Comparative performance of scFv-based anti-BCMA CAR formats for improved T cell therapy in multiple myeloma.

In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.

Prior flavivirus immunity skews the yellow fever vaccine response to cross-reactive antibodies with potential to enhance dengue virus infection.

The yellow fever 17D vaccine (YF17D) is highly effective but is frequently administered to individuals with pre-existing cross-reactive immunity, potentially impacting their immune responses. Here, we investigate the impact of pre-existing flavivirus immunity induced by the tick-borne encephalitis virus (TBEV) vaccine on the response to YF17D vaccination in 250 individuals up to 28 days post-vaccination (pv) and 22 individuals sampled one-year pv. Our findings indicate that previous TBEV vaccination does not affect the early IgM-driven neutralizing response to YF17D. However, pre-vaccination sera enhance YF17D virus infection in vitro via antibody-dependent enhancement (ADE). Following YF17D vaccination, TBEV-pre-vaccinated individuals develop high amounts of cross-reactive IgG antibodies with poor neutralizing capacity. In contrast, TBEV-unvaccinated individuals elicit a non-cross-reacting neutralizing response. Using YF17D envelope protein mutants displaying different epitopes, we identify quaternary dimeric epitopes as the primary target of neutralizing antibodies. Additionally, TBEV-pre-vaccination skews the IgG response towards the pan-flavivirus fusion loop epitope (FLE), capable of mediating ADE of dengue and Zika virus infections in vitro. Together, we propose that YF17D vaccination conceals the FLE in individuals without prior flavivirus exposure but favors a cross-reactive IgG response in TBEV-pre-vaccinated recipients directed to the FLE with potential to enhance dengue virus infection.

Association between bisphosphonate use and COVID-19 related outcomes.

Background: Although there are several efficacious vaccines against COVID-19, vaccination rates in many regions around the world remain insufficient to prevent continued high disease burden and emergence of viral variants. Repurposing of existing therapeutics that prevent or mitigate severe COVID-19 could help to address these challenges. The objective of this study was to determine whether prior use of bisphosphonates is associated with reduced incidence and/or severity of COVID-19. Methods: A retrospective cohort study utilizing payer-complete health insurance claims data from 8,239,790 patients with continuous medical and prescription insurance January 1, 2019 to June 30, 2020 was performed. The primary exposure of interest was use of any bisphosphonate from January 1, 2019 to February 29, 2020. Bisphosphonate users were identified as patients having at least one bisphosphonate claim during this period, who were then 1:1 propensity score-matched to bisphosphonate non-users by age, gender, insurance type, primary-care-provider visit in 2019, and comorbidity burden. Main outcomes of interest included: (a) any testing for SARS-CoV-2 infection; (b) COVID-19 diagnosis; and (c) hospitalization with a COVID-19 diagnosis between March 1, 2020 and June 30, 2020. Multiple sensitivity analyses were also performed to assess core study outcomes amongst more restrictive matches between BP users/non-users, as well as assessing the relationship between BP-use and other respiratory infections (pneumonia, acute bronchitis) both during the same study period as well as before the COVID outbreak. Results: A total of 7,906,603 patients for whom continuous medical and prescription insurance information was available were selected. A total of 450,366 bisphosphonate users were identified and 1:1 propensity score-matched to bisphosphonate non-users. Bisphosphonate users had lower odds ratios (OR) of testing for SARS-CoV-2 infection (OR = 0.22; 95%CI:0.21-0.23; p<0.001), COVID-19 diagnosis (OR = 0.23; 95%CI:0.22-0.24; p<0.001), and COVID-19-related hospitalization (OR = 0.26; 95%CI:0.24-0.29; p<0.001). Sensitivity analyses yielded results consistent with the primary analysis. Bisphosphonate-use was also associated with decreased odds of acute bronchitis (OR = 0.23; 95%CI:0.22-0.23; p<0.001) or pneumonia (OR = 0.32; 95%CI:0.31-0.34; p<0.001) in 2019, suggesting that bisphosphonates may protect against respiratory infections by a variety of pathogens, including but not limited to SARS-CoV-2. Conclusions: Prior bisphosphonate-use was associated with dramatically reduced odds of SARS-CoV-2 testing, COVID-19 diagnosis, and COVID-19-related hospitalizations. Prospective clinical trials will be required to establish a causal role for bisphosphonate-use in COVID-19-related outcomes. Funding: This study was supported by NIH grants, AR068383 and AI155865, a grant from MassCPR (to UHvA) and a CRI Irvington postdoctoral fellowship, CRI2453 (to PH).

The COVID-19 pandemic challenged the world to rapidly develop strategies to combat the virus responsible for the disease. While several effective vaccines and new drugs have since become available, these therapies are not always easy to access and take time to generate and distribute. To address these challenges, researchers have tried to find ways to repurpose existing medications that are already commonly used and known to be safe. One potential candidate are bisphosphonates, a family of drugs used to reduce bone loss in patients with osteoporosis. Bisphosphonates have been shown to boost the immune response to viral infections, and it has been observed that patients prescribed these drugs are less likely to develop or die from pneumonia. But whether bisphosphonates are effective against COVID-19 had not been fully explored. To investigate, Thompson, Wang et al. analyzed insurance claims data from about 8 million patients between January 2019 and June 2020, including around 450,000 individuals that had filled a prescription for bisphosphonates. Patients prescribed bisphosphonates were then compared to non-users that were similar in terms of their gender, age, the type of health insurance they had, their access to healthcare, and other health comorbidities. The study revealed that bisphosphonate users were around three to five times less likely to be tested for, diagnosed with, or hospitalized for COVID-19 during the first four months of the pandemic. They were also less commonly diagnosed with other respiratory infections in 2019, like bronchitis or pneumonia. Although the results suggest that bisphosphonates provide some protection against COVID-19, they cannot directly prove it. Verifying that bisphosphonates can treat or prevent COVID-19 and/or other respiratory infections requires more studies that follow patients in real-time rather than studying previously collected data. If such studies confirm the link, bisphosphonates could be a helpful tool to protect against COVID-19 or other virus outbreaks. The drugs are widely available, safe, and affordable, and therefore may provide an alternative for patients who cannot access other medications or vaccines.

Rational design of PD-1-CD28 immunostimulatory fusion proteins for CAR T cell therapy.

BACKGROUND: In many situations, the therapeutic efficacy of CAR T cells is limited due to immune suppression and poor persistence. Immunostimulatory fusion protein (IFP) constructs have been advanced as a tool to convert suppressive signals into stimulation and thus promote the persistence of T cells, but no universal IFP design has been established so far. We now took advantage of a PD-1-CD28 IFP as a clinically relevant structure to define key determinants of IFP activity. METHODS: We compared different PD-1-CD28 IFP variants in a human leukemia model to assess the impact of distinctive design choices on CAR T cell performance in vitro and a xenograft mouse model. RESULTS: We observed that IFP constructs that putatively exceed the extracellular length of PD-1 induce T-cell response without CAR target recognition, rendering them unsuitable for tumour-specific therapy. IFP variants with physiological PD-1 length ameliorated CAR T cell effector function and proliferation in response to PD-L1+ tumour cells in vitro and prolonged survival in vivo. Transmembrane or extracellular CD28 domains were found to be replaceable by corresponding PD-1 domains for in vivo efficacy. CONCLUSION: PD-1-CD28 IFP constructs must mimic the physiological interaction of PD-1 with PD-L1 to retain selectivity and mediate CAR-conditional therapeutic activity.

Mechanisms and strategies for safe chimeric antigen receptor T-cell activity control.

The clinical application of chimeric antigen receptor (CAR) T-cell therapy has rapidly changed the treatment options for terminally ill patients with defined blood-borne cancer types. However, CAR T-cell therapy can lead to severe therapy-associated toxicities including CAR-related hematotoxicity, ON-target OFF-tumor toxicity, cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Just as CAR T-cell therapy has evolved regarding receptor design, gene transfer systems and production protocols, the management of side effects has also improved. However, because of measures taken to abrogate adverse events, CAR T-cell viability and persistence might be impaired before complete remission can be achieved. This has fueled efforts for the development of extrinsic and intrinsic strategies for better control of CAR T-cell activity. These approaches can mediate a reversible resting state or irreversible T-cell elimination, depending on the route chosen. Control can be passive or active. By combination of CAR T-cells with T-cell inhibiting compounds, pharmacologic control, mostly independent of the CAR construct design used, can be achieved. Other strategies involve the genetic modification of T-cells or further development of the CAR construct by integration of molecular ON/OFF switches such as suicide genes. Alternatively, CAR T-cell activity can be regulated intracellularly through a self-regulation function or extracellularly through titration of a CAR adaptor or of a priming small molecule. In this work, we review the current strategies and mechanisms to control activity of CAR T-cells reversibly or irreversibly for preventing and for managing therapy-associated toxicities.

Bispecific antibodies redirect synthetic agonistic receptor modified T cells against melanoma.

BACKGROUND: Melanoma is an immune sensitive disease, as demonstrated by the activity of immune check point blockade (ICB), but many patients will either not respond or relapse. More recently, tumor infiltrating lymphocyte (TIL) therapy has shown promising efficacy in melanoma treatment after ICB failure, indicating the potential of cellular therapies. However, TIL treatment comes with manufacturing limitations, product heterogeneity, as well as toxicity problems, due to the transfer of a large number of phenotypically diverse T cells. To overcome said limitations, we propose a controlled adoptive cell therapy approach, where T cells are armed with synthetic agonistic receptors (SAR) that are selectively activated by bispecific antibodies (BiAb) targeting SAR and melanoma-associated antigens. METHODS: Human as well as murine SAR constructs were generated and transduced into primary T cells. The approach was validated in murine, human and patient-derived cancer models expressing the melanoma-associated target antigens tyrosinase-related protein 1 (TYRP1) and melanoma-associated chondroitin sulfate proteoglycan (MCSP) (CSPG4). SAR T cells were functionally characterized by assessing their specific stimulation and proliferation, as well as their tumor-directed cytotoxicity, in vitro and in vivo. RESULTS: MCSP and TYRP1 expression was conserved in samples of patients with treated as well as untreated melanoma, supporting their use as melanoma-target antigens. The presence of target cells and anti-TYRP1 × anti-SAR or anti-MCSP × anti-SAR BiAb induced conditional antigen-dependent activation, proliferation of SAR T cells and targeted tumor cell lysis in all tested models. In vivo, antitumoral activity and long-term survival was mediated by the co-administration of SAR T cells and BiAb in a syngeneic tumor model and was further validated in several xenograft models, including a patient-derived xenograft model. CONCLUSION: The SAR T cell-BiAb approach delivers specific and conditional T cell activation as well as targeted tumor cell lysis in melanoma models. Modularity is a key feature for targeting melanoma and is fundamental towards personalized immunotherapies encompassing cancer heterogeneity. Because antigen expression may vary in primary melanoma tissues, we propose that a dual approach targeting two tumor-associated antigens, either simultaneously or sequentially, could avoid issues of antigen heterogeneity and deliver therapeutic benefit to patients.

Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia.

Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development.

T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis.

Although T cells can exert potent anti-tumor immunity, a subset of T helper (Th) cells producing interleukin-22 (IL-22) in breast and lung tumors is linked to dismal patient outcome. Here, we examined the mechanisms whereby these T cells contribute to disease. In murine models of lung and breast cancer, constitutional and T cell-specific deletion of Il22 reduced metastases without affecting primary tumor growth. Deletion of the IL-22 receptor on cancer cells decreases metastasis to a degree similar to that seen in IL-22-deficient mice. IL-22 induced high expression of CD155, which bound to the activating receptor CD226 on NK cells. Excessive activation led to decreased amounts of CD226 and functionally impaired NK cells, which elevated the metastatic burden. IL-22 signaling was also associated with CD155 expression in human datasets and with poor patient outcomes. Taken together, our findings reveal an immunosuppressive circuit activated by T cell-derived IL-22 that promotes lung metastasis.

Beyond direct killing-novel cellular immunotherapeutic strategies to reshape the tumor microenvironment.

The clinical use of cellular immunotherapies is gaining momentum and the number of approved indications is steadily increasing. One class of cellular therapies-chimeric antigen receptor (CAR)-modified T cells-has achieved impressive results in distinct blood cancer indications. These existing cellular therapies treating blood cancers face significant relapse rates, and their application beyond hematology has been underwhelming, especially in solid oncology. Major reasons for resistance source largely in the tumor microenvironment (TME). The TME in fact functionally suppresses, restricts, and excludes adoptive immune cells, which limits the efficacy of cellular immunotherapies from the onset. Many promising efforts are ongoing to adapt cellular immunotherapies to address these obstacles, with the aim of reshaping the tumor microenvironment to ameliorate function and to achieve superior efficacy against both hematological and solid malignancies.

PD-1-CD28 fusion protein strengthens mesothelin-specific TRuC T cells in preclinical solid tumor models.

BACKGROUND: T cell receptor fusion constructs (TRuC) consist of an antibody-based single chain variable fragment (scFv) fused to a T cell receptor chain (TCR) and allow recognition of cancer cells in an HLA-independent manner. Unlike chimeric antigen receptors (CAR), TRuC are integrated into the TCR complex resulting in a functional chimera with novel specificity, whilst retaining TCR signaling. To further enhance anti-tumor function, we expressed a PD-1-CD28 fusion receptor in TRuC T cells aiming to prevent tumor-induced immune suppression and T cell anergy. METHODS: The activation level of engineered T cells was investigated in co-culture experiments with tumor cells followed by quantification of released cytokines using ELISA. To study T cell-mediated tumor cell lysis in vitro, impedance-based real-time tumor cell killing and LDH release was measured. Finally, two xenograft mouse cancer models were employed to explore the therapeutic potential of engineered T cells. RESULTS: In co-culture assays, co-expression of PD-1-CD28 enhanced cytokine production of TRuC T cells. This effect was dependent on PD-L1 to PD-1-CD28 interactions, as blockade of PD-L1 amplified IFN-γ production in unmodified TRuC T cells to a greater level compared to TRuC-PD-1-CD28 T cells. In vivo, PD-1-CD28 co-expression supported the anti-tumor efficacy of TRuC T cells in two xenograft mouse cancer models. CONCLUSION: Together, these results demonstrate the therapeutic potential of PD-1-CD28 co-expression in TRuC T cells to prevent PD-L1-induced T cell hypofunction.

Migratory Engineering of T Cells for Cancer Therapy.

Adoptive cell therapy (ACT) and chimeric antigen receptor (CAR) T cell therapy in particular represents an adaptive, yet versatile strategy for cancer treatment. Convincing results in the treatment of hematological malignancies have led to FDA approval for several CAR T cell therapies in defined refractory diseases. In contrast, the treatment of solid tumors with adoptively transferred T cells has not demonstrated convincing efficacy in clinical trials. One of the main reasons for ACT failure in solid tumors is poor trafficking or access of transferred T cells to the tumor site. Tumors employ a variety of mechanisms shielding themselves from immune cell infiltrates, often translating to only fractions of transferred T cells reaching the tumor site. To overcome this bottleneck, extensive efforts are being undertaken at engineering T cells to improve ACT access to solid tumors. In this review, we provide an overview of the immune cell infiltrate in human tumors and the mechanisms tumors employ toward immune exclusion. We will discuss ways in which T cells can be engineered to circumvent these barriers. We give an outlook on ongoing clinical trials targeting immune cell migration to improve ACT and its perspective in solid tumors.

Impact of the selective A2AR and A2BR dual antagonist AB928/etrumadenant on CAR T cell function.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has been successfully translated to clinical practice for the treatment of B cell malignancies. The suppressive microenvironment of many malignancies is a bottleneck preventing treatment success of CAR T cells in a broader range of tumours. Among others, the immunosuppressive metabolite adenosine is present in high concentrations within many tumours and dampens anti-tumour function of immune cells and consequently therapeutic response. METHODS: Here, we present the impact of the selective adenosine A2A and A2B receptor antagonist AB928/etrumadenant on CAR T cell cytokine secretion, proliferation, and cytotoxicity. Using phosphorylation-specific flow cytometry, we evaluated the capability of AB928 to shield CAR T cells from adenosine-mediated signalling. The effect of orally administered AB928 on CAR T cells was assessed in a syngeneic mouse model of colon carcinoma. RESULTS: We found that immunosuppressive signalling in CAR T cells in response to adenosine was fully blocked by the small molecule inhibitor. AB928 treatment enhanced CAR T cell cytokine secretion and proliferation, granted efficient cytolysis of tumour cells in vitro and augmented CAR T cell activation in vivo. CONCLUSIONS: Together our results suggest that combination therapy with AB928 represents a promising approach to improve adoptive cell therapy.

Chimeric antigen receptor T cells engineered to recognize the P329G-mutated Fc part of effector-silenced tumor antigen-targeting human IgG1 antibodies enable modular targeting of solid tumors.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has proven its clinical utility in hematological malignancies. Optimization is still required for its application in solid tumors. Here, the lack of cancer-specific structures along with tumor heterogeneity represent a critical barrier to safety and efficacy. Modular CAR T cells indirectly binding the tumor antigen through CAR-adaptor molecules have the potential to reduce adverse events and to overcome antigen heterogeneity. We hypothesized that a platform utilizing unique traits of clinical grade antibodies for selective CAR targeting would come with significant advantages. Thus, we developed a P329G-directed CAR targeting the P329G mutation in the Fc part of tumor-targeting human antibodies containing P329G L234A/L235A (LALA) mutations for Fc silencing. METHODS: A single chain variable fragment-based second generation P329G-targeting CAR was retrovirally transduced into primary human T cells. These CAR T cells were combined with IgG1 antibodies carrying P329G LALA mutations in their Fc part targeting epidermal growth factor receptor (EGFR), mesothelin (MSLN) or HER2/neu. Mesothelioma, pancreatic and breast cancer cell lines expressing the respective antigens were used as target cell lines. Efficacy was evaluated in vitro and in vivo in xenograft mouse models. RESULTS: Unlike CD16-CAR T cells, which bind human IgG in a non-selective manner, P329G-targeting CAR T cells revealed specific effector functions only when combined with antibodies carrying P329G LALA mutations in their Fc part. P329G-targeting CAR T cells cannot be activated by an excess of human IgG. P329G-directed CAR T cells combined with a MSLN-targeting P329G-mutated antibody mediated pronounced in vitro and in vivo antitumor efficacy in mesothelioma and pancreatic cancer models. Combined with a HER2-targeting antibody, P329G-targeting CAR T cells showed substantial in vitro activation, proliferation, cytokine production and cytotoxicity against HER2-expressing breast cancer cell lines and induced complete tumor eradication in a breast cancer xenograft mouse model. The ability of the platform to target multiple antigens sequentially was shown in vitro and in vivo. CONCLUSIONS: P329G-targeting CAR T cells combined with antigen-binding human IgG1 antibodies containing the P329G Fc mutation mediate pronounced in vitro and in vivo effector functions in different solid tumor models, warranting further clinical translation of this concept.

Utilizing chemokines in cancer immunotherapy.

Immune checkpoint inhibition and chimeric antigen receptor (CAR) T cell therapy have demonstrated stunning clinical efficacy in many cancer types. However, most patients do not respond to immunotherapies or relapse after an initial response, stressing the need for improved strategies. Chemokines, as mediators of immune cell trafficking, play an important role in the composition of the tumor microenvironment and exert both pro- and antitumorigenic functions. Here, chemokines may represent valuable prognostic biomarkers of response to immunotherapy and a strategy to improve immunotherapies. In this review, the pleiotropic functions of chemokines in the tumor microenvironment (TME) and strategies of utilizing chemokines or chemokine antagonism in immunotherapy are discussed. The review highlights preclinical and clinical studies that apply or target chemokines in monotherapy or in combination therapies.

FluoRNT: A robust, efficient assay for the detection of neutralising antibodies against yellow fever virus 17D.

There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), and recurring viral outbreaks like the yellow fever outbreaks in Angola and the Democratic Republic of Congo (2016) and in Brazil (2016-2018). Current assays to determine neutralising activity against viral infections in sera are costly in time and equipment and suffer from high variability. Therefore, both basic infection research and diagnostic population screenings would benefit from improved methods to determine virus-neutralising activity in patient samples. Here we describe a robust, objective, and scalable Fluorescence Reduction Neutralisation Test (FluoRNT) for yellow fever virus, relying on flow cytometric detection of cells infected with a fluorescent Venus reporter containing variant of the yellow fever vaccine strain 17D (YF-17D-Venus). It accurately measures neutralising antibody titres in human serum samples within as little as 24 h. Samples from 32 vaccinees immunised with YF-17D were tested for neutralising activity by both a conventional focus reduction neutralisation test (FRNT) and FluoRNT. Both types of tests proved to be equally reliable for the detection of neutralising activity, however, FluoRNT is significantly more precise and reproducible with a greater dynamic range than conventional FRNT. The FluoRNT assay protocol is substantially faster, easier to control, and cheaper in per-assay costs. FluoRNT additionally reduces handling time minimising exposure of personnel to patient samples. FluoRNT thus brings a range of desirable features that can accelerate and standardise the measurement of neutralising anti-yellow fever virus antibodies. It could be used in applications ranging from vaccine testing to large cohort studies in systems virology and vaccinology. We also anticipate the potential to translate the methodology and analysis of FluoRNT to other flaviviruses such as West Nile, Dengue and Zika or to RNA viruses more generally.

Enhanced Chimeric Antigen Receptor T Cell Therapy through Co-Application of Synergistic Combination Partners.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable response rates and revolutionized the treatment of patients suffering from defined hematological malignancies. However, many patients still do not respond to this therapy or relapse after an initial remission, underscoring the need for improved efficacy. Insufficient in vivo activity, persistence, trafficking, and tumor infiltration of CAR T cells, as well as antigen escape and treatment-associated adverse events, limit the therapeutic success. Multiple strategies and approaches have been investigated to further improve CAR T cell therapy. Besides genetic modification of the CAR itself, the combination with other treatment modalities has the potential to improve this approach. In particular, combining CAR T cells with clinically approved compounds such as monoclonal antibodies and small molecule inhibitors might be a promising strategy. Combination partners could already be applied during the production process to influence the cellular composition and immunophenotype of the final CAR T cell product. Alternatively, simultaneous administration of clinically approved compounds with CAR T cells would be another feasible avenue. In this review, we will discuss current strategies to combine CAR T cells with compounds to overcome recent limitations and further enhance this promising cancer therapy, potentially broadening its application beyond hematology.

Flow cytometry detection and quantification of CAR T cells into solid tumors.

Adoptive T cell therapy (ACT) is a therapeutic approach which employs genetically manipulated autologous T cells to target and eliminate a patient's malignancy. This novel therapeutic approach, when employing a chimeric antigen receptor (CAR) targeting CD19-expressing B cells, has shown remarkable success in treating acute B-cell lymphocytic leukemia. However, blood born malignancies represent only a fraction of cancers which affect patients. Unfortunately, the utilization of ACT to target solid malignancies has only shown marginal success rates. There are many known obstacles which hinder CAR T cell therapy in patients suffering from solid cancer, one notable obstacle is the effective trafficking of CAR T cells to the tumor site. With the rapid advancement of novel approaches and targets which may enhance CAR T cell infiltration into solid tumors, a standardized approach to assess and measure CAR T cell infiltration becomes imperative in order to compare these different approaches across platforms. Here we describe a flow cytometry method which enables the rapid detection and quantification of CAR T cells which have reached and entered the tumor mass following intravenous injection. Competence with single cell preparation and flow cytometry is required for optimal results.

Prodrug-Activating Chain Exchange (PACE) converts targeted prodrug derivatives to functional bi- or multispecific antibodies.

Driven by the potential to broaden the target space of conventional monospecific antibodies, the field of multi-specific antibody derivatives is growing rapidly. The production and screening of these artificial proteins entails a high combinatorial complexity. Antibody-domain exchange was previously shown to be a versatile strategy to produce bispecific antibodies in a robust and efficient manner. Here, we show that the domain exchange reaction to generate hybrid antibodies also functions under physiological conditions. Accordingly, we modified the exchange partners for use in therapeutic applications, in which two inactive prodrugs convert into a product with additional functionalities. We exemplarily show the feasibility for generating active T cell bispecific antibodies from two inactive prodrugs, which per se do not activate T cells alone. The two complementary prodrugs harbor antigen-targeting Fabs and non-functional anti-CD3 Fvs fused to IgG-CH3 domains engineered to drive chain-exchange reactions between them. Importantly, Prodrug-Activating Chain Exchange (PACE) could be an attractive option to conditionally activate therapeutics at the target site. Several examples are provided that demonstrate the efficacy of PACE as a new principle of cancer immunotherapy in vitro and in a human xenograft model.

CAR T cell therapy in solid tumors: a short review.

Chimeric antigen receptor (CAR) T cell therapy has been established in the treatment of hematological malignancies. However, in solid tumors its efficacy remains limited. The aim of this article is to give an overview of the field of cell therapy itself, to introduce the underlying concepts of CAR T cell-based treatment approaches and to address its limitations in advancing the treatment for solid malignancies.