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CD70 has emerged as a promising target in acute myeloid leukemia (AML), and we have previously demonstrated the potency of an optimized CD70-targeted ligand-based CAR. However, here, we identify in vivo CD70 antigen escape as a limitation of single antigen targeting. Combination targeting of CD70 and CD33 may overcome AML antigen heterogeneity. We hypothesized that modifying our CD70 CAR platform to secrete a bispecific T cell engaging antibody molecule ("TEAM") targeting CD33 (7033) would create a therapeutic window whereby AML heterogeneity could be addressed without increasing tissue toxicity. We found that CD33 TEAMs mediated specific cytotoxicity across AML cell lines, including CD33 or CD70 single-antigen knockout tumors. 7033 CAR-T cells eradicated tumor in an in vivo mixed tumor model of CD70 antigen escape and outperformed the previously optimized CD70 CAR in a patient-derived xenograft. In vivo gene expression profiling of CAR-T cells revealed enhanced 7033 CAR-T cell pathway scoring for persistence, activation, and TCR signaling. Additionally, CD33 TEAMs successfully redirected T cells isolated from AML patients to activate, secrete cytokines, and kill tumor targets despite exposure to substantial prior cytotoxic therapies. In summary, our findings demonstrate the feasibility of our 7033 CAR to overcome AML heterogeneity and leverage the bystander T cells of patients; this approach warrants further study in patients with this dire clinical need., (Copyright © 2024 American Society of Hematology.)

Purpose: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells)., Experimental Design: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids., Results: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors., Conclusions: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer., (©2024 American Association for Cancer Research.)

Tumors evolve together with the tumor microenvironment (TME) and reshape it towards immunosuppression. Immunostimulating cytokines can be used to revert this state leading to effective antitumor immune responses, but their exploitation as anticancer drugs has been hampered by severe toxicity associated with systemic administration. Local, TME-targeted delivery of immune activating cytokines can deploy their antitumoral function more effectively than systemic administration while, at the same time, avoiding exposure of healthy organs and limiting toxicity. Here, we review different gene and cell therapy platforms developed for tumor-directed cytokine delivery highlighting their potential for clinical translation., Competing Interests: Declaration of interests L.N. is an inventor on patents on lentiviral vector technology filed by the Telethon Foundation and San Raffaele Scientific Institute. According to the respective institutional policies, inventors may be entitled to receive some financial benefits from the licensing of such patents. L.N. is a founder, owns equity, and is a consultant and member of the scientific advisory board of Genenta Science, a biotechnology company aiming at developing cancer gene therapy by targeted cytokine delivery by tumor-infiltrating monocytes. The other authors have no interests to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor characterized by a strongly immunosuppressive tumor microenvironment (TME) that represents a barrier also for the development of effective immunotherapies. The possibility to revert this hostile TME by immunoactivating cytokines is hampered by the severe toxicity associated with their systemic administration. Here, we exploited a lentiviral vector-based platform to engineer hematopoietic stem cells ex vivo with the aim of releasing, via their tumor-infiltrating monocyte/macrophage progeny, interferon-α (IFN-α) or interleukin-12 (IL-12) at the tumor site with spatial and temporal selectivity. Taking advantage of a syngeneic GBM mouse model, we showed that inducible release of IFN-α within the TME achieved robust tumor inhibition up to eradication and outperformed systemic treatment with the recombinant protein in terms of efficacy, tolerability, and specificity. Single-cell RNA sequencing of the tumor immune infiltrate revealed reprogramming of the immune microenvironment toward a proinflammatory and antitumoral state associated with loss of a macrophage subpopulation shown to be associated with poor prognosis in human GBM. The spatial and temporal control of IL-12 release was critical to overcome an otherwise lethal hematopoietic toxicity while allowing to fully exploit its antitumor activity. Overall, our findings demonstrate a potential therapeutic approach for GBM and set the bases for a recently launched first-in-human clinical trial in patients with GBM.

The receptor tyrosine kinase ERBB2 interacts with HSP90 and is overexpressed in aggressive breast cancers. Therapeutic HSP90 inhibitors, i.e. Geldanamycin (GA), target ERBB2 to degradation. We have previously shown that HSP90 is responsible for the missorting of recycling ERBB2 to degradation compartments. In this study, we used biochemical, immunofluorescence and electron microscopy techniques to demonstrate that in SKBR3 human breast cancer cells, GA strongly induces polyubiquitination and internalization of the full-length p185-ERBB2, and promotes its cleavage, with the formation of a p116-ERBB2 form in EEA1-positive endosomes (EE). p116-ERBB2 corresponds to a non-ubiquitinated, signaling-impaired, membrane-bound fragment, which is readily sorted to lysosomes and degraded. To define the sequence of events leading to p116-ERBB2 degradation, we first blocked the EE maturation/trafficking to late endosomes/lysosomes with wortmannin, and found an increase in GA-dependent formation of p116-ERBB2; we then inhibited the proteasome activity with MG-132 or lactacystin, and observed an efficient block of p185-ERBB2 cleavage, and its accumulation in EE, suggesting that p185-ERBB2 polyubiquitination is necessary for proteasome-dependent p116-ERBB2 generation occurring in EE. As polyubiquitination has also been implicated in autophagy-mediated degradation of ERBB2 under different experimental conditions, we exploited this possibility and demonstrate that GA strongly inhibits early autophagy, and reduces the levels of the autophagy markers atg5-12 and LC3-II, irrespective of GA-induced ERBB2 polyubiquitination, ruling out a GA-dependent autophagic degradation of ERBB2. In conclusion, we propose that HSP90 inhibition fosters ERBB2 polyubiquitination and proteasome-dependent generation of a non-ubiquitinated and inactive p116-ERBB2 form in EE, which is trafficked from altered EE to lysosomes.

Medical decision making is sometimes considered as a relatively simple process in which a decision may be made by the physician, by the patient, or by both patient and physician working together. There are three main models of decision making--paternalism, patient informed choice, and shared decision-making (SDM), having each one of these drawbacks and limitations. Historically, the most adopted one was the paternalism (strongly 'Doctor knows best'), where the professional made the decision based on what he/she considered to be as the patient's best interest, not necessarily contemplating patient's will and wishes. Currently, at the antipodes, the patient informed choice, where the patient makes his/her decision based on information received from the physician with no possible interference of professional's own preferences, seems to be the preferred relationship standard. SDM represents an intermediate approach between the two above-mentioned opposite models, being a medical process that involves actively the doctor and the patient who both bring their own facts and preferences to reach an agreement on the decision on if, when and how to treat a disease. This model, being characterized by elements pertaining to both the others, is gaining popularity in several medical and surgical scenarios whenever a competent patient is able to actively participate into the decisional process. On this basis can this model be implemented also in a Neonatology Intensive Care Unit where little patients are--by nature--incompetent, being the diagnostic/therapeutic choices taken by parents? We focused on this complex item considering four possible different scenarios and it seems to us that it could be possible to introduce such an approach, providing that parents' empowerment, a good physician's communication skill and consideration of all cultural, religious, economic, and ethic values of every single actor have been fairly taken into account.

Aim: The specific aim of the study was to determine the correlation between the severity of pathology, the amount of blood removed for diagnostic purposes in the 1st week of life and the incidence of early anaemia in very low birth weight (VLBW) infants., Methods: We recorded the level of haemoglobin (Hb) and haematocrit (Ht) in each of the 50 infants entered in the study at their admission in our neonatal intensive care unit (NICU) and at the age of 8 days. We quantified for each infant the blood drawn for clinical purpose during the 1(st)week of life, using microanalytic techniques for all types of analysis performed. Using the neonatal therapeutic intensive score system (NTISS) we divided all patients into 2 groups: group A= mild light pathology; group B= severe pathology., Results: There was statistically significant difference between the percent decrease of Hb and Ht with reference to the birth weight in the 2 groups. Logistic regression analysis indicated a strong correlation (P = 0.009) between higher degree of illness severity and higher percent decrease of Hb and Ht. The difference due to the amount of phlebotomy losses was not significant., Conclusions: To our knowledge, this study is the first that strongly suggest that phlebotomy losses is not the main cause of anaemia in VLBW preterm infants in the 1st week of life, when a policy of strictly attention to the amount of blood removed is performed.

The specific aim of the study was to assess the safety and efficacy of recombinant human erythropoietin (rHuEpo) in reducing the need for blood transfusions in preterm infants after the 15th day of life. Between 1 October 1994 and 1 October 1995, 107 preterm infants, gestational age < or = 34 weeks, were admitted to the Neonatal Intensive Care Unit and received rHuEpo subcutaneously, 900 U/kg week-1, 3 times weekly, supplemented with iron and vitamin E. Treatment was started at 8 days of life and lasted from a minimum of 6 weeks to a maximum of 3 months. A total of 116 preterm infants of the same gestational age, admitted to the Neonatal Intensive Care Unit from 1 January 1992 to 31 December 1992, served as controls. Entry criteria were gestational age < or = 34 weeks and no major congenital malformation. There were no differences in routine care between the two groups. Hematological measurements and transfusion requirements were followed during therapy. The infants were divided into two groups according to birth weight (< 1500 g and > or = 1500 g), and for each group the number of patients who received blood transfusions and when blood transfusions occurred, before or after the 15th day of life, was recorded. There was a statistically significant difference only for transfusions carried out after the 15th day of life (p < 0.002). No adverse effects attributable to rHuEpo during the treatment were noted. The results indicate that early rHuEpo treatment, in combination with iron supplements, is effective in reducing the need for blood transfusions in preterm infants after the 15th day of life.