CD20 expression regulates CD37 levels in B-cell lymphoma - implications for immunotherapies.

Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies.

CD37 is a safe chimeric antigen receptor target to treat acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells in the bone marrow and the peripheral blood. Nearly half of the AML patients relapse after standard induction therapy, and new forms of therapy are urgently needed. Chimeric antigen receptor (CAR) T therapy has so far not been successful in AML due to lack of efficacy and safety. Indeed, the most attractive antigen targets are stem cell markers such as CD33 or CD123. We demonstrate that CD37, a mature B cell marker, is expressed in AML samples, and its presence correlates with the European LeukemiaNet (ELN) 2017 risk stratification. We repurpose the anti-lymphoma CD37CAR for the treatment of AML and show that CD37CAR T cells specifically kill AML cells, secrete proinflammatory cytokines, and control cancer progression in vivo. Importantly, CD37CAR T cells display no toxicity toward hematopoietic stem cells. Thus, CD37 is a promising and safe CAR T cell AML target.

Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16.

BACKGROUND: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and the evasion of host immune responses. Currently, debulking surgery and platinum-based chemotherapy are the treatment cornerstones, although recurrence is common. As the clinical efficacy of immune checkpoint blockade is low, new immunotherapeutic strategies are needed. Chimeric antigen receptor (CAR) T cell therapy empowers patients' own T cells to fight and eradicate cancer, and has been tested against various targets in OC. A promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC. CA125 itself has not been tested as a possible CAR target. In this study, we examined the suitability of the CA125 as a target for CAR T cell therapy. METHODS: We tested a series of antibodies raised against the CA125 extracellular repeat domain of MUC16 and adapted them to the CAR format. Comparisons between these candidates, and against an existing CAR targeting the MUC16 ectodomain, identified K101 as having high potency and specificity. The K101CAR was subjected to further biochemical and functional tests, including examination of the effect of soluble CA125 on its activity. Finally, we used cell lines and advanced orthotopic patient-derived xenograft (PDX) models to validate, in vivo, the efficiency of our K101CAR construct. RESULTS: We observed a high efficacy of K101CAR T cells against cell lines and patient-derived tumors, in vitro and in vivo. We also demonstrated that K101CAR functionality was not impaired by the soluble antigen. Finally, in direct comparisons, K101CAR, which targets the CA125 extracellular repeat domains, was shown to have similar efficacy to the previously validated 4H11CAR, which targets the MUC16 ectodomain. CONCLUSIONS: Our in vitro and in vivo results, including PDX studies, demonstrate that the CA125 domain of MUC16 represents an excellent target for treating MUC16-positive malignancies.

Transient TCR-based T cell therapy in a patient with advanced treatment-resistant MSI-high colorectal cancer.

We previously demonstrated the antitumor effectiveness of transiently T cell receptor (TCR)-redirected T cells recognizing a frameshift mutation in transforming growth factor beta receptor 2. We here describe a clinical protocol using mRNA TCR-modified T cells to treat a patient with progressive, treatment-resistant metastatic microsatellite instability-high (MSI-H) colorectal cancer. Following 12 escalating doses of autologous T cells electroporated with in-vitro-transcribed Radium-1 TCR mRNA, we assessed T cell cytotoxicity, phenotype, and cytokine production. Tumor markers and growth on computed tomography scans were evaluated and immune cell tumor infiltrate at diagnosis assessed. At diagnosis, tumor-infiltrating CD8+ T cells had minimal expression of exhaustion markers, except for PD-1. Injected Radium-1 T cells were mainly naive and effector memory T cells with low expression of exhaustion markers, except for TIGIT. We confirmed cytotoxicity of transfected Radium-1 T cells against target cells and found key cytokines involved in tumor metastasis, growth, and angiogenesis to fluctuate during treatment. The treatment was well tolerated, and despite his advanced cancer, the patient obtained a stable disease with 6 months survival post-treatment. We conclude that treatment of metastatic MSI-H colorectal cancer with autologous T cells electroporated with Radium-1 TCR mRNA is feasible, safe, and well tolerated and that it warrants further investigation in a phase 1/2 study.

In vitro re-challenge of CAR T cells.

Chimeric antigen receptor (CAR) T cells (CAR T) have emerged as a potential therapy for cancer patients. CAR T cells are capable of recognizing membrane proteins on cancer cells which initiates a downstream signaling in T cells that ends in cancer cell death. Continuous antigen exposure over time, activation of inhibitory signaling pathways and/or chronic inflammation can lead to CAR T cell exhaustion. In this context, the design of CARs can have a great impact on the functionality of CAR T cells, on their potency and exhaustion. Here, using CD19CAR as model, we provide a re-challenge protocol where CAR T cells are cultured weekly with malignant lymphoid cell lines BL-41 and Nalm-6 to simulate them with continuous antigen pressure over a four-week period. This protocol can be value for assessing CAR T cell functionality and for the comparison of different CAR constructs.

Determination of CAR T cell metabolism in an optimized protocol.

Adoptive transfer of T cells modified to express chimeric antigenic receptors (CAR) has emerged as a solution to cure refractory malignancies. However, although CAR T cell treatment of haematological cancers has now shown impressive improvement in outcome, solid tumours have been more challenging to control. The latter type is protected by a strong tumour microenvironment (TME) which might impact cellular therapeutic treatments. Indeed, the milieu around the tumour can become particularly inhibitory to T cells by directly affecting their metabolism. Consequently, the therapeutic cells become physically impeded before being able to attack the tumour. It is therefore extremely important to understand the mechanism behind this metabolic break in order to develop TME-resistant CAR T cells. Historically, the measurement of cellular metabolism has been performed at a low throughput which only permitted a limited number of measurements. However, this has been changed by the introduction of real-time technologies which have lately become more popular to study CAR T cell quality. Unfortunately, the published protocols lack uniformity and their interpretation become confusing. We herein tested the essential parameters to perform a metabolic study on CAR T cells and propose a check list of factors that should be set in order to draw sound conclusion.

Efficient chimeric antigen receptor targeting of a central epitope of CD22.

Chimeric antigen receptor (CAR) T-cell therapy has had considerable success in the treatment of B-cell malignancies. Targeting the B-lineage marker CD19 has brought great advances to the treatment of acute lymphoblastic leukemia and B-cell lymphomas. However, relapse remains an issue in many cases. Such relapse can result from downregulation or loss of CD19 from the malignant cell population or expression of alternate isoforms. Consequently, there remains a need to target alternative B-cell antigens and diversify the spectrum of epitopes targeted within the same antigen. CD22 has been identified as a substitute target in cases of CD19-negative relapse. One anti-CD22 antibody-clone m971-targets a membrane-proximal epitope of CD22 and has been widely validated and used in the clinic. Here, we have compared m971-CAR with a novel CAR derived from IS7, an antibody that targets a central epitope on CD22. The IS7-CAR has superior avidity and is active and specific against CD22-positive targets, including B-acute lymphoblastic leukemia patient-derived xenograft samples. Side-by-side comparisons indicated that while IS7-CAR killed less rapidly than m971-CAR in vitro, it remains efficient in controlling lymphoma xenograft models in vivo. Thus, IS7-CAR presents a potential alternative candidate for the treatment of refractory B-cell malignancies.

ALPL-1 is a target for chimeric antigen receptor therapy in osteosarcoma.

Osteosarcoma (OS) remains a dismal malignancy in children and young adults, with poor outcome for metastatic and recurrent disease. Immunotherapies in OS are not as promising as in some other cancer types due to intra-tumor heterogeneity and considerable off-target expression of the potentially targetable proteins. Here we show that chimeric antigen receptor (CAR) T cells could successfully target an isoform of alkaline phosphatase, ALPL-1, which is highly and specifically expressed in primary and metastatic OS. The target recognition element of the second-generation CAR construct is based on two antibodies, previously shown to react against OS. T cells transduced with these CAR constructs mediate efficient and effective cytotoxicity against ALPL-positive cells in in vitro settings and in state-of-the-art in vivo orthotopic models of primary and metastatic OS, without unexpected toxicities against hematopoietic stem cells or healthy tissues. In summary, CAR-T cells targeting ALPL-1 show efficiency and specificity in treating OS in preclinical models, paving the path for clinical translation.

Mechanism of CD79A and CD79B Support for IgM+ B Cell Fitness through B Cell Receptor Surface Expression.

The BCR consists of surface-bound Ig and a heterodimeric signaling unit comprised of CD79A and CD79B. Upon cognate Ag recognition, the receptor initiates important signals for B cell development and function. The receptor also conveys Ag-independent survival signals termed tonic signaling. Although the requirement of a CD79A/CD79B heterodimer for BCR complex assembly and surface expression is well established based on mice models, few studies have investigated this in human mature B cells. In this study, we found that human tonsillar B cells with high surface expression of IgM or IgG had potentiated BCR signaling compared with BCRlow cells, and high IgM expression in germinal center B cells was associated with reduced apoptosis. We explored the mechanism for IgM surface expression by CRISPR/Cas9-induced deletion of CD79A or CD79B in four B lymphoma cell lines. Deletion of either CD79 protein caused loss of surface IgM in all cell lines and reduced fitness in three. From two cell lines, we generated stable CD79A or CD79B knockout clones and demonstrated that loss of CD79A or CD79B caused a block in N-glycan maturation and accumulation of immature proteins, compatible with retention of BCR components in the endoplasmic reticulum. Rescue experiments with CD79B wild-type restored surface expression of CD79A and IgM with mature glycosylation, whereas a naturally occurring CD79B G137S mutant disrupting CD79A/CD79B heterodimerization did not. Our study highlights that CD79A and CD79B are required for surface IgM expression in human B cells and illuminates the importance of the IgM expression level for signaling and fitness.

A phase I/II escalation trial design T-RAD: Treatment of metastatic lung cancer with mRNA-engineered T cells expressing a T cell receptor targeting human telomerase reverse transcriptase (hTERT).

Background: Adoptive cellular therapy (ACT) with genetically modified T cells aims to redirect T cells against resistant cancers through introduction of a T cell receptor (TCR). The Radium-4 TCR was isolated from a responding patient in a cancer vaccination study and recognizes the enzymatic component of human Telomerase Reverse Transcriptase (hTERT) presented on MHC class II (HLA-DP04). hTERT is a constitutively overexpressed tumor-associated antigen present in most human cancers, including non-small-cell lung cancer (NSCLC), which is the second most common type of cancer worldwide. Treatment alternatives for relapsing NSCLC are limited and survival is poor. To improve patient outcome we designed a TCR-based ACT study targeting hTERT. Methods: T-RAD is a phase I/II study to evaluate the safety and efficacy of Radium-4 mRNA electroporated autologous T cells in the treatment of metastatic NSCLC with no other treatment option. Transient TCR expression is applied for safety considerations. Participants receive two intravenous injections with escalating doses of redirected T cells weekly for 6 consecutive weeks. Primary objectives are safety and tolerability. Secondary objectives include progression-free survival, time to progression, overall survival, patient reported outcomes and overall radiological response. Discussion: Treatment for metastatic NSCLC is scarce and new personalized treatment options are in high demand. hTERT is a tumor target applicable to numerous cancer types. This proof-of-concept study will explore for the first time the safety and efficacy of TCR mRNA electroporated autologous T cells targeting hTERT. The T-RAD study will thus evaluate an attractive candidate for future immunotherapy of solid tumors.

How CAR T Cells Breathe.

The manufacture of efficacious CAR T cells represents a major challenge in cellular therapy. An important aspect of their quality concerns energy production and consumption, known as metabolism. T cells tend to adopt diverse metabolic profiles depending on their differentiation state and their stimulation level. It is therefore expected that the introduction of a synthetic molecule such as CAR, activating endogenous signaling pathways, will affect metabolism. In addition, upon patient treatment, the tumor microenvironment might influence the CAR T cell metabolism by compromising the energy resources. The access to novel technology with higher throughput and reduced cost has led to an increased interest in studying metabolism. Indeed, methods to quantify glycolysis and mitochondrial respiration have been available for decades but were rarely applied in the context of CAR T cell therapy before the release of the Seahorse XF apparatus. The present review will focus on the use of this instrument in the context of studies describing the impact of CAR on T cell metabolism and the strategies to render of CAR T cells more metabolically fit.

In vivo experimental mouse model to test CD19CAR T cells generated with different methods.

Pre-clinical evaluation of CAR T cells includes procedures testing T-cell efficacy and safety in as close to real world conditions as possible. An important step in efficacy testing is the in vivo study, most often using immunodeficient murine models into which both the poison and the cure are injected; namely a human cancer cell line and genetically modified human T cells. The capacity of the T cells to control the cancer progression will provide information about the CAR T-cell efficacy. We here provide a protocol to test CAR T cells in vivo using the validated anti-B-cell marker CD19CAR and an aggressive lymphoma model.

CAR-Ts: new perspectives in cancer therapy.

Chimeric antigen receptor (CAR)-T-cell therapy is a promising anticancer treatment that exploits the host's immune system to fight cancer. CAR-T cell therapy relies on immune cells being modified to express an artificial receptor targeting cancer-specific markers, and infused into the patients where they will recognize and eliminate the tumour. Although CAR-T cell therapy has produced encouraging outcomes in patients with haematologic malignancies, solid tumours remain challenging to treat, mainly due to the lack of cancer-specific molecular targets and the hostile, often immunosuppressive, tumour microenvironment. CAR-T cell therapy also depends on the quality of the injected product, which is closely connected to CAR design. Here, we explain the technology of CAR-Ts, focusing on the composition of CARs, their application, and limitations in cancer therapy, as well as on the current strategies to overcome the challenges encountered. We also address potential future targets to overcome the flaws of CAR-T cell technology in the treatment of cancer, emphasizing glycan antigens, the aberrant forms of which attain high tumour-specific expression, as promising targets for CAR-T cell therapy.

PD-L1 CAR effector cells induce self-amplifying cytotoxic effects against target cells.

BACKGROUND: Immune checkpoint inhibitors and chimeric antigen receptor (CAR)-based therapies have transformed cancer treatment. Recently, combining these approaches into a strategy of PD-L1-targeted CAR has been proposed to target PD-L1high tumors. Our study provides new information on the efficacy of such an approach against PD-L1low targets. METHODS: New atezolizumab-based PD-L1-targeted CAR was generated and introduced into T, NK, or NK-92 cells. Breast cancer MDA-MB-231 and MCF-7 cell lines or non-malignant cells (HEK293T, HMEC, MCF-10A, or BM-MSC) were used as targets to assess the reactivity or cytotoxic activity of the PD-L1-CAR-bearing immune effector cells. Stimulation with IFNγ or with supernatants from activated CAR T cells were used to induce upregulation of PD-L1 molecule expression on the target cells. HER2-CAR T cells were used for combination with PD-L1-CAR T cells against MCF-7 cells. RESULTS: PD-L1-CAR effector cells responded vigorously with degranulation and cytokine production to PD-L1high MDA-MB-231 cells, but not to PD-L1low MCF-7 cells. However, in long-term killing assays, both MDA-MB-231 and MCF-7 cells were eliminated by the PD-L1-CAR cells, although with a delay in the case of PD-L1low MCF-7 cells. Notably, the coculture of MCF-7 cells with activated PD-L1-CAR cells led to bystander induction of PD-L1 expression on MCF-7 cells and to the unique self-amplifying effect of the PD-L1-CAR cells. Accordingly, PD-L1-CAR T cells were active not only against MDA-MD-231 and MCF-7-PD-L1 but also against MCF-7-pLVX cells in tumor xenograft models. Importantly, we have also observed potent cytotoxic effects of PD-L1-CAR cells against non-malignant MCF-10A, HMEC, and BM-MSC cells, but not against HEK293T cells that initially did not express PD-L1 and were unresponsive to the stimulation . Finally, we have observed that HER-2-CAR T cells stimulate PD-L1 expression on MCF-7 cells and therefore accelerate the functionality of PD-L1-CAR T cells when used in combination. CONCLUSIONS: In summary, our studies show that CAR-effector cells trigger the expression of PD-L1 on target cells, which in case of PD-L1-CAR results in the unique self-amplification phenomenon. This self-amplifying effect could be responsible for the enhanced cytotoxicity of PD-L1-CAR T cells against both malignant and non-malignant cells and implies extensive caution in introducing PD-L1-CAR strategy into clinical studies.

"Built-in" PD-1 blocker to rescue NK-92 activity from PD-L1-mediated tumor escape mechanisms.

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.

Targeting KRAS mutations with HLA class II-restricted TCRs for the treatment 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.

Pharmacologic Control of CAR T Cells.

Chimeric antigen receptor (CAR) therapy is a promising modality for the treatment of advanced cancers that are otherwise incurable. During the last decade, different centers worldwide have tested the anti-CD19 CAR T cells and shown clinical benefits in the treatment of B cell tumors. However, despite these encouraging results, CAR treatment has also been found to lead to serious side effects and capricious response profiles in patients. In addition, the CD19 CAR success has been difficult to reproduce for other types of malignancy. The appearance of resistant tumor variants, the lack of antigen specificity, and the occurrence of severe adverse effects due to over-stimulation of the therapeutic cells have been identified as the major impediments. This has motivated a growing interest in developing strategies to overcome these hurdles through CAR control. Among them, the combination of small molecules and approved drugs with CAR T cells has been investigated. These have been exploited to induce a synergistic anti-cancer effect but also to control the presence of the CAR T cells or tune the therapeutic activity. In the present review, we discuss opportunistic and rational approaches involving drugs featuring anti-cancer efficacy and CAR-adjustable effect.

Targeting Telomerase with an HLA Class II-Restricted TCR for Cancer Immunotherapy.

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.

Combinatorial CAR design improves target restriction.

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.

T cell receptor therapy against melanoma-Immunotherapy for the future?

Malignant melanoma has seen monumental changes in treatment options the last decade from the very poor results of dacarbazine treatment to the modern-day use of targeted therapies and immune checkpoint inhibitors. Melanoma has a high mutational burden making it more capable of evoking immune responses than many other tumours. Even when considering double immune checkpoint blockade with anti-CTLA-4 and anti-PD-1, we still have far to go in melanoma treatment as 50% of patients with metastatic disease do not respond to current treatment. Alternative immunotherapy should therefore be considered. Since melanoma has a high mutational burden, it is considered more immunogenic than many other tumours. T cell receptor (TCR) therapy could be a possible way forward, either alone or in combination, to improve the response rates of this deadly disease. Melanoma is one of the cancers where TCR therapy has been frequently applied. However, the number of antigens targeted remains fairly limited, although advanced personalized therapies aim at also targeting private mutations. In this review, we look at possible aspects of targeting TCR therapy towards melanoma and provide an implication of its use in the future.