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Whilst treatment of multiple myeloma (MM) with daratumumab significantly extend patient lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/rMM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T-cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT®) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38 including those with a lower sensitivity to daratumumab. In a killing assay, wherein multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity towards MM cells compared to daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in two therapeutic mouse models, unlike daratumumab. Lastly, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/rMM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently developed in a phase 1 clinical study.

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Central memory CD8

Significance Cancer immunotherapy demonstrates clinical efficacy in selected cancer types. Yet, as the majority of patients do not respond to the most effective immunotherapeutics, novel immunotherapy combinations are extensively investigated. There is increasing clinical interest in combining cancer immunotherapies with antiangiogenic agents, particularly VEGFA pathway inhibitors. Here we show that anti-CD40 immunotherapy increases CD8+ T cell infiltration in the tumor, yet tumor regression is achieved more robustly when anti-CD40 is combined with dual Ang2 and VEGFA blockade, but not VEGFA inhibition alone. Tumor regression was associated with proinflammatory skewing of the tumor microenvironment and intratumoral redistribution of CD8+ T cells. These data emphasize the rationale for blocking Ang2 as a vascular-modulatory strategy in combination with T cell-targeting immunotherapies., Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8+ T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.

Central memory CD8 + T cells (Tcm) control systemic secondary infections and can protect from chronic infection and cancer as a result of their stem-cell-like capacity to expand, differentiate, and self-renew. Central memory is generally thought to emerge following pathogen clearance and to form based on the de-differentiation of cytolytic effector cells. Here, we uncovered rare effector-phase CD8 + T cells expressing high amounts of the transcription factor Tcf7 (Tcf1) that showed no evidence of prior cytolytic differentiation and that displayed key hallmarks of Tcm cells. These effector-phase Tcf7 hi cells quantitatively yielded Tcm cells based on lineage tracing. Mechanistically, Tcf1 counteracted the differentiation of Tcf7 hi cells and sustained the expression of conserved adult stem-cell genes that were critical for CD8 + T cell stemness. The discovery of stem-cell-like CD8 + T cells during the effector response to acute infection provides an opportunity to optimize Tcm cell formation by vaccination.

Checkpoint blockade mediates a proliferative response of tumor-infiltrating CD8

Chronic infections promote the terminal differentiation (or "exhaustion") of T cells and are thought to preclude the formation of memory T cells. In contrast, we discovered a small subpopulation of virus-specific CD8(+) T cells that sustained the T cell response during chronic infections. These cells were defined by, and depended on, the expression of the transcription factor Tcf1. Transcriptome analysis revealed that this population shared key characteristics of central memory cells but lacked an effector signature. Unlike conventional memory cells, Tcf1-expressing T cells displayed hallmarks of an "exhausted" phenotype, including the expression of inhibitory receptors such as PD-1 and Lag-3. This population was crucial for the T cell expansion that occurred in response to inhibitory receptor blockade during chronic infection. These findings identify a memory-like T cell population that sustains T cell responses and is a prime target for therapeutic interventions to improve the immune response in chronic infections.

Relatório final do estágio profissionalizante do 6.º ano

Checkpoint blockade mediates a proliferative response of tumor-infiltrating CD8 + T lymphocytes (TILs). The origin of this response has remained elusive because chronic activation promotes terminal differentiation or exhaustion of tumor-specific T cells. Here we identified a subset of tumor-reactive TILs bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcf1. Tcf1 + PD-1 + TILs mediated the proliferative response to immunotherapy, generating both Tcf1 + PD-1 + and differentiated Tcf1 - PD-1 + cells. Ablation of Tcf1 + PD-1 + TILs restricted responses to immunotherapy. Tcf1 was not required for the generation of Tcf1 + PD-1 + TILs but was essential for the stem-like functions of these cells. Human TCF1 + PD-1 + cells were detected among tumor-reactive CD8 + T cells in the blood of melanoma patients and among TILs of primary melanomas. Thus, immune checkpoint blockade relies not on reversal of T cell exhaustion programs, but on the proliferation of a stem-like TIL subset.

Mestrado em Biomedicina Molecular Angiogenesis is crucial for breast cancer growth, progression and dissemination, making it a promising therapeutic target. Given the dominant role of Vascular Endothelial Growth Factor (VEGF) signaling in tumor angiogenesis, the majority of clinical studies with anti-angiogenic therapies in breast cancer were based on VEGF blockade. However, lack of clinical efficacy was observed in all clinical studies with antiangiogenic therapies for breast cancer. Since angiopoietin 2 (ANG-2) has been reported to induce therapeutic resistance after VEGF-targeted therapies, breast cancer patients may benefit from a therapeutic strategy that simultaneously blocks ANG-2 and VEGF-A. Indeed, using the bispecific ANG-2/VEGF-A targeting antibody (A2V), durable inhibition of tumor progression, as well as increased survival, could be observed after dual ANG-2/VEGF-A blockade in the aggressive mouse model of breast cancer, the MMTV-PyMT transgenic mouse. Consistent with tumor inhibition, increased tumor necrosis and a drastic reduction of vascular density was observed after A2V therapy, with little evidence of tumor revascularization or resistance to therapy. Moreover, A2V led to increased perivascular coverage of the remaining tumor blood vessels, favoring vascular normalization, which is known to facilitate the development of an immunosupportive microenvironment. These findings suggested that breast cancer immunotherapy may benefit from the combination with A2V. Therefore the A2V mAb was combined with a CD40 agonistic antibody (FGK45). The combined therapeutic approach led to remarkable tumor growth inhibition in an orthotopic MMTV-PyMT model, with the achievement of cases of stable disease and further increase in tumor necrosis. Increased infiltration of CD8+ cells after FGK45 therapy, either alone or in combination with A2V, could also be observed. Of note, a correlation between the extent of tumor inhibition and increased tumor infiltration by CD8+ cells was detected, suggesting that the influx of CD8+ cells in the combination therapy-treated tumors was responsible for the remarkable anti-tumor responses observed. In summary, these results point to a significant synergistic activity of anti-ANG-2/VEGF-A and CD40 agonistic therapy in the MMTV-PyMT model of breast carcinoma. This combinatorial approach may benefit breast cancer patients, so further studies that explore the mechanistic bases of the cooperative activity of antiangiogenic and immunostimulatory therapy are warranted. A angiogénese é um processo crucial para o crescimento, progressão e disseminação do cancro da mama, tornando-se assim um alvo terapêutico promissor. A maioria dos estudos clínicos de terapias anti-angiogénicas em cancro da mama baseiam-se na neutralização do fator de crescimento endotelial vascular (VEGF), uma vez que este apresenta um papel dominante na angiogénese tumoral. No entanto, todos os estudos clínicos baseados em terapias anti-angiogénicas têm-se revelado ineficazes no tratamento de pacientes com cancro da mama. Uma vez que a Angiopoietina 2 (ANG-2), outro fator de crescimento vascular, tem sido reportada como responsável pela resistência a terapias baseadas no bloqueio de função do VEGF, é possível que pacientes com cancro da mama beneficiem de uma estratégia terapêutica que neutralize simultaneamente ANG-2 e VEGF-A. De facto, através do uso de um anticorpo ‘biespecífico’, capaz de neutralizar tanto ANG-2 como VEGF-A e denominado A2V, observou-se uma forte inibição da progressão tumoral e aumento da sobrevivência num modelo pré-clínico de cancro da mama agressivo, baseado em ratinhos transgénicos e denominado MMTV-PyMT. Consistentemente, esta terapia levou ao aumento de necrose tumoral e a uma drástica redução na densidade vascular, sem evidência de revascularização ou resistência à terapia. A administração de A2V induziu ainda o aumento da associação entre células perivasculares (‘pericytes’) e células endoteliais dos restantes vasos sanguíneos tumorais, favorecendo a maturação e normalização vascular, o que facilita o desenvolvimento de um microambiente que suporta respostas imunológicas. Estes resultados sugeriam assim que imunoterapia em cancro da mama poderia beneficiar da combinação com A2V, tendo-se por conseguinte combinado o anticorpo A2V com o anticorpo agonista de CD40 (FGK45), no modelo ortotópico de MMTV-PyMT. Esta terapia induziu uma notável inibição do crescimento tumoral, com obtenção de casos estáveis, e aumento adicional de necrose tumoral. Observou-se ainda o aumento de infiltração de células CD8+ após terapia com FGK45, quer isoladamente ou em combinação com A2V, e uma correlação entre a extensão da inibição do tumor e o aumento da infiltração tumoral por células CD8+. Isto sugere que o influxo de células CD8+ nos tumores tratados com A2V e FGK45 foi o principal responsável pelas notáveis respostas anti-tumorais observadas. Em resumo, estes resultados apontam para uma sinergia entre a neutralização simultânea de ANG-2/VEGF-A e imunoterapia baseada em agonistas do recetor CD40, no modelo pré-clínico de cancro da mama MMTV-PyMT. Esta abordagem combinatória pode beneficiar pacientes com cancro da mama, por isso, mais estudos são necessários para explorar os mecanismos subjacentes à atividade cooperativa da terapia anti-angiogénica e a imunoterapia.