Adoptive immunotherapy for cancer or viruses.

Adoptive immunotherapy, or the infusion of lymphocytes, is a promising approach for the treatment of cancer and certain chronic viral infections. The application of the principles of synthetic biology to enhance T cell function has resulted in substantial increases in clinical efficacy. The primary challenge to the field is to identify tumor-specific targets to avoid off-tumor, on-target toxicity. Given recent advances in efficacy in numerous pilot trials, the next steps in clinical development will require multicenter trials to establish adoptive immunotherapy as a mainstream technology.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.

Dominant-Negative TGF-ß Receptor Enhances PSMA-Targeted Human CAR T Cell Proliferation And Augments Prostate Cancer Eradication.

Cancer has an impressive ability to evolve multiple processes to evade therapies. While immunotherapies and vaccines have shown great promise, particularly in certain solid tumors such as prostate cancer, they have been met with resistance from tumors that use a multitude of mechanisms of immunosuppression to limit effectiveness. Prostate cancer, in particular, secretes transforming growth factor ß (TGF-ß) as a means to inhibit immunity while allowing for cancer progression. Blocking TGF-ß signaling in T cells increases their ability to infiltrate, proliferate, and mediate antitumor responses in prostate cancer models. We tested whether the potency of chimeric antigen receptor (CAR) T cells directed to prostate-specific membrane antigen (PSMA) could be enhanced by the co-expression of a dominant-negative TGF-ßRII (dnTGF-ßRII). Upon expression of the dominant-negative TGF-ßRII in CAR T cells, we observed increased proliferation of these lymphocytes, enhanced cytokine secretion, resistance to exhaustion, long-term in vivo persistence, and the induction of tumor eradication in aggressive human prostate cancer mouse models. Based on our observations, we initiated a phase I clinical trial to assess these CAR T cells as a novel approach for patients with relapsed and refractory metastatic prostate cancer (ClinicalTrials.gov: NCT03089203).

A prospective, randomized study on hepatotoxicity of anastrozole compared with tamoxifen in women with breast cancer.

Tamoxifen and anastrozole are widely used as adjuvant treatment for early stage breast cancer, but their hepatotoxicity is not fully defined. We aimed to compare hepatotoxicity of anastrozole with tamoxifen in the adjuvant setting in postmenopausal breast cancer patients. Three hundred and fifty-three Chinese postmenopausal women with hormone receptor-positive early breast cancer were randomized to anastrozole or tamoxifen after optimal primary therapy. The primary end-point was fatty liver disease, defined as a liver-spleen ratio <0.9 as determined using a computed tomography scan. The secondary end-points included abnormal liver function and treatment failure during the 3-year follow up. The cumulative incidence of fatty liver disease after 3 years was lower in the anastrozole arm than that of tamoxifen (14.6% vs 41.1%, P < 0.0001; relative risk, 0.30; 95% CI, 0.21-0.45). However, there was no difference in the cumulative incidence of abnormal liver function (24.6% vs 24.7%, P = 0.61). Interestingly, a higher treatment failure rate was observed in the tamoxifen arm compared with anastrozole and median times to treatment failure were 15.1 months and 37.1 months, respectively (P < 0.0001; HR, 0.27; 95% CI, 0.20-0.37). The most commonly reported adverse events were 'reproductive system disorders' in the tamoxifen group (17.1%), and 'musculoskeletal disorders' in the anastrozole group (14.6%). Postmenopausal women with hormone receptor-positive breast cancer receiving adjuvant anastrozole displayed less fatty liver disease, suggesting that this drug had a more favorable hepatic safety profile than tamoxifen and may be preferred for patients with potential hepatic dysfunction.

Engineered T cells for cancer therapy.

It is now well established that the immune system can control and eliminate cancer cells. Adoptive T cell transfer has the potential to overcome the significant limitations associated with vaccine-based strategies in patients who are often immune compromised. Application of the emerging discipline of synthetic biology to cancer, which combines elements of genetic engineering and molecular biology to create new biological structures with enhanced functionalities, is the subject of this focused research review.

TCR affinity and specificity requirements for human regulatory T-cell function.

We investigated whether TCRs restricted to the more ubiquitously expressed MHC class I molecules could be used to redirect human regulatory T cells (Tregs). Using a series of HLA-A2-restricted TCRs that recognize the same peptide-MHC class I complex (pMHC) with affinities varying up to 3500 fold, we observed that TCR affinity had no effect on the ability of the introduced TCRs to confer potent Ag-specific suppressive activity. Surprisingly, we found a naturally occurring, low-affinity MHC class I-restricted TCR specific for an NY-ESO-1 epitope that was unable to redirect a functional CD4 T-effector cell response could confer potent antigen-specific suppressive activity when expressed in Tregs and severely impair the expansion of highly functional HIV-1(GAG)-specific CD8 T cells expressing a high-affinity TCR. This suppressive activity was only observed when both Ags were presented by the same cell, and no suppression was observed when the target Ags were put in distinct cells. These studies underscore the clinical utility of using MHC class I-restricted TCRs to endow Tregs with specificity to control autoimmune disease and highlight the conditions in which this approach would have most therapeutic benefit.

Relation of clinical culture method to T-cell memory status and efficacy in xenograft models of adoptive immunotherapy.

BACKGROUND AIMS: Cytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in pre-clinical models, and this efficacy has translated to success in several clinical trials. Many early trials were disappointing in large part because of the lack of proliferation and subsequent persistence of transferred cells. Recent investigations have pointed to the importance of delivering highly proliferative cells, whether of naive or early memory phenotypes. METHODS: We investigated the influence of two common cell culturing methods used in early trials and their relationship to T-cell phenotype and pre-clinical efficacy. RESULTS: We observed that stimulation with soluble anti-CD3 antibody OKT-3 and high-dose interleukin-2 produces more effector memory-type T cells with shorter average telomeres when compared with cells generated with the use of CD3/CD28 beads. When used in xenograft models of leukemia, bead-stimulated cells proliferated earlier and to a higher degree than those generated with the use of OKT-3/IL2 and resulted in better disease control despite no difference in distribution or migration throughout the mouse. Inclusion of the known successful clinical 4-1BB endodomain in the CAR could not rescue the function of OKT-3/IL-2-cultured cells. T cells isolated from animals that survived long-term (>120 days) retained a central memory-like phenotype and demonstrated a memory response to a large re-challenge of CD19-positive leukemia. CONCLUSIONS: In summary, we confirm that cells with a younger phenotype or higher proliferative capacity perform better in pre-clinical models and that cell culturing influences cell phenotype seemingly independent of the 4-1BB endodomain in the CAR structure.

Mutant presenilin-1 deregulated peripheral immunity exacerbates Alzheimer-like pathology.

Mutations in the presenilin-1 (PS1) gene are independent causes of familial Alzheimer's disease (AD). AD patients have dysregulated immunity, and PS1 mutant mice exhibit abnormal systemic immune responses. To test whether immune function abnormality caused by a mutant human PS1 gene (mhPS1) could modify AD-like pathology, we reconstituted immune systems of AD model mice carrying a mutant human amyloid precursor protein gene (mhAPP; Tg2576 mice) or both mhAPP and mhPS1 genes (PSAPP mice) with allogeneic bone marrow cells. Here, we report a marked reduction in amyloid-ß (Aß) levels, ß-amyloid plaques and brain inflammatory responses in PSAPP mice following strain-matched wild-type PS1 bone marrow reconstitution. These effects occurred with immune switching from pro-inflammatory T helper (Th) 1 to anti-inflammatory Th2 immune responses in the periphery and in the brain, which likely instructed microglia to phagocytose and clear Aß in an ex vivo assay. Conversely, Tg2576 mice displayed accelerated AD-like pathology when reconstituted with mhPS1 bone marrow. These data show that haematopoietic cells bearing the mhPS1 transgene exacerbate AD-like pathology, suggesting a novel therapeutic strategy for AD based on targeting PS1 in peripheral immune cells.

A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity.

To generate chimeric Ag receptors (CARs) for the adoptive immunotherapy of cancer patients with ErbB2-expressing tumors, a single-chain Ab derived from the humanized mAb 4D5 Herceptin (trastuzumab) was initially linked to T cell signaling domains derived from CD28 and the CD3zeta to generate a CAR against ErbB2. Human PBLs expressing the 4D5 CAR demonstrated Ag-specific activities against ErbB2(+) tumors. However, a gradual loss of transgene expression was noted for PBLs transduced with this 4D5 CAR. When the CD3zeta signaling domain of the CAR was truncated or mutated, loss of CAR expression was not observed, suggesting that the CD3zeta signaling caused the transgene decrease, which was supported by the finding that T cells expressing 4D5 CARs with CD3zeta ITAM mutations were less prone to apoptosis. By adding 4-1BB cytoplasmic domains to the CD28-CD3zeta signaling moieties, we found increased transgene persistence in 4D5 CAR-transduced PBLs. Furthermore, constructs with 4-1BB sequences demonstrated increased cytokine secretion and lytic activity in 4D5 CAR-transduced T cells. More importantly, PBLs expressing this new version of the 4D5 CAR could not only efficiently lyse the autologous fresh tumor digests, but they could strongly suppress tumor growth in a xenogenic mouse model.

CCR5-edited CD4+ T cells augment HIV-specific immunity to enable post-rebound control of HIV replication.

BackgroundWe conducted a phase I clinical trial that infused CCR5 gene-edited CD4+ T cells to determine how these T cells can better enable HIV cure strategies.MethodsThe aim of trial was to develop RNA-based approaches to deliver zinc finger nuclease (ZFN), evaluate the effect of CCR5 gene-edited CD4+ T cells on the HIV-specific T cell response, test the ability of infused CCR5 gene-edited T cells to delay viral rebound during analytical treatment interruption, and determine whether individuals heterozygous for CCR5 Δ32 preferentially benefit. We enrolled 14 individuals living with HIV whose viral load was well controlled by antiretroviral therapy (ART). We measured the time to viral rebound after ART withdrawal, the persistence of CCR5-edited CD4+ T cells, and whether infusion of 10 billion CCR5-edited CD4+ T cells augmented the HIV-specific immune response.ResultsInfusion of the CD4+ T cells was well tolerated, with no serious adverse events. We observed a modest delay in the time to viral rebound relative to historical controls; however, 3 of the 14 individuals, 2 of whom were heterozygous for CCR5 Δ32, showed post-viral rebound control of viremia, before ultimately losing control of viral replication. Interestingly, only these individuals had substantial restoration of HIV-specific CD8+ T cell responses. We observed immune escape for 1 of these reinvigorated responses at viral recrudescence, illustrating a direct link between viral control and enhanced CD8+ T cell responses.ConclusionThese findings demonstrate how CCR5 gene-edited CD4+ T cell infusion could aid HIV cure strategies by augmenting preexisting HIV-specific immune responses.REGISTRATIONClinicalTrials.gov NCT02388594.FundingNIH funding (R01AI104400, UM1AI126620, U19AI149680, T32AI007632) was provided by the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute on Drug Abuse (NIDA), the National Institute of Mental Health (NIMH), and the National Institute of Neurological Disorders and Stroke (NINDS). Sangamo Therapeutics also provided funding for these studies.

Tim-4 in Health and Disease: Friend or Foe?

T-cell immunoglobulin and mucin domain containing 4 (Tim-4) is a phosphatidylserine receptor and is selectively expressed on antigen presenting cells. Recently, Tim-4 was reported to be expressed on iNKT cells, B1 cells, and tumor cells, suggesting it has multiple biological functions. In this review, we mainly summarize the expression and regulation of Tim-4 in immune cells including T cells, macrophages, dendritic cells, NKT cells, B cells, and mast cells. The expression of Tim-4 in these cells implies that Tim-4 might participate in immune related diseases. Emerging evidence emphasizes a substantial role for Tim-4 in maintaining homeostasis by regulating various immune responses, including viral infection, allergy, autoimmunity, and tumor immunity. Here, we collectively evaluated the role of Tim-4 in health and diseases. This summary will be extremely useful to fully understand the function of Tim-4 in the pathogenesis of immune related diseases, which would provide novel clues for the diagnosis and treatment of diseases.

A rational mouse model to detect on-target, off-tumor CAR T cell toxicity.

Off-tumor targeting of human antigens is difficult to predict in preclinical animal studies and can lead to serious adverse effects in patients. To address this, we developed a mouse model with stable and tunable human Her2 (hHer2) expression on normal hepatic tissue and compared toxicity between affinity-tuned Her2 chimeric antigen receptor T cells (CARTs). In mice with hHer2-high livers, both the high-affinity (HA) and low-affinity (LA) CARTs caused lethal liver damage due to immunotoxicity. In mice with hHer2-low livers, LA-CARTs exhibited less liver damage and lower systemic levels of IFN-γ than HA-CARTs. We then compared affinity-tuned CARTs for their ability to control a hHer2-positive tumor xenograft in our model. Surprisingly, the LA-CARTs outperformed the HA-CARTs with superior antitumor efficacy in vivo. We hypothesized that this was due, in part, to T cell trafficking differences between LA and HA-CARTs and found that the LA-CARTs migrated out of the liver and infiltrated the tumor sooner than the HA-CARTs. These findings highlight the importance of T cell targeting in reducing toxicity of normal tissue and also in preventing off-tumor sequestration of CARTs, which reduces their therapeutic potency. Our model may be useful to evaluate various CARTs that have conditional expression of more than 1 single-chain variable fragment (scFv).

CRISPR-engineered T cells in patients with refractory cancer.

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRß (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

Recognition of NY-ESO-1+ tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression.

The therapeutic use of T cell receptor (TCR)-transduced peripheral blood lymphocytes (PBL) targeting tumor-associated antigens is emerging as a promising investigational treatment for patients with cancer. Initial response rates to therapy were low, suggesting the need to improve the function of TCR-transduced PBL. We constructed standard bicistronic retroviral vectors using an internal promoter or internal ribosomal entry site element as well as vectors incorporating coding sequences for 2A linker peptides between coding sequences for alpha and beta chains targeting the cancer-testis (CT) antigen, NY-ESO-1. Incorporation of coding sequences for 2A linker peptides in the bicistronic TCR expression cassette resulted in up to a fourfold increase in TCR expression and a significant improvement in effector function as measured by interferon-gamma release following co-culture with peptide-pulsed targets and NY-ESO-1+ tumors. We also sought to enhance reactivity of TCR-transduced PBL against tumor targets by modulation of tumor antigen expression on target cells. Induction of NY-ESO-1 expression on tumor targets using the demethylating agent 5-aza-2'-deoxycytidine (alone or in combination with the histone deacetylase inhibitor depsipeptide) resulted in enhanced interferon-gamma secretion by the TCR-transduced PBL on culture with treated targets. Taken together, these results indicate that design of TCR-based vectors incorporating 2A linker peptides improves TCR expression and effector function of transduced PBL. Furthermore, induction of CT antigen expression through treatment of tumor targets with chromatin-remodeling agents may augment TCR-based immunotherapy targeting these antigens. These results have relevance for TCR-based gene therapies targeting common epithelial malignancies.

Extrathymic generation of tumor-specific T cells from genetically engineered human hematopoietic stem cells via Notch signaling.

Adoptive cell transfer (ACT) of tumor-reactive lymphocytes has been shown to be an effective treatment for cancer patients. Studies in murine models of ACT indicated that antitumor efficacy of adoptively transferred T cells is dependent on the differentiation status of the cells, with lymphocyte differentiation inversely correlated with in vivo antitumor effectiveness. T-cell in vitro development technologies provide a new opportunity to generate naive T cells for the purpose of ACT. In this study, we genetically modified human umbilical cord blood-derived hematopoietic stem cells (HSCs) to express tumor antigen-specific T-cell receptor (TCR) genes and generated T lymphocytes by coculture with a murine cell line expressing Notch-1 ligand, Delta-like-1 (OP9-DL1). Input HSCs were differentiated into T cells as evidenced by the expression of T-cell markers, such as CD7, CD1a, CD4, CD8, and CD3, and by detection of TCR excision circles. We found that such in vitro differentiated T cells expressed the TCR and showed HLA-A2-restricted, specific recognition and killing of tumor antigen peptide-pulsed antigen-presenting cells but manifested additional natural killer cell-like killing of tumor cell lines. The genetic manipulation of HSCs has broad implications for ACT of cancer.

iGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity.

Genome engineering methods have advanced greatly with the development of programmable nucleases, but methods for quantifying on- and off-target cleavage sites and associated deletions remain nascent. Here, we report an improvement of the GUIDE-seq method, iGUIDE, which allows filtering of mispriming events to clarify the true cleavage signal. Using iGUIDE, we specify the locations of Cas9-guided cleavage for four guide RNAs, characterize associated deletions, and show that naturally occurring background DNA double-strand breaks are associated with open chromatin, gene dense regions, and chromosomal fragile sites. iGUIDE is available from https://github.com/cnobles/iGUIDE .

[Application of gene therapy in tumor adoptive immunotherapy].

Adoptive cell transfer of tumor-infiltrating lymphocyte (TIL) has resulted in clear and reproducible responses in a substantial percentage (approximately 50%) of patients with metastatic melanoma. The availability of tumor reactive TIL limits the use of adoptive cell transfer for the treatment of most non-melanoma cancer patients. Recent report indicated that adoptive transfer of T lymphocytes genetically modified with T-cell receptor (TCR) against a tumor antigen resulted in objective response in melanoma patients, thus shedding light on the use of this strategy for the treatment of common epithelial cancers beyond melanoma. In this review, the current status and potential use of genetic modification in the adoptive immunotherapy of cancer patients are be discussed.

CRISPR/Cas9 genome editing: Fueling the revolution in cancer immunotherapy.

The development of genomic editing technologies expands the landscape of T cell engineering for adoptive cell therapy. Among the multiple tools that can be used, CRISPR/Cas9 has been shown to be relatively easy to use, simple to design and cost effective with highly efficient multiplex genome engineering capabilities. Allogeneic universal chimeric antigen receptor (CAR) T cells can be produced by disrupting T cell receptor (TCR) and beta-2-microglobulin (B2M) in CAR T cells or by directly knocking in a CAR at the disrupted TRAC locus. The anti-tumor function can be further boosted by simultaneous ablation of PD-1 and CTLA-4. The anti-tumor activities and safety of TCR-transferred T cells can be improved by knocking out endogenous TCR, which avoids the use of affinity-enhanced TCRs that may lose specificity and cause severe adverse effects. Therefore, CRISPR/Cas9 technology holds enormous promise to advance the field of adoptive cell therapy.