Engineered T cells secreting anti-BCMA T cell engagers control multiple myeloma and promote immune memory in vivo.

Multiple myeloma is the second most common hematological malignancy in adults and remains an incurable disease. B cell maturation antigen (BCMA)-directed immunotherapy, including T cells bearing chimeric antigen receptors (CARs) and systemically injected bispecific T cell engagers (TCEs), has shown remarkable clinical activity, and several products have received market approval. However, despite promising results, most patients eventually become refractory and relapse, highlighting the need for alternative strategies. Engineered T cells secreting TCE antibodies (STAb) represent a promising strategy that combines the advantages of adoptive cell therapies and bispecific antibodies. Here, we undertook a comprehensive preclinical study comparing the therapeutic potential of T cells either expressing second-generation anti-BCMA CARs (CAR-T) or secreting BCMAxCD3 TCEs (STAb-T) in a T cell-limiting experimental setting mimicking the conditions found in patients with relapsed/refractory multiple myeloma. STAb-T cells recruited T cell activity at extremely low effector-to-target ratios and were resistant to inhibition mediated by soluble BCMA released from the cell surface, resulting in enhanced cytotoxic responses and prevention of immune escape of multiple myeloma cells in vitro. These advantages led to robust expansion and persistence of STAb-T cells in vivo, generating long-lived memory BCMA-specific responses that could control multiple myeloma progression in xenograft models, outperforming traditional CAR-T cells. These promising preclinical results encourage clinical testing of the BCMA-STAb-T cell approach in relapsed/refractory multiple myeloma.

Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers.

Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.

Synapse topology and downmodulation events determine the functional outcome of anti-CD19 T cell-redirecting strategies.

Cancer immunotherapy strategies based on the endogenous secretion of T cell-redirecting bispecific antibodies by engineered T lymphocytes (STAb-T) are emerging as alternative or complementary approaches to those based on chimeric antigen receptors (CAR-T). The antitumor efficacy of bispecific anti-CD19 × anti-CD3 (CD19×CD3) T cell engager (BiTE)-secreting STAb-T cells has been demonstrated in several mouse models of B-cell acute leukemia. Here, we have investigated the spatial topology and downstream signaling of the artificial immunological synapses (IS) that are formed by CAR-T or STAb-T cells. Upon interaction with CD19-positive target cells, STAb-T cells form IS with structure and signal transduction, which more closely resemble those of physiological cognate IS, compared to IS formed by CAR-T cells expressing a second-generation CAR bearing the same CD19-single-chain variable fragment. Importantly, while CD3 is maintained at detectable levels on the surface of STAb-T cells, indicating sustained activation mediated by the secreted BiTE, the anti-CD19 CAR was rapidly downmodulated, which correlated with a more transient downstream signaling. Furthermore, CAR-T cells, but not STAb-T cells, provoke an acute loss of CD19 in target cells. Such differences might represent advantages of the STAb-T strategy over the CAR-T approach and should be carefully considered in order to develop more effective and safer treatments for hematological malignancies.

Trispecific T-cell engagers for dual tumor-targeting of colorectal cancer.

Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR-EpCAM-) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity.

Bispecific Antibody Format and the Organization of Immunological Synapses in T Cell-Redirecting Strategies for Cancer Immunotherapy.

T cell-redirecting strategies have emerged as effective cancer immunotherapy approaches. Bispecific antibodies (bsAbs) are designed to specifically recruit T cells to the tumor microenvironment and induce the assembly of the immunological synapse (IS) between T cells and cancer cells or antigen-presenting cells. The way that the quality of the IS might predict the effectiveness of T cell-redirecting strategies, including those mediated by bsAbs or by chimeric antigen receptors (CAR)-T cells, is currently under discussion. Here we review the organization of the canonical IS assembled during natural antigenic stimulation through the T cell receptor (TCR) and to what extent different bsAbs induce T cell activation, canonical IS organization, and effector function. Then, we discuss how the biochemical parameters of different formats of bsAbs affect the effectivity of generating an antigen-induced canonical IS. Finally, the quality of the IS assembled by bsAbs and monoclonal antibodies or CAR-T cells are compared, and strategies to improve bsAb-mediated T cell-redirecting strategies are discussed.

Effect of Pharmacological Inhibition of the Catalytic Activity of Phosphatases of Regenerating Liver in Early T Cell Receptor Signaling Dynamics and IL-2 Production.

We have previously shown the delivery of phosphatase of regenerating liver-1 (PRL-1) to the immunological synapse (IS) and proposed a regulatory role of the catalytic activity of PRLs (PRL-1, PRL-2 and PRL-3) in antigen-induced IL-2 production. Nonetheless, the expression in T cells and delivery to the IS of the highly homologous PRL-3, as well as the role of the catalytic activity of PRLs in antigen-induced early signaling, has not been investigated. Here, the expression of PRL-3 protein was detected in primary CD4 T cells and in the CD4 T cell line Jurkat (JK), in which an overexpressed GFP-PRL-3 fluorescent fusion protein trafficked through the endosomal recycling compartment and co-localized with PLCγ1 signaling sites at the IS. Pharmacological inhibition was used to compare the role of the catalytic activity of PRLs in antigen-induced early signaling and late IL-2 production. Although the phosphatase activity of PRLs was not critical for early signaling triggered by antigen, it seemed to regulate signaling dynamics and was necessary for proper IL-2 production. We propose that enzymatic activity of PRLs has a higher significance for cytokine production than for early signaling at the IS. However, further research will be necessary to deeply understand the regulatory role of PRLs during lymphocyte activation and effector function.