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T cell receptor (TCR)-engineered T cell therapy is a promising cancer treatment approach. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of tumors and a potential target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR-redirected primary CD4+ and CD8+ T cells demonstrated in vitro efficacy, producing inflammatory cytokines and killing hTERT+ melanoma cells in both 2D and 3D settings, as well as malignant, patient-derived ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. Notably, Radium-4 TCR+ T cells also significantly reduced tumor growth and improved survival in a xenograft mouse model. Since hTERT is a universal cancer antigen, and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumors.

T-cell receptor (TCR) redirected T cells are considered as the next generation of care for the treatment of numerous solid tumors. KRAS mutations are driver neoantigens that are expressed in over 25% of all cancers and are thus regarded as ideal targets for Adoptive Cell Therapy (ACT). We have isolated four KRAS-specific TCRs from a long-term surviving pancreatic cancer patient vaccinated with a mix of mutated KRAS peptides. The sequence of these TCRs could be identified and expressed in primary cells. We demonstrated stable expression of all TCRs as well as target-specific functionality when expressing T cells were co-incubated with target cells presenting KRAS peptides. In addition, these TCRs were all partially co-receptor independent since they were functional in both CD4 and CD8 T cells, thus indicating high affinity. Interestingly, we observed that certain TCRs were able to recognize several KRAS mutations in complex with their cognate Human leukocyte antigen (HLA), suggesting that, here, the point mutations were less important for the HLA binding and TCR recognition, whereas others were single-mutation restricted. Finally, we demonstrated that these peptides were indeed processed and presented, since HLA-matched antigen presenting cells exogenously loaded with KRAS proteins were recognized by TCR-transduced T cells. Taken together, our data demonstrate that KRAS mutations are immunogenic for CD4 T cells and are interesting targets for TCR-based cancer immunotherapy.

Success of adoptive cell therapy mainly depends on the ability of immune cells to persist and function optimally in the immunosuppressive tumor microenvironment. Although present at the cancer site, immune cells become exhausted and/or inhibited, due to the presence of inhibitory receptors such as PD-L1 on malignant cells. Novel genetic strategies to manipulate the PD1/PD-L1 axis comprise (i) PD-1 reversion where the receptor intracellular domain is replaced with an activating unit, (ii) the use of anti-PD-L1 CAR or (iii) the disruption of the PD-1 gene. We here present an alternative strategy to equip therapeutic cells with a truncated PD-1 (tPD-1) to abrogate PD-1/PD-L1 inhibition. We show that engagement of tPD-1 with PD-L1-positive tumor unleashes NK-92 activity in vitro. Furthermore, this binding was sufficiently strong to induce killing of targets otherwise not recognized by NK-92, thus increasing the range of targets. In vivo treatment with NK-92 tPD-1 cells led to reduced tumor growth and improved survival. Importantly, tPD-1 did not interfere with tumor recognition in PD-L1 negative conditions. Thus, tPD-1 represents a straightforward method for improving antitumor immunity and revealing new targets through PD-L1 positivity.

Off-target toxicity due to the expression of target antigens in normal tissue or TCR cross-reactivity represents a major risk when using T cell receptor (TCR)-engineered T cells for treatment of solid tumours. Due to the inherent cross-reactivity of TCRs it is difficult to accurately predict their target recognition pre-clinically. It has become evident that direct testing in a human being represents the best evaluation of the risks. There is, therefore, a clear unmet need for assessing the safety of a therapeutic TCR in a more controllable manner than by the injection of permanently modified cellular products. Using transiently modified T cells combined with dose escalation has already been shown feasible for chimeric antigen receptor (CAR)-engineered T cells, but nothing is yet reported for TCR. We performed a preclinical evaluation of a therapeutic TCR transiently expressed in T cells by mRNA electroporation. We analyzed if the construct was active in vitro, how long it was detectable for and if this expression format was adapted to in vivo efficacy assessment. Our data demonstrate the potential of mRNA engineered T cells, although less powerful than permanent redirection, to induce a significant response. Thus, these findings support the development of mRNA based TCR-therapy strategies as a feasible and efficacious method for evaluating TCR safety and efficacy in first-in-man testing.

Off-target toxicity due to the expression of target antigens in normal tissue or TCR cross-reactivity represents a major risk when using T cell receptor (TCR)-engineered T cells for treatment of solid tumours. Due to the inherent cross-reactivity of TCRs it is difficult to accurately predict their target recognition pre-clinically. It has become evident that direct testing in a human being represents the best evaluation of the risks. There is, therefore, a clear unmet need for assessing the safety of a therapeutic TCR in a more controllable manner than by the injection of permanently modified cellular products. Using transiently modified T cells combined with dose escalation has already been shown feasible for chimeric antigen receptor (CAR)-engineered T cells, but nothing is yet reported for TCR. We performed a preclinical evaluation of a therapeutic TCR transiently expressed in T cells by mRNA electroporation. We analyzed if the construct was active in vitro, how long it was detectable for and if this expression format was adapted to in vivo efficacy assessment. Our data demonstrate the potential of mRNA engineered T cells, although less powerful than permanent redirection, to induce a significant response. Thus, these findings support the development of mRNA based TCR-therapy strategies as a feasible and efficacious method for evaluating TCR safety and efficacy in first-in-man testing. Electronic supplementary material The online version of this article (10.1007/s00262-019-02356-2) contains supplementary material, which is available to authorized users.

T cells modified to express chimeric antigen receptor (CAR) targeting CD19 (CD19CAR) have produced remarkable clinical responses in patients with relapsed/refractory B-cell acute lymphoblastic leukemia. CD19CAR T-cell therapy has also demonstrated prominent effects in B-cell non-Hodgkin lymphoma (B-NHL) patients. However, a subset of patients who relapse after CD19CAR T-cell therapy have outgrowth of CD19− tumor cells. Hence, development of alternative CARs targeting other B-cell markers represents an unmet medical need for B-cell acute lymphoblastic leukemia and B-NHL. Here, we confirmed previous data by showing that, overall, B-NHL has high expression of CD37. A second-generation CD37CAR was designed, and its efficacy in T cells was compared with that of CD19CAR. In vitro assessment of cytotoxicity and T-cell function upon coculture of the CAR T cells with different target B-cell lymphoma cell lines demonstrated comparable efficacy between the 2 CARs. In an aggressive B-cell lymphoma xenograft model, CD37CAR T cells were as potent as CD19CAR T cells in controlling tumor growth. In a second xenograft model, using U2932 lymphoma cells containing a CD19− subpopulation, CD37CAR T cells efficiently controlled tumor growth and prolonged survival, whereas CD19CAR T cells had limited effect. We further show that, unlike CD19CAR, CD37CAR was not sensitive to antigen masking. Finally, CD37CAR reactivity was restricted to B-lineage cells. Collectively, our results demonstrated that CD37CAR T cells also can effectively eradicate B-cell lymphoma tumors when CD19 antigen expression is lost and support further clinical testing for patients with relapsed/refractory B-NHL.

Currently the greatest challenge in oncology is the lack of homogeneity of the lesions where different cell components respond differently to treatment. There is growing consensus that monotherapies are insufficient to eradicate the disease and there is an unmet need for more potent combinatorial treatments. We have previously shown that hypericin photodynamic therapy (HYP-PDT) triggers electron transport chain (ETC) inhibition in cell mitochondria. We have also shown that tamoxifen (TAM) enhances cytotoxicity in cells with high respiration, when combined with ETC inhibitors. Herein we introduce a synergistic treatment based on TAM chemotherapy and HYP-PDT. We tested this novel combinatorial treatment (HYPERTAM) in two metabolically different breast cancer cell lines, the triple-negative MDA-MB-231 and the estrogen-receptor-positive MCF7, the former being quite sensitive to HYP-PDT while the latter very responsive to TAM treatment. In addition, we investigated the mode of death, effect of lipid peroxidation, and the effect on cell metabolism. The results were quite astounding. HYPERTAM exhibited over 90% cytotoxicity in both cell lines. This cytotoxicity was in the form of both necrosis and autophagy, while high levels of lipid peroxidation were observed in both cell lines. We, consequently, translated our research to an in vivo pilot study encompassing the MDA-MB-231 and MCF7 tumor models in NOD SCID-γ immunocompromised mice. Both treatment cohorts responded very positively to HYPERTRAM, which significantly prolonged mice survival. HYPERTAM is a potent, synergistic modality, which may lay the foundations for a novel, composite anticancer treatment, effective in diverse tumor types.

Immunotherapy, in particular Immune Checkpoint Blockade (ICB) has been regarded as the next standard of care for many solid tumor indications. Yet, a significant proportion of patients do not respond to therapy. More specifically, failure of ICB has been linked to the absence of an anti-tumor immune response that can be boosted or revitalized. Cancer vaccines that are designed to elicit such response by educating the host immune system to recognize tumor antigens, are therefore considered a key next generation therapeutic modality for the treatment of human cancers. Various vaccine platforms have been developed over the years, but recent advances in delivery technologies combined with the intrinsic qualities of the DNA matrix have positioned DNA vaccines as a safe and flexible alternative to other types of vaccine technologies. Vaccibody is developing DNA vaccines that allows specific targeting of tumors antigens to Antigen Presenting Cells (APCs), thus maximizing the elicited immune response. Here we present a second-generation version of our DNA platform in which our vaccibody molecule can be co-expressed with immune-stimulatory proteins from one plasmid using a multicistronic design. Compared to the vaccibody molecule alone the simultaneous expression of selected immune stimulatory cytokines was shown to boost the overall immune response almost 3-fold to drive a potent anti-tumor response. These data demonstrate the flexibility and potential of DNA vaccines as well as the advantages of combining an APC targeted delivery of tumor specific antigens together with a local production of immune stimulatory proteins. Citation Format: Audun Beraas, Pierre Dillard, Judith Jing Wong, Christian Winther Wold, Eirik Solbakken, Linn Guro Oslen, Joel Benjamin Heim, Stalin Chellappa, Agnete Fredriksen, Mikkel Wandahl Pedersen, Stine Granum. A novel and versatile cytokine empowered DNA vaccine platform with superior immune activating potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3558.

CAR T cells targeting the B-lymphocyte antigen CD19 have led to remarkable clinical results in B-cell leukemia and lymphoma, but eliminate all B-lineage cells, leading to increased susceptibility to severe infections. As malignant B cells will express either immunoglobulin (Ig) light chain κ or λ, we designed a second-generation CAR targeting Igκ, IGK CAR. This construct demonstrated high target specificity, but displayed reduced efficacy in the presence of serum IgG. Since CD19 CAR is insensitive to serum IgG, we designed various combinatorial CAR constructs in order to maintain the CD19 CAR T cell efficacy, but with IGK CAR target selectivity. The Kz-19BB design, combining CD19 CAR containing a 4-1BB co-stimulatory domain with an IGK CAR containing a CD3zeta stimulatory domain, maintained the target specificity of IgK CAR and was resistant to the presence of soluble IgG. Our results demonstrate that a combinatorial CAR approach can improve target selectivity and efficacy.

CAR T cells targeting the B lymphocyte antigen CD19 have led to remarkable clinical results in B cell leukemia and lymphoma but eliminate all B lineage cells, leading to increased susceptibility to severe infections. As malignant B cells will express either immunoglobulin (Ig) light chain κ or λ, we designed a second-generation CAR targeting Igκ, IGK CAR. This construct demonstrated high target specificity but displayed reduced efficacy in the presence of serum IgG. Since CD19 CAR is insensitive to serum IgG, we designed various combinatorial CAR constructs in order to maintain the CD19 CAR T cell efficacy, but with IGK CAR target selectivity. The Kz-19BB design, combining CD19 CAR containing a 4-1BB costimulatory domain with an IGK CAR containing a CD3zeta stimulatory domain, maintained the target specificity of IgK CAR and was resistant to the presence of soluble IgG. Our results demonstrate that a combinatorial CAR approach can improve target selectivity and efficacy.

Background Treatment of cancers has largely benefited from the development of immunotherapy. In particular, Chimeric Antigen Receptor (CAR) redirected T cells have demonstrated impressive efficacy against B-cell malignancies and continuous efforts are made to adapt this new therapy to solid tumors, where the immunosuppressive tumor microenvironment is a barrier for delivery. CAR T-cell validation relies on in vitro functional assays using monolayer or suspension cells and in vivo xenograft models in immunodeficient animals. However, the efficacy of CAR therapies remains difficult to predict with these systems, in particular when challenged against 3D organized solid tumors with highly intricate microenvironment. An increasing number of reports have now included an additional step in the development process in which redirected T cells are tested against tumor spheres. Results Here, we report a method to produce 3D structures, or cysts, out of a colorectal cancer cell line, Caco-2, which has the ability to form polarized spheroids as a validation tool for adoptive cell therapy in general. We used CD19CAR T cells to explore this method and we show that it can be adapted to various platforms including high resolution microscopy, bioluminescence assays and high-throughput live cell imaging systems. Conclusion We developed an affordable, reliable and practical method to produce cysts to validate therapeutic CAR T cells. The integration of this additional layer between in vitro and in vivo studies could be an important tool in the pre-clinical workflow of cell-based immunotherapy.

Background Although chemo-immunotherapy has led to an improved overall survival for most B-cell lymphoma types, relapsed and refractory disease remains a challenge. The malaria drug artesunate has previously been identified as a growth suppressor in some cancer types and was tested as a new treatment option in B-cell lymphoma. Methods We included artesunate in a cancer sensitivity drug screen in B lymphoma cell lines. The preclinical properties of artesunate was tested as single agent in vitro in 18 B-cell lymphoma cell lines representing different histologies and in vivo in an aggressive B-cell lymphoma xenograft model, using NSG mice. Artesunate-treated B lymphoma cell lines were analyzed by functional assays, gene expression profiling, and protein expression to identify the mechanism of action. Results Drug screening identified artesunate as a highly potent anti-lymphoma drug. Artesunate induced potent growth suppression in most B lymphoma cells with an IC50 comparable to concentrations measured in serum from artesunate-treated malaria patients, while leaving normal B-cells unaffected. Artesunate markedly inhibited highly aggressive tumor growth in a xenograft model. Gene expression analysis identified endoplasmic reticulum (ER) stress and the unfolded protein response as the most affected pathways and artesunate-induced expression of the ER stress markers ATF-4 and DDIT3 was specifically upregulated in malignant B-cells, but not in normal B-cells. In addition, artesunate significantly suppressed the overall cell metabolism, affecting both respiration and glycolysis. Conclusions Artesunate demonstrated potent apoptosis-inducing effects across a broad range of B-cell lymphoma cell lines in vitro, and a prominent anti-lymphoma activity in vivo, suggesting it to be a relevant drug for treatment of B-cell lymphoma.

Mechanosensing by T cells through the T cell receptor (TCR) is at the heart of immune recognition. While the mechanobiology of the TCR at the molecular level is increasingly well documented, its link to cell-scale response is poorly understood. Here we explore T cell spreading response as a function of substrate rigidity and show that remarkably, depending on the surface receptors stimulated, the cellular response may be either biphasic or monotonous. When adhering solely via the TCR complex, T cells respond to environmental stiffness in an unusual fashion, attaining maximal spreading on an optimal substrate stiffness comparable to that of professional antigen-presenting cells. However, in the presence of additional ligands for the integrin LFA-1, this biphasic response is abrogated and the cell spreading increases monotonously with stiffness up to a saturation value. This ligand-specific mechanosensing is effected through an actin-polymerization-dependent mechanism. We construct a mesoscale semianalytical model based on force-dependent bond rupture and show that cell-scale biphasic or monotonous behavior emerges from molecular parameters. As the substrate stiffness is increased, there is a competition between increasing effective stiffness of the bonds, which leads to increased cell spreading and increasing bond breakage, which leads to decreased spreading. We hypothesize that the link between actin and the receptors (TCR or LFA-1), rather than the ligand/receptor linkage, is the site of this mechanosensing.

Eradication of tumors by the immune system relies on the efficient activation of a T-cell response. For many years, the main focus of cancer immunotherapy has been on cytotoxic CD8 T-cell. However, stimulation of CD4 helper T cells is critical for the promotion and maintenance of immune memory, thus a good vaccine should evoke a two-dimensional T-cell response. The invariant chain (Ii) is required for the MHC class II heterodimer to be correctly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells (APC). We previously showed that by replacing the Ii CLIP peptide by an MHC-I cancer peptide, we could efficiently load MHC-I. This prompted us to test whether longer cancer peptides could be loaded on both MHC classes and whether such peptides could be accommodated in the CLIP region of Ii. We here present data showing that expanding the CLIP replacement size leads to T-cell activation. We demonstrate by using long peptides that APCs can present peptides from the same Ii molecule on both MHC-I and -II. In addition, we present evidence that antigen presentation after Ii-loading was superior to an ER-targeted minigene construct, suggesting that ER-localization was not sufficient to obtain efficient MHC-II loading. Finally, we verified that Ii-expressing dendritic cells could prime CD4+ and CD8+ T cells from a naïve population. Taken together our study demonstrates that CLIP peptide replaced Ii constructs fulfill some of the major requirements for an efficient vector for cancer vaccination.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

Background Adoptive T-cell transfer of therapeutic TCR holds great promise to specifically kill cancer cells, but relies on modifying the patient's own T cells ex vivo before injection. The manufacturing of T cells in a tailor-made setting is a long and expensive process which could be resolved by the use of universal cells. Currently, only the Natural Killer (NK) cell line NK-92 is FDA approved for universal use. In order to expand their recognition ability, they were equipped with Chimeric Antigen Receptors (CARs). However, unlike CARs, T-cell receptors (TCRs) can recognize all cellular proteins, which expand NK-92 recognition to the whole proteome. Methods We herein genetically engineered NK-92 to express the CD3 signaling complex, and showed that it rendered them able to express a functional TCR. Functional assays and in vivo efficacy were used to validate these cells. Findings This is the first demonstration that a non-T cell can exploit TCRs. This TCR-redirected cell line, termed TCR-NK-92, mimicked primary T cells phenotypically, metabolically and functionally, but retained its NK cell effector functions. Our results demonstrate a unique manner to indefinitely produce TCR-redirected lymphocytes at lower cost and with similar therapeutic efficacy as redirected T cells. Interpretation These results suggest that an NK cell line could be the basis for an off-the-shelf TCR-based cancer immunotherapy solution. Fund This work was supported by the Research Council of Norway (#254817), South-Eastern Norway Regional Health Authority (#14/00500-79), by OUS-Radiumhospitalet (Gene Therapy program) and the department of Oncology at the University of Lausanne .

Force sensitivity of the T cell receptor (TCR) is now believed to be essential for immune recognition, but cellular mechanosensitivity of T cells is still poorly understood. Here we show that T cells adhering via the TCR-complex respond to environmental stiffness in an unusual biphasic fashion. As the stiffness increases, adhesion and spreading first increase, then decrease, attaining their maximal values on an optimally stiff surface, with stiffness comparable to certain antigen presenting cells. Remarkably, in presence of additional ligands for the integrin LFA-1, spreading increases monotonously with stiffness up to a saturation value. Using a mesoscopic semi-analytical model linking spreading to molecular characteristics of bonds, we identify force sensitivity of the off-rate and the effective bond stiffness as the crucial parameters that determine monotonic or biphasic mechanosensitive behavior.

International audience

The first Chimeric antigen receptor (CAR) T-cell therapies have been approved for treatment of B-cell malignancies. This is mainly due to the success of CAR T cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Since CD19 is a general B-cell antigen, CAR-T cells eliminate all B-lineage cells, including non-malignant B cells. Therefore, the patients suffer from the impaired humoral immune response, increasing susceptibility to severe infections.Since B-cell lymphoma and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa positive tumor cells can be targeted while sparing normal Ig-lambda positive B-cells. In this respect, we isolated the sequence encoding the antigen-binding parts of an anti-Ig kappa antibody and designed a second-generation CAR construct (IGK CAR). Expression of IGK CAR in expanded peripheral blood T cells and subsequent testing of the CAR T cells in various in vitro assay with target cells, demonstrated cytokine production and potent killing of Ig-kappa expressing B-cell lines such as BL-41, whereas no response was observed against Ig lambda positive B-cell lines such as Granta-519. We compared IGK CAR with a clinical CD19 CAR (fmc63) and observed similar potency in target cell killing. Previous reports have shown that the presence of free immunoglobulins present in human serum could inhibit IGK CAR T cells, and our tests confirmed this. To improve IGK CAR T cells in the presence of IgGs while maintaining the specificity, we utilized a combinatorial CAR system, where the signaling domains were split. Our design demonstrated efficient killing of Ig-kappa positive cells and was less sensitive to free IgG as compared to IGK CAR T cells. Additionally, we observed a trade-off between specificity and cytotoxic potential. Increasing one individual component of the combinatorial system made the cells less prone to serum IgG inhibition but demonstrated somewhat higher cytotoxic activity against Ig-kappa negative targets. Our fully adjustable design, therefore, brings another perspective to the field by regulating the individual expression levels according to the treatment needs. Hence, enabling T cells to be either more aggressive or specific depending on the treatment efficiency and on the on-target toxicity in patients. Taken together, our in vitro data demonstrate that IGK-CD19 CAR combination is as potent as IGK or CD19 CAR T cells, and provides an alternative by combining their benefits into one design and thus reduces on-target toxicity. Citation Format: Hakan Köksal, Pierre Dillard, Sólrún M. Maggadóttir, Gunnar Kvalheim, Erlend B. Smeland, June H. Myklebust, Else Marit Inderberg, Sébastien Walchli. Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2318.

T cells modified to express chimeric antigen receptor (CAR) targeting CD19 have produced remarkable clinical responses in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). CD19CAR T-cell therapy has also demonstrated prominent effects in B-cell non-Hodgkin lymphoma (B-NHL) patients. However, a subset of patients who relapse after CD19CAR T-cell therapy have outgrowth of CD19-negative tumor cells. Hence, development of alternative CARs targeting other B-cell markers represents an unmet medical need for B-ALL and B-NHL. Here, we confirmed previous data by showing that B-NHL overall have high expression of CD37. A second generation CD37CAR was designed and its efficacy in T cells was compared to that of CD19CAR. In vitro assessment of cytotoxicity and T-cell function upon co-culture of the CAR T cells with different target B-cell lymphoma cell lines demonstrated comparable efficacy between the two CARs. In an aggressive B-cell lymphoma xenograft model, CD37CAR T cells were as potent as CD19CAR T cells in controlling tumor growth. In a second xenograft model, using U2932 lymphoma cells containing a CD19-negative subpopulation, CD37CAR T cells efficiently controlled tumors and cured the mice while CD19CAR T cells had limited effect. We further show that, unlike CD19CAR, CD37CAR was not sensitive to antigen masking. Finally, CD37CAR reactivity was restricted to B-lineage cells. Collectively, our results demonstrated that CD37CAR T cells effectively can eradicate B-cell lymphoma tumors also when CD19 antigen expression is lost, and support further clinical testing for patients with relapsed/refractory B-NHL. Citation Format: Pierre Dillard, Hakan Köksal, Sarah Josefsson, Solrun Melkorka Maggadottir, Sylvie Pollmann, Anne Fåne, Yngvild Nuvin Blaker, Klaus Beiske, Kanutte Huse, Ane Kolstad, Harald Holte, Gunnar Kvalheim, Erlend Bremertun Smeland, June Myklebust, Else Marit Inderberg, Sebastien Wälchli. Preclinical development of CD37CAR T-cell therapy for treatment of B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1422.

The first chimeric antigen receptor (CAR) T-cell therapies have been approved for treatment of B-cell malignancies. This is mainly due to the success of CAR T-cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Since CD19 is a general B-cell antigen, CAR-T-cells eliminate all B-lineage cells, including nonmalignant B cells. Therefore, the patients suffer from the impaired humoral immune response, increasing susceptibility to severe infections. Since B-cell lymphoma and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa positive tumor cells can be targeted while sparing normal Ig-lambda positive B-cells. In this respect, we isolated the sequence encoding the antigen-binding parts of an anti-Ig kappa antibody and designed a second-generation CAR construct (IGK CAR). Expression of IGK CAR in expanded peripheral blood T-cells and subsequent testing of the CAR T-cells in various in vitro assay with targeT-cells demonstrated cytokine production and potent killing of Ig-kappa expressing B-cell lines such as BL-41, whereas no response was observed against Ig lambda positive B-cell lines such as Granta-519. We compared IGK CAR with a clinical CD19 CAR (fmc63) and observed similar potency in targeT-cell killing. Previous reports have shown that the presence of free immunoglobulins present in human serum could inhibit IGK CAR T-cells, and our tests confirmed this. To improve IGK CAR T-cells in the presence of IgGs while maintaining the specificity, we utilized a combinatorial CAR system, where the signaling domains were split. Our design demonstrated efficient killing of Ig-kappa positive cells and was less sensitive to free IgG as compared to IGK CAR T-cells. Additionally, we observed a trade-off between specificity and cytotoxic potential. Increasing one individual component of the combinatorial system made the cells less prone to serum IgG inhibition but demonstrated somewhat higher cytotoxic activity against Ig-kappa negative targets. Our fully adjustable design, therefore, brings another perspective to the field by regulating the individual expression levels according to the treatment needs, hence enabling T-cells to be either more aggressive or specific depending on the treatment efficiency and on the on-target toxicity in patients. Taken together, our in vitro data demonstrate that IGK-CD19 CAR combination is as potent as IGK or CD19 CAR T-cells, and provides an alternative by combining their benefits into one design and thus reduces on-target toxicity. Citation Format: Hakan Köksal, Pierre Dillard, Sólrún Melkorka Maggadóttir, Gunnar Kvalheim, Erlend Bremertun Smeland, June Helen Myklebust, Else Marit Inderberg, Sébastien Wälchli. Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A035.

International audience; We investigate the adhesion and molecular organization of the plasma membrane of T lymphocytes interacting with a surrogate antigen presenting cell comprising glass supported ordered arrays of antibody (alpha-CD3) nano-dots dispersed in a non-adhesive matrix of polyethylene glycol (PEG). The local membrane adhesion and topography, as well as the distribution of the T cell receptors (TCRs) and the kinase ZAP-70, are influenced by dot-geometry, whereas the cell spreading area is determined by the overall average density of the ligands rather than specific characteristics of the dots. TCR clusters are recruited preferentially to the nano-dots and the TCR cluster size distribution has a weak dot-size dependence. On the patterns, the clusters are larger, more numerous, and more enriched in TCRs, as compared to the homogeneously distributed ligands at comparable concentrations. These observations support the idea that non-ligated TCRs residing in the non-adhered parts of the proximal membrane are able to diffuse and enrich the existing clusters at the ligand dots. However, long distance transport is impaired and cluster centralization in the form of a central supramolecular cluster (cSMAC) is not observed. Time-lapse imaging of early cell-surface contacts indicates that the ZAP-70 microclusters are directly recruited to the site of the antibody dots and this process is concomitant with membrane adhesion. These results together point to a complex interplay of adhesion, molecular organization and activation in response to spatially modulated stimulation.

International audience; Arrays of protein nano-dots with dot-size tuned independently of spacing (e.g. ~100 to 600 nm diameter for 900 nm spacing) are fabricated. The mechanism of size control is demonstrated, by numerical simulations, to arise from shadow effects during deposition of a sacrificial metal mask. We functionalize the nano-dots with antibodies and embed them in a polymer-cushion or in lipid-bilay-ers, or transfer them to soft elastomers. Their ability to influence cell architecture and local membrane organization is demonstrated in T-lymphocytes, using reflection interference contrast and total internal reflection fluorescence microscopy. TEXT Nano-patterning of bio-molecules on surfaces is currently an important tool for bio-engineering and cutting-edge cell biology studies. 1-9 Experiments on cellular interactions with such patterned surfaces provide insights into the mechanisms of cell adhesion, and this knowledge in turn is used to optimize the performance of artificial soft tissue grafts and prosthesis (see reviews 10-12 and references

We present a simple cost-effective benchtop protocol to functionalize glass and polydimethylsiloxane (PDMS) with nanometric protein patches for cell adhesion studies. Evaporation masks, covering macroscopic areas on glass, were made using improved strategies for self-assembly of colloidal microbeads which then served as templates for creating the protein patch arrays via the intermediate steps of organo-aminosilane deposition and polyethylene-glycol grafting. The diameter of the patches could be varied down to about 80 nm. The glass substrates were used for advanced optical imaging of T-lymphocytes to explore adhesion by reflection interference contrast microscopy and the possible colocalization of T-cell receptor microclusters and the activating protein patches by total internal reflection fluorescence microscopy. The selectively functionalized glass could also serve as template for transferring the protein nanopatches to the surface of a soft elastomer. We demonstrated successful reverse contact printing onto the surface of thin layers of PDMS with stiffness ranging from 30 KPa to 3 MPa.

Spreading of T cells on antigen presenting cells is a crucial initial step in immune response. Spreading occurs through rapid morphological changes concomitant with the reorganization of surface receptors and of the cytoskeleton. Ligand mobility and frictional coupling of receptors to the cytoskeleton were separately recognized as important factors but a systematic study to explore their biophysical role in spreading was hitherto missing. To explore the impact of ligand mobility, we prepared chemically identical substrates on which molecules of anti-CD3 (capable of binding and activating the T cell receptor complex), were either immobilized or able to diffuse. We quantified the T cell spreading area and cell edge dynamics using quantitative reflection interference contrast microscopy, and imaged the actin distribution. On mobile ligands, as compared to fixed ligands, the cells spread much less, the actin is centrally, rather than peripherally distributed and the edge dynamics is largely altered. Blocking myosin-II or adding molecules of ICAM1 on the substrate largely abrogates these differences. We explain these observations by building a model based on the balance of forces between activation-dependent actin polymerization and actomyosin-generated tension on one hand, and on the frictional coupling of the ligand-receptor complexes with the actin cytoskeleton, the membrane and the substrate, on the other hand. Introducing the measured edge velocities in the model, we estimate the coefficient of frictional coupling between T Cell receptors or LFA-1 and the actin cytoskeleton. Our results provide for the first time, to our knowledge, a quantitative framework bridging T cell-specific biology with concepts developed for integrin-based mechanisms of spreading.