Enhanced potency of immunotherapy against B-cell precursor acute lymphoblastic leukemia by combination of an Fc-engineered CD19 antibody and CD47 blockade.

CD19-directed immunotherapy has become a cornerstone in the therapy of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). CD19-directed cellular and antibody-based therapeutics have entered therapy of primary and relapsed disease and contributed to improved outcomes in relapsed disease and lower therapy toxicity. However, efficacy remains limited in many cases due to a lack of therapy response, short remission phases, or antigen escape. Here, BCP-ALL cell lines, patient-derived xenograft (PDX) samples, human macrophages, and an in vivo transplantation model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were used to examine the therapeutic potency of a CD19 antibody Fc-engineered for improved effector cell recruitment (CD19-DE) and antibody-dependent cellular phagocytosis (ADCP), in combination with a novel modified CD47 antibody (Hu5F9-IgG2σ). For the in vivo model, only samples refractory to CD19-DE monotherapy were chosen. Hu5F9-IgG2σ enhanced ADCP by CD19-DE in various BCP-ALL cell line models with varying CD19 surface expression and cytogenetic backgrounds, two of which contained the KMT2A-AFF1 fusion. Also, the antibody combination was efficient in inducing ADCP by human macrophages in pediatric PDX samples with and adult samples with and without KMT2A-rearrangement in vitro. In a randomized phase 2-like PDX trial using seven KMT2A-rearranged BCP-ALL samples in NSG mice, the CD19/CD47 antibody combination proved highly efficient. Our findings support that the efficacy of Fc-engineered CD19 antibodies may be substantially enhanced by a combination with CD47 blockade. This suggests that the combination may be a promising therapy option for BCP-ALL, especially in relapsed patients and/or patients refractory to CD19-directed therapy.

Venetoclax enhances the efficacy of therapeutic antibodies in B-cell malignancies by augmenting tumor cell phagocytosis.

Immunotherapy has evolved as a powerful tool for the treatment of B-cell malignancies, and patient outcomes have improved by combining therapeutic antibodies with conventional chemotherapy. Overexpression of antiapoptotic B-cell lymphoma 2 (Bcl-2) is associated with a poor prognosis, and increased levels have been described in patients with "double-hit" diffuse large B-cell lymphoma, a subgroup of Burkitt's lymphoma, and patients with pediatric acute lymphoblastic leukemia harboring a t(17;19) translocation. Here, we show that the addition of venetoclax (VEN), a specific Bcl-2 inhibitor, potently enhanced the efficacy of the therapeutic anti-CD20 antibody rituximab, anti-CD38 daratumumab, and anti-CD19-DE, a proprietary version of tafasitamab. This was because of an increase in antibody-dependent cellular phagocytosis by macrophages as shown in vitro and in vivo in cell lines and patient-derived xenograft models. Mechanistically, double-hit lymphoma cells subjected to VEN triggered phagocytosis in an apoptosis-independent manner. Our study identifies the combination of VEN and therapeutic antibodies as a promising novel strategy for the treatment of B-cell malignancies.

Combining daratumumab with CD47 blockade prolongs survival in preclinical models of pediatric T-ALL.

Acute lymphoblastic leukemia (ALL) is the most common malignant disease affecting children. Although therapeutic strategies have improved, T-cell acute lymphoblastic leukemia (T-ALL) relapse is associated with chemoresistance and a poor prognosis. One strategy to overcome this obstacle is the application of monoclonal antibodies. Here, we show that leukemic cells from patients with T-ALL express surface CD38 and CD47, both attractive targets for antibody therapy. We therefore investigated the commercially available CD38 antibody daratumumab (Dara) in combination with a proprietary modified CD47 antibody (Hu5F9-IgG2σ) in vitro and in vivo. Compared with single treatments, this combination significantly increased in vitro antibody-dependent cellular phagocytosis in T-ALL cell lines as well as in random de novo and relapsed/refractory T-ALL patient-derived xenograft (PDX) samples. Similarly, enhanced antibody-dependent cellular phagocytosis was observed when combining Dara with pharmacologic inhibition of CD47 interactions using a glutaminyl cyclase inhibitor. Phase 2-like preclinical in vivo trials using T-ALL PDX samples in experimental minimal residual disease-like (MRD-like) and overt leukemia models revealed a high antileukemic efficacy of CD47 blockade alone. However, T-ALL xenograft mice subjected to chemotherapy first (postchemotherapy MRD) and subsequently cotreated with Dara and Hu5F9-IgG2σ displayed significantly reduced bone marrow infiltration compared with single treatments. In relapsed and highly refractory T-ALL PDX combined treatment with Dara and Hu5F9-IgG2σ was required to substantially prolong survival compared with single treatments. These findings suggest that combining CD47 blockade with Dara is a promising therapy for T-ALL, especially for relapsed/refractory disease harboring a dismal prognosis in patients.

Enhancement of epidermal growth factor receptor antibody tumor immunotherapy by glutaminyl cyclase inhibition to interfere with CD47/signal regulatory protein alpha interactions.

Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a "don't eat me" signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N-terminal pyro-glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell-mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab-mediated direct growth inhibition, complement-dependent cytotoxicity, or Ab-dependent cell-mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα-Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose-dependent manner, suggesting that pyro-glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab-dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte-mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell-mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors.

Modelling the transmission dynamics of bovine digital dermatitis in New Zealand pastoral dairy production systems.

Bovine digital dermatitis (DD) is an important infectious cause of cattle lameness worldwide that has become increasingly prevalent in New Zealand pastoral dairy herds. In this study, a simplified DD scoring system after considering both M and Iowa DD scoring systems was applied to explore the transmission dynamics of DD in a typical spring-calving pastoral New Zealand dairy herd. The modified model only included three compartments: normal skin, early stage lesions and advanced lesions. Lesions regressing after treatment were excluded as DD lesions are rarely treated in New Zealand. Furthermore, sub-classes within each lesion class were not defined due to the lack of variability in DD lesion presentations within New Zealand. The model was validated based on longitudinal field data from three dairy herds in the Waikato region during one lactation season (2017-18). The model suggested that in infected dairy herds, although DD prevalence will tend to increase year-on-year it is likely to remain relatively low (< 18%) even after 10 years of within-herd transmission. It is likely that the low transmission rate during the late lactation (model assumption) results in more cases resolving than developing during this period and therefore results in the low prevalence of infectious cattle at the start of each subsequent lactation. Cattle with advanced lesions had a stronger influence on the establishment and maintenance of DD than cattle with early stage lesions highlighting the importance of targeting these animals for intervention. On-going monitoring of DD is highly recommended to assess the long-term progression of the disease in affected dairy herds.

Bayesian analysis of herd-level risk factors for bovine digital dermatitis in New Zealand dairy herds.

BACKGROUND: Bovine digital dermatitis (BDD) is considered the most important infectious cause of lameness in dairy cattle worldwide, but has only recently been observed in New Zealand. Although many studies have investigated the risk factors for BDD in confined dairy systems, information on risk factors in pasture-based system is limited. Therefore a cross-sectional study including 59,849 animals from 127 dairy herds in four regions of New Zealand was conducted to identify the herd-level factors associated with the probability of a herd being BDD-lesion positive and with within-herd BDD prevalence. RESULTS: Purchasing heifers was associated with increased odds of a herd being BDD-lesion positive (odds ratio [OR]: 2.33, 95% probability interval [PI]: 1.26-4.42) and a cow being BDD affected (OR: 3.76, 95%PI: 1.73-8.38), respectively. Higher odds of a herd being BDD-lesion positive (OR: 2.06, 95%PI: 1.17-3.62) and a cow being BDD affected (OR: 2.87, 95%PI: 1.43-5.94) were also seen in herds where heifers co-grazed with cattle from other properties. In addition, using outside staff to treat lameness was associated with higher odds of a cow being BDD affected (OR: 2.18, 95%PI: 0.96-4.98). CONCLUSION: This study highlighted that movements of heifers are significantly associated with the spread of BDD within and between dairy herds in New Zealand. To minimise the risk of disease introductions in herds where moving heifers cannot be avoided, it is best to purchase heifers only from herds where BDD-freedom has been confirmed and, if heifers have to graze-off a farm, they should be reared as a single biosecure management group, especially since animals may be BDD-infected without having clinically obvious lesions.