TCR-engaging scaffolds selectively expand antigen-specific T-cells with a favorable phenotype for adoptive cell therapy.

BACKGROUND: Adoptive cell therapy (ACT) has shown promising results for the treatment of cancer and viral infections. Successful ACT relies on ex vivo expansion of large numbers of desired T-cells with strong cytotoxic capacity and in vivo persistence, which constitutes the greatest challenge to current ACT strategies. Here, in this study, we present a novel technology for ex vivo expansion of antigen-specific T-cells; artificial antigen-presenting scaffolds (Ag-scaffolds) consisting of a dextran-polysaccharide backbone, decorated with combinations of peptide-Major Histocompatibility Complex (pMHC), cytokines and co-stimulatory molecules, enabling coordinated stimulation of antigen-specific T-cells. METHODS: The capacity of Ag-scaffolds to expand antigen-specific T-cells was explored in ex vivo cultures with peripheral blood mononuclear cells from healthy donors and patients with metastatic melanoma. The resulting T-cell products were assessed for phenotypic and functional characteristics. RESULTS: We identified an optimal Ag-scaffold for expansion of T-cells for ACT, carrying pMHC and interleukin-2 (IL-2) and IL-21, with which we efficiently expanded both virus-specific and tumor-specific CD8+ T cells from peripheral blood of healthy donors and patients, respectively. The resulting T-cell products were characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion, a multifunctional cytokine profile upon antigen-challenge and superior tumor killing capacity. This demonstrates that the coordinated stimuli provided by an optimized stoichiometry of TCR engaging (pMHC) and stimulatory (cytokine) moieties is essential to obtain desired T-cell characteristics. To generate an 'off-the-shelf' multitargeting Ag-scaffold product of relevance to patients with metastatic melanoma, we identified the 30 most frequently recognized shared HLA-A0201-restricted melanoma epitopes in a cohort of 87 patients. By combining these in an Ag-scaffold product, we were able to expand tumor-specific T-cells from 60-70% of patients with melanoma, yielding a multitargeted T-cell product with up to 25% specific and phenotypically and functionally improved T cells. CONCLUSIONS: Taken together, the Ag-scaffold represents a promising new technology for selective expansion of antigen-specific CD8+ T cells directly from blood, yielding a highly specific and functionally enhanced T-cell product for ACT.

Engineering T-cells with chimeric antigen receptors to combat hematological cancers: an update on clinical trials.

Chimeric antigen receptor (CAR) redirected T-cells has shown efficacy in the treatment of B-cell leukemia/lymphoma, however, high numbers of relapses occur due to loss of targeted antigen or intrinsic failure of the CAR T-cells. In this situation modifications of the basic strategy are envisaged to reduce the risk of relapse, some of them are in early clinical exploration. These include simultaneous targeting of multiple antigens or combination of CAR T-cell therapy with other treatment modalities such as checkpoint inhibitors. The review evaluates and discusses these modified advanced therapies and pre-clinical approaches with respect to their potential to control leukemia and lymphoma in the long-term.

Augmenting engineered T-cell strategies in solid cancers through epigenetic priming.

T-cell receptor (TCR)- and chimeric antigen receptor (CAR)-based adoptive cell transfer (ACT) has shown promising results in hematological malignancies, but remains immature in solid cancers. The challenges associated with identification of tumor-specific targets, the heterogenic antigen expression, limited T-cell trafficking to tumor sites and the hostile tumor microenvironment (TME), are all factors contributing to the limited efficacy of ACT therapies against solid tumors. Epigenetic priming of tumor cells and the microenvironment may be a way of overcoming these obstacles and improving the clinical efficacy of adoptive T-cell therapies in the future. Here, we review the current literature and suggest combining epigenetic modulators and ACT strategies as a way of augmenting the efficacy of TCR- and CAR-engineered T cells against solid tumors.

Rational design of a trimeric APRIL-based CAR-binding domain enables efficient targeting of multiple myeloma.

Chimeric antigen receptor (CAR) T cells (CARTs) have shown tremendous potential for the treatment of certain B-cell malignancies, including patients with relapsed/refractory multiple myeloma (MM). Targeting the B-cell maturation antigen (BCMA) has produced the most promising results for CART therapy of MM to date, but not all remissions are sustained. Emergence of BCMA escape variants has been reported under the selective pressure of monospecific anti-BCMA CART treatment. Thus, there is a clinical need for continuous improvement of CART therapies for MM. Here, we show that a novel trimeric APRIL (a proliferation-inducing ligand)-based CAR efficiently targets both BCMA+ and BCMA- MM. Modeled after the natural ligand-receptor pair, APRIL-based CARs allow for bispecific targeting of the MM-associated antigens BCMA and transmembrane activator and CAML interactor (TACI). However, natural ligands as CAR antigen-binding domains may require further engineering to promote optimal binding and multimerization to adequately trigger T-cell activation. We found that using a trimeric rather than a monomeric APRIL format as the antigen-binding domain enhanced binding to BCMA and TACI and CART activity against MM in vitro and in vivo. Dual-specific, trimeric APRIL-based CAR are a promising therapeutic approach for MM with potential for preventing and treating BCMA escape.

Chimeric Antigen Receptor T Cells Targeting CD79b Show Efficacy in Lymphoma with or without Cotargeting CD19.

PURPOSE: T cells engineered to express a chimeric antigen receptor (CAR) against CD19 have recently been FDA approved for the treatment of relapsed or refractory large B-cell lymphoma. Despite the success and curative potential of CD19 CAR T cells, several reports describing disease relapse due to antigen loss are now emerging. EXPERIMENTAL DESIGN: We developed a novel CAR construct directed against CD79b, a critical receptor for successful B-cell development that remains highly expressed in several subtypes of B-cell lymphoma, including mantle cell lymphoma (MCL). We tested CAR T cells directed against CD79b alone or in combination with CD19 targeting in a single construct, against cell line- and patient-derived xenograft models. RESULTS: We demonstrate CAR79b antigen-specific recognition and cytotoxicity against a panel of cell lines and patient-derived xenograft models of MCL. Importantly, we show that downregulation of CD19 does not influence surface expression of CD79b and that anti-CD79b CAR T cells alone or arranged in a dual-targeting format with a CD19 single-chain variable fragment (scFv) are able to recognize and eliminate CD19+, CD19-, and mixed CD19+/CD19-B-cell lymphoma. CONCLUSIONS: Our findings demonstrate that CAR T cells targeting CD79b alone or in combination have promise for treating and preventing CD19 antigen escape in B-cell lymphomas.

Anti-CD37 chimeric antigen receptor T cells are active against B- and T-cell lymphomas.

Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

CARs in the Lead Against Multiple Myeloma.

The recent clinical success of CD19-directed chimeric antigen receptor (CAR) T cell therapy in chronic and acute leukemia has led to increased interest in broadening this technology to other hematological malignancies and solid tumors. Now, advances are being made using CAR T cell technology to target myeloma antigens such as B cell maturation antigen (BCMA), CD138, and kappa-light chain as well as CD19 on putative myeloma stem cells. To date, only a limited number of multiple myeloma patients have received CAR T cell therapy but preliminary results have been encouraging. In this review, we summarize the recently reported results of clinical trials conducted utilizing CAR T cell therapy in multiple myeloma (MM).

Low Constitutive Cell Surface Expression of HLA-B Is Caused by a Posttranslational Mechanism Involving Glu180 and Arg239.

HLA class I cell surface expression is crucial for normal immune responses, and variability in HLA expression may influence the course of infections. We have previously shown that classical HLA class I expression on many human cell types is biased with greatly reduced expression of HLA-B compared with HLA-A in the absence of inflammatory signals. In the search for the mechanisms responsible for this discrepancy, we have recently reported that the regulation is mainly posttranslational and that the C-terminal part of the α2 domain and the α3 domain contain the molecular determinants that explain most of the variability of expression between common HLA-A and -B allomorphs. In this study, we present a fine mapping of the structural determinants that allow such variability by exchanging key amino acids located within the C-terminal part of the α2 domain and the α3 domain of HLA-A2 and -B8, including Glu/Asp at position 177, Gln/Glu at position 180, Gly/Arg at position 239, and Pro/Ser at position 280. We found that the HLA-A2 and -B8 expression profiles could be interconverted to a large extent by mutual exchange of Gln/Glu at position 180 or by Gly/Arg at position 239. The presence of Gln180 and Gly239, as in HLA-A2, led to higher cell surface expression levels when compared with the presence of Glu180 and Arg239, as in HLA-B8. This indicates that the amino acids at positions 180 and 239 determine the level of cell surface expression of common HLA-A and -B allomorphs, probably by affecting HLA processing in the Ag presentation pathway.

Protective effects of surfactant protein D treatment in 1,3-ß-glucan-modulated allergic inflammation.

Surfactant protein D (SP-D) is a pulmonary collectin important in lung immunity. SP-D-deficient mice (Sftpd(-/-)) are reported to be susceptible to ovalbumin (OVA)- and fungal allergen-induced pulmonary inflammation, while treatment with exogenous SP-D has therapeutic effects in such disease models. ß-Glucans are a diverse group of polysaccharides previously suggested to serve as fungal ligands for SP-D. We set out to investigate if SP-D could interact with 1,3-ß-glucan and attenuate allergic pulmonary inflammation in the presence of 1,3-ß-glucan. Allergic airway disease was induced in Sftpd(-/-) and Sftpd(+/+) mice by OVA sensitization and subsequent challenge with OVA, 1,3-ß-glucan, or OVA/1,3-ß-glucan together. Mice in the combined treatment group were further treated with a high dose of recombinant fragment of human SP-D (rfhSP-D). We demonstrated direct interaction between SP-D and 1,3-ß-glucan. OVA-induced mucous cell metaplasia was increased in Sftpd(-/-) mice, supporting previously reported protective effects of endogenous SP-D in allergy. OVA-induced parenchymal CCL11 levels and eosinophilic infiltration in bronchoalveolar lavage were unaffected by 1,3-ß-glucan, but were reversed with rfhSP-D treatment. 1,3-ß-Glucan treatment did, however, induce pulmonary neutrophilic infiltration and increased TNF-α levels in bronchoalveolar lavage, independently of OVA-induced allergy. This infiltration was also reversed by treatment with rfhSP-D. 1,3-ß-Glucan reduced OVA-induced mucous cell metaplasia, T helper 2 cytokines, and IFN-γ production. rfhSP-D treatment further reduced mucous metaplasia and T helper 2 cytokine secretion to background levels. In summary, rfhSP-D treatment resulted in attenuation of both allergic inflammation and 1,3-ß-glucan-mediated neutrophilic inflammation. Our data suggest that treatment with high-dose SP-D protects from mold-induced exacerbations of allergic asthma.

Characterization of spontaneous air space enlargement in mice lacking microfibrillar-associated protein 4.

Microfibrillar-associated protein 4 (MFAP4) is localized to elastic fibers in blood vessels and the interalveolar septa of the lungs and is further present in bronchoalveolar lavage. Mfap4 has been previously suggested to be involved in elastogenesis in the lung. We tested this prediction and aimed to characterize the pulmonary function changes and emphysematous changes that occur in Mfap4-deficient (Mfap4(-/-)) mice. Significant changes included increases in total lung capacity and compliance, which were evident in Mfap4(-/-) mice at 6 and 8 mo but not at 3 mo of age. Using in vivo breath-hold gated microcomputed tomography (micro-CT) in 8-mo-old Mfap4(-/-) mice, we found that the mean density of the lung parenchyma was decreased, and the low-attenuation area (LAA) was significantly increased by 14% compared with Mfap4(+/+) mice. Transmission electron microscopy (TEM) did not reveal differences in the organization of elastic fibers, and there was no difference in elastin content, but a borderline significant increase in elastin mRNA expression in 3-mo-old mice. Stereological analysis showed that alveolar surface density in relation to the lung parenchyma and total alveolar surface area inside of the lung were both significantly decreased in Mfap4(-/-) mice by 25 and 15%, respectively. The data did not support an essential role of MFAP4 in pulmonary elastic fiber organization or content but indicated increased turnover in young Mfap4(-/-) mice. However, Mfap4(-/-) mice developed a spontaneous loss of lung function, which was evident at 6 mo of age, and moderate air space enlargement, with emphysema-like changes.

Systemically administered anti-TNF therapy ameliorates functional outcomes after focal cerebral ischemia.

BACKGROUND: The innate immune system contributes to the outcome after stroke, where neuroinflammation and post-stroke systemic immune depression are central features. Tumor necrosis factor (TNF), which exists in both a transmembrane (tm) and soluble (sol) form, is known to sustain complex inflammatory responses associated with stroke. We tested the effect of systemically blocking only solTNF versus blocking both tmTNF and solTNF on infarct volume, functional outcome and inflammation in focal cerebral ischemia. METHODS: We used XPro1595 (a dominant-negative inhibitor of solTNF) and etanercept (which blocks both solTNF and tmTNF) to test the effect of systemic administration on infarct volume, functional recovery and inflammation after focal cerebral ischemia in mice. Functional recovery was evaluated after one, three and five days, and infarct volumes at six hours, 24 hours and five days after ischemia. Brain inflammation, liver acute phase response (APR), spleen and blood leukocyte profiles, along with plasma microvesicle analysis, were evaluated. RESULTS: We found that both XPro1595 and etanercept significantly improved functional outcomes, altered microglial responses, and modified APR, spleen T cell and microvesicle numbers, but without affecting infarct volumes. CONCLUSIONS: Our data suggest that XPro1595 and etanercept improve functional outcome after focal cerebral ischemia by altering the peripheral immune response, changing blood and spleen cell populations and decreasing granulocyte infiltration into the brain. Blocking solTNF, using XPro1595, was just as efficient as blocking both solTNF and tmTNF using etanercept. Our findings may have implications for future treatments with anti-TNF drugs in TNF-dependent diseases.