plus intra-nodal injection. A 52% decrease in tumor volume was observed in the first patient 4 days post-IV infusion, coinciding with an 82% increase in T cell frequency within the malignant lymph node. CAR T cells were administered via intra-nodal injection into the largest malignant node of the second dog, which stayed stable in volume over two weeks while all other non-injected malignant nodes doubled in size. The third dog received the largest number of CAR T cells administered IV. In this dog, CAR T cells have expanded and persisted for three weeks and malignant nodes have remained below the limit of accurate measurement.These data demonstrate the ability of canine CAR T cells to function in vitro and to halt and even temporarily reverse solid tumor progression in canine patients, mirroring what has been observed in human disease and treatment. This work shows the feasibility of this approach in dogs and offers a spontaneous cancer model for pre-clinical testing of novel CAR targets and next generation CAR design that aims to inform human clinical trials.

Chimeric antigen receptor (CAR) therapy has demonstrated great promise in treating human leukemias, but preclinical murine models are limited in their ability to predict safety and efficacy in humans. Given the rapid and on-going advances in CAR T cell technology in the laboratory, it now becomes necessary to identify and develop an outbred, large animal spontaneous cancer model in which the safety of novel targets and therapeutic effectiveness of re-directed T cells can be evaluated and optimized. Canines naturally develop spontaneous B cell lymphoma, and we have previously shown the feasibility of evaluating CAR T cell therapy in dogs using CD20-targeting RNA CAR T cells. To parallel human CAR T cell approaches, we have now optimized primary canine T cell transduction with lentivirus. Once peak T cell activation and artificial antigen presenting cell elimination timeframes were established, transduction yielded up to 26% CAR+ canine T cells. Canine T cells transduced with a second generation CD20-targeting CAR (CD20-28-ζ) demonstrated CAR-mediated, antigen-specific proliferation and efficient cytolysis of a CD20+ canine B cell lymphoma line in vitro. Three relapsed canine B cell lymphoma patients were treated with autologous CD20-28-ζ CAR T cells. Despite the administration of low numbers of CAR T cells, transient anti-tumor effects were observed in the first 2 patients. Following administration of ~700,000 CAR T cells/kg intravenously to the third patient, CAR T cells were found to persist and expand in the periphery and malignant nodes and disease progression was halted. This work establishes the feasibility of using the dogs with spontaneous cancer as pre-clinical models for advancing human CAR T cell therapy.