Christina Angeliki Tsiverioti

Multispecific antibodies have emerged as versatile therapeutic agents, and therefore, approaches to optimize and streamline their design and assembly are needed. Here we report on the modular and programmable assembly of IgG antibodies, F(ab) and scFv fragments on DNA origami nanocarriers. We screened 105 distinct quadruplet antibody variants in vitro for the ability to activate T cells in the presence of target cells. T-cell engagers were identified, which in vitro showed the specific and efficient T-cell-mediated lysis of five distinct target cell lines. We used these T-cell engagers to target and lyse tumour cells in vivo in a xenograft mouse tumour model. Our approach enables the rapid generation, screening and testing of bi- and multispecific antibodies to facilitate preclinical pharmaceutical development from in vitro discovery to in vivo proof of concept.

Chimeric antigen receptor (CAR)-T cell therapy has led to remarkable clinical outcomes in the treatment of hematological malignancies. However, challenges remain, such as limited infiltration into solid tumors, inadequate persistence, systemic toxicities, and manufacturing insufficiencies. The use of alternative cell sources for CAR-based therapies, such as natural killer cells (NK), macrophages (MΦ), invariant Natural Killer T (iNKT) cells, γδT cells, neutrophils, and induced pluripotent stem cells (iPSC), has emerged as a promising avenue. By harnessing these cells' inherent cytotoxic mechanisms and incorporating CAR technology, common CAR-T cell-related limitations can be effectively mitigated. We herein present an overview of the tumoricidal mechanisms, CAR designs, and manufacturing processes of CAR-NK cells, CAR-MΦ, CAR-iNKT cells, CAR-γδT cells, CAR-neutrophils, and iPSC-derived CAR-cells, outlining the advantages, limitations, and potential solutions of these therapeutic strategies.

ABSTRACT: The success of targeted therapies for hematological malignancies has heralded their potential as both salvage treatment and early treatment lines, reducing the need for high-dose, intensive, and often toxic chemotherapeutic regimens. For young patients with classic Hodgkin lymphoma (cHL), immunotherapies provide the possibility to lessen long-term, treatment-related toxicities. However, suitable therapeutic targets are lacking. By integrating single-cell dissection of the tumor landscape and an in-depth, single-cell-based off-tumor antigen prediction, we identify CD86 as a promising therapeutic target in cHL. CD86 is highly expressed on Hodgkin and Reed-Sternberg cancer cells and cHL-specific tumor-associated macrophages. We reveal CD86-CTLA-4 as a key suppressive pathway in cHL, driving T-cell exhaustion. Cellular therapies targeting CD86 had extraordinary efficacy in vitro and in vivo and were safe in immunocompetent mouse models without compromising bacterial host defense in sepsis models. Our results prove the potential value of anti-CD86 immunotherapies for treating cHL.

The success of targeted immunotherapies for hematological malignancies has heralded their potential as salvage therapies as well as in earlier treatment lines (Cappell & Kochenderfer, 2023). While conventional chemotherapy-based treatments can achieve long-term survival in up to 90 % of treated patients with classic Hodgkin lymphoma (cHL), these therapies are associated with treatment-related comorbidities, calling for more tailored and specific approaches (Schaapveld et al., 2015; Shanbhag & Ambinder, 2018). While targeted treatments, especially immunotherapies are taking oncology by storm, the utility in cHL is so far limited to CD30 and PD-1-targeting strategies and there is a clear lack of drugable relevant target structures in this disease. This can be partly attributed to technical difficulties of analyzing the malignant Hodgkin-Reed-Sternberg (HRS) cells specifically. Capitalizing on our previous work using large scale data mining to inform target discovery, we hypothesized that combining different analytical methods with large single-cell RNA-Sequencing (scRNA-Seq) datasets would permit selective target definition with functional relevance to the disease and thereby allow the development of novel immunotherapeutic strategies. Leveraging microarray profiles of laser-dissected HRS cells and a scRNA-Seq cohort of cHL patients (total of n = 44 primary samples; n = 34 cHL samples; n = 10 RLN (reactive lymph node) control samples), we screened for novel target antigens highly expressed on HRS cells with functional relevance in the tumor microenvironement (TME) of cHL. Unbiased in silico analyses revealed CD80, CD86 and PD-L1 as most suitable candidate target antigens with CD86 showing the highest expression on HRS cells. ScRNA-Seq analyses unveiled a shift of the CD80-CD86-CTLA-4-CD28 towards the immunosuppressive CTLA-4 axis in the TME of cHL compared to RLN controls. In advanced cell culture models, including iPSC-derived organoid models, blockage of CD86 lead to the decreased expression of PD-1 and CTLA-4 and an overall reversal of the exhaustive phenotype of cHL-associated T cells. High protein expression of CD86 on HRS cells and in the TME (cHL-infiltrating tumor-associated macrophages (cHL-TAM), B cells) was confirmed in different validation cohorts including relapsed and refractory cHL (r/r cHL) patients by conventional immunohistochemistry and multiplexed immunofluorescence (n = 34 cHL patients). Following target identification, CAR T cells redirected against CD86 were developed and the functionality of these CAR T cells was investigated in preclinical models both in vitro and in vivo. Anti-CD86 CAR T cells effectively deplete cHL-TAM and are highly effective in various in vitro and in vivo models of cHL, including models of CD30-negative disease. Given the fundamental role of the CD80-CD86-CTLA-4-CD28 axis in the generation of the adaptive immune response, detailed toxicity assessments were carried out leveraging murine surrogate anti-CD86 CAR T cells, with similar binding and activation thresholds as their human counterpart. These anti-mCD86 CAR T cells did not cause toxicities in lymphodepleted, immunocompetent mice. In addition, the impact of anti-CD86-directed immunotherapies (e.g. anti-CD86-blocking antibodies, anti-mCD86 CAR T cells) on bacterial host defense and formation of antigen-specific adaptive immunity was investigated in syngeic mouse models. Anti-CD86 immunotherapy did not lead to enhanced bacteremia in a model of gram-negative sepsis, while preclinical vaccination models revealed a mildy reduced formation of antigen-specific T cell development in mice. In summary, we provide a framework for unbiased, multi-dimensional target screening and highlight the functional relevance of the immunosuppressive CD86-CTLA-4 axis in cHL. CD86-directed immunotherapy could reverse the exhaustive phenotype of cHL-associated T cells, while demonstrating strong treatment efficacy in xenograft mouse models. Importantly, elaborate toxicity assessments of anti-CD86-targeted immunotherapies utilizing syngenic mouse models did not reveal measureable toxicity in mice. Overall, our data emphasizes the vast translational potential of CD86-targeted immunotherapies in cHL and provide a strong rationale for further clinical investigations.