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Acute myeloid leukemia (AML) is characterized by accumulation of abnormal blast cells in the bone marrow and blood. Although treatments like chemotherapy, allogeneic stem cell transplantation and new biotherapies improve the survival of patients, relapse is common and may be due to residual leukemic stem cells (LSCs). Since LSCs express high levels of CD123 (interleukin-3 receptor), CD123 represents an attractive target for relapsed/refractory (R/R) AML patients. Here, the expression of CD123 on leukemic cells and LSCs from fresh AML patient samples was confirmed, with a similar receptor density ranging from 350 to 12,500 and from 1,200 to 29,000 sites per cell, respectively. Also, the cytotoxic activity of the bispecific T-cell engager (TCE) CD123-CODV-TCE (that binds to CD3 on T cells and CD123 on blast cells) against primary blast cells isolated from the blood of 30 AML patients was evaluated. CD123-CODV-TCE induced high killing of AML blast cells with both high and low CD123 antigen density (from 298 to 18,719 sites per cell). The Effector: Target (E:T) ratio was highly variable among all patients, ranging from less than 1 to more than 100 blast cells for one T cell, and did not impact the activity of CD123-CODV-TCE. Since cytokine release syndrome is associated with TCE therapies, the release of proinflammatory cytokines was monitored in parallel to cytotoxicity. CD123-CODV-TCE induced the release of all tested cytokines (IL-6, IFNγ, TNFα and IL-8) without clear correlation with blast killing efficacy level. Since a similar CD123 expression pattern in human and cynomolgus monkey tissues was found, and CD123-CODV-TCE binds to human and cynomolgus CD3ε and CD123, non-human primates were used for the evaluation of pharmacodynamic effects and safety. A repeat-dose monkey study was performed using a progressive, weekly (X4) intra-monkey dose escalation scheme. The depletion of CD123 positive cells in blood was evidenced from the lowest dose tested (0.1 μg/kg) to the highest (5 µg/kg) with no adverse effects observed at 0.1 µg/kg. The main adverse effect consisted of a marked increase of the level of cytokines at 5 hours following compound administration at 1 μg/kg and above. Importantly, following the dose-escalation scheme 0.1/1/3/3 µg/kg, only limited cytokine release after the 4th administration was observed, suggesting that mitigation of cytokine release can be obtained by progressive intra-monkey dose escalation. Taken together, these results indicate that CD123-CODV-TCE leads to potent and specific leukemic cancer cell killing independently of CD123 receptor density and E:T ratio, and that studies monitoring cytokine release in monkeys can be useful for future clinical development of TCEs in the setting of R/R AML. Citation Format: Helene Bonnevaux, Jacqueline Courta, Wilfried Passe-Coutrin, Anne-Laure Bauchet, Annelies Roobrouck, Celine Amara, Eric Beys, Marielle Balzano, Maxime Moulard, Marco Meloni, Melissa Dullaers, Marielle Chiron, Angela Virone-Oddos. CD123-CODV-TCE bispecific T-cell engager for acute myeloid leukemia (AML): Activity on primary AML and safety in non-human primates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2909.

Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudomonas aeruginosa PcrV. PcrV is an essential part of the P. aeruginosa type III secretion system (T3SS), and its oligomeric nature allows for multiple complex binding and blocking options. The library screening yielded a large number of promising biparatopic lead candidates, revealing significant (and non-trivial) preferences in terms of Nanobody building block and epitope bin combinations and orientations. Excellent potencies were confirmed upon further characterization in two different P. aeruginosa T3SS-mediated cytotoxicity assays. Three biparatopic Nanobodies were evaluated in a lethal mouse P. aeruginosa challenge pneumonia model, conferring 100% survival upon prophylactic administration and reducing lung P. aeruginosa burden by up to 2 logs. At very low doses, they protected the mice from P. aeruginosa infection-related changes in lung histology, myeloperoxidase production, and lung weight. Importantly, the most potent Nanobody still conferred protection after therapeutic administration up to 24 h post-infection. The concept of screening such formatting libraries for potency improvement is applicable to other targets and biological therapeutic platforms.

Hepatitis B surface antigen, when produced in yeast (rHBsAg), is capable of binding to cells that express the lipopolysaccharide coreceptor CD14. This interaction is enhanced by a serum protein, the lipopolysaccharide binding protein (LBP). Here we report that most of the rHBsAg particles that attached to monocytes at 0 degrees C, were not endocytosed but were released back into the serum-containing binding buffer at 37 degrees C. Additionally, serum-dependent binding at 37 degrees C was weak when compared to the serum-dependent attachment at 0 degrees C. Pre-incubation at 37 degrees C of cells together with serum did not abolish binding of freshly added rHBsAg at 0 degrees C. However, pre-incubation of rHBsAg with serum at 37 degrees C reduced attachment to cells following incubation at 0 degrees C. Soluble CD14 and LBP, two serum proteins which can act as phospholipid transfer molecules, were shown not to be responsible for the inhibitory effect. Pre-incubation at 37 degrees C of rHBsAg in serum-free hepatoma cell line-conditioned media resulted in a pronounced reduction in subsequent binding to cells at 0 degrees C. These observations suggest that the temperature-dependent inhibitory effect is caused by serum factors that are probably secreted by hepatocytes.

It was observed recently that recombinant yeast-derived hepatitis B surface antigen (rHBsAg) particles, which contain the S protein only, bind almost exclusively to monocytes. It is shown here that binding requires the presence of the LPS receptor CD14. Furthermore, evidence is presented that a domain on CD14 that is identical to or largely overlaps with the LPS-binding pocket is instrumental for the attachment of rHBsAg. Additionally, it is shown that the heat-labile LPS-binding protein (LBP) catalyses the binding of rHBsAg to the cells. Remarkably, natural plasma-derived HBsAg (pHBsAg) does not have this property. pHBsAg devoid of its lipids and reconstituted with phosphatidylserine or phosphatidylglycerol acquires the characteristic of yeast-derived HBsAg. Clearly, the interaction of rHBsAg with the cell membrane is determined by the presence of charged phospholipids that are absent in pHBsAg. Although a lipid–receptor interaction is suggested, antibody-inhibition experiments suggest a possible involvement of the C-terminal region of the S protein in the interaction with monocytes. The possible implications of these observations for hepatitis B virus (HBV) infection and HBV vaccine efficiency are discussed.

Yeast expressed Hepatitis B surface antigen (rHBsAg) binds to monocytes through interaction with the LPS binding protein (LBP) and the LPS receptor CD14. Charged phospholipids of rHBsAg determine the interaction with these proteins. Although attachment of rHBsAg resembles the pro-inflammatory binding of LPS to CD14, rHBsAg does not activate monocytes and even reduces the expression of pro-inflammatory cytokines by LPS-stimulated monocytes. It is reported here that addition of rHBsAg to LPS-stimulated PBMC often results in increased secretion of IL-10, suggesting a similarity between the interaction of monocytes with apoptotic cells and rHBsAg. Using THP-1 cells, it is shown that IL-10 is not necessary to reduce TNFalpha protein levels. Addition of rHBsAg to LPS-stimulated cells reduces TNFalpha mRNA levels, but does not affect phosphorylation of p65 NF-kappaB and p38 MAP kinase. Instead, a reduced phosphorylation of ERK-1/2 and JNK-1/2 MAP kinases is observed.