225Ac-labeled CD33-targeting antibody reverses resistance to Bcl-2 inhibitor venetoclax in acute myeloid leukemia models.

PURPOSE: Despite the availability of new drugs, many patients with acute myeloid leukemia (AML) do not achieve remission and outcomes remain poor. Venetoclax is a promising new therapy approved for use in combination with a hypomethylating agent or with low-dose cytarabine for the treatment of newly diagnosed older AML patients or those ineligible for intensive chemotherapy. 225 Actinium-lintuzumab (225 Ac-lintuzumab) is a clinical stage radioimmunotherapy targeting CD33 that has shown evidence of single-agent activity in relapsed/refractory AML. Increased expression of MCL-1 is a mediator of resistance to venetoclax in cancer. EXPERIMENTAL DESIGN: Here we investigated the potential for 225 Ac-lintuzumab-directed DNA damage to suppress MCL-1 levels as a possible mechanism of reversing resistance to venetoclax in two preclinical in vivo models of AML. RESULTS: We demonstrated that 225 Ac-lintuzumab in combination with venetoclax induced a synergistic increase in tumor cell killing compared to treatment with either drug alone in venetoclax-resistant AML cell lines through both an induction of double-stranded DNA breaks (DSBs) and depletion of MCL-1 protein levels. Further, this combination led to significant tumor growth control and prolonged survival benefit in venetoclax-resistant in vivo AML models. CONCLUSIONS: There results suggest that the combination of 225 Ac-lintuzumab with venetoclax is a promising therapeutic strategy for the treatment of patients with venetoclax-resistant AML. Clinical trial of this combination therapy (NCT03867682) is currently ongoing.

Targeted lymphodepletion with a CD45-directed antibody radioconjugate as a novel conditioning regimen prior to adoptive cell therapy.

Chimeric antigen receptor (CAR) T cell therapies, and adoptive cell therapy (ACT) in general, represent one of the most promising anti-cancer strategies. Conditioning has been shown to improve the immune homeostatic environment to enable successful ACT or CAR-T engraftment and expansion in vivo following infusion, and represents potential point of intervention to decrease serious toxicities following CAR-T treatment. In contrast to relatively non-specific chemotherapy-derived lymphodepletion, targeted lymphodepletion with radioimmunotherapy (RIT) directed to CD45 may be a safer and more effective alternative to target and deplete immune cells. Here we describe the results of preclinical studies with an anti-mouse CD45 antibody 30F11, labeled with two different beta-emitters 131Iodine (131I) and 177Lutetium (177Lu), to investigate the effect of anti-CD45 RIT lymphodepletion on immune cell types and on tumor control in a model of adoptive cell therapy. Treatment of mice with 3.7 MBq 131I-30F11 or 1.48 MBq 177Lu-30F11 safely depleted immune cells such as spleen CD4+ and CD8+ T Cells, B and NK cells as well as Tregs in OT I tumor model while sparing RBC and platelets and enabled E. G7 tumor control. Our results support the application of CD45-targeted RIT lymphodepletion with a non-myeloablative dose of 131I-30F11 or 177Lu-30F11 antibody prior to adoptive cell therapy.

Surface (S) Layer Proteins of Lactobacillus acidophilus Block Virus Infection via DC-SIGN Interaction.

Alphaviruses and flaviviruses are important human pathogens that include Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV), which can cause diseases in humans ranging from arthralgia to hemorrhagic fevers and microcephaly. It was previously shown that treatment with surface layer (S-layer) protein, present on the bacterial cell-envelope of Lactobacillus acidophilus, is able to inhibit viral and bacterial infections by blocking the pathogen's interaction with DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), a trans-membrane protein that is a C-type calcium-dependent lectin. DC-SIGN is known to act as an attachment factor for several viruses including alphaviruses and flaviviruses. In the present study, we used alphaviruses as a model system to dissect the mechanism of S-layer inhibition. We first evaluated the protective effect of S-layer using 3T3 cells, either wild type or stably expressing DC-SIGN, and infecting with the alphaviruses Semliki Forest virus (SFV) and CHIKV and the flaviviruses ZIKV and DENV. DC-SIGN expression significantly enhanced infection by all four viruses. Treatment of the cells with S-layer prior to infection decreased infectivity of all viruses only in cells expressing DC-SIGN. In vitro ELISA experiments showed a direct interaction between S-layer and DC-SIGN; however, confocal microscopy and flow cytometry demonstrated that S-layer binding to the cells was independent of DC-SIGN expression. S-layer protein prevented SFV binding and internalization in DC-SIGN-expressing cells but had no effect on virus binding to DC-SIGN-negative cells. Inhibition of virus binding occurred in a time-dependent manner, with a significant reduction of infection requiring at least a 30-min pre-incubation of S-layer with DC-SIGN-expressing cells. These results suggest that S-layer has a different mechanism of action compared to mannan, a common DC-SIGN-binding compound that has an immediate effect in blocking viral infection. This difference could reflect slower kinetics of S-layer binding to the DC-SIGN present at the plasma membrane (PM). Alternatively, the S-layer/DC-SIGN interaction may trigger the activation of signaling pathways that are required for the inhibition of viral infection. Together our results add important information relevant to the potential use of L. acidophilus S-layer protein as an antiviral therapy.

Characterization of BK Polyomaviruses from Kidney Transplant Recipients Suggests a Role for APOBEC3 in Driving In-Host Virus Evolution.

BK polyomavirus (BKV) frequently causes nephropathy (BKVN) in kidney transplant recipients (KTRs). BKV has also been implicated in the etiology of bladder and kidney cancers. We characterized BKV variants from two KTRs who developed BKVN followed by renal carcinoma. Both patients showed a swarm of BKV sequence variants encoding non-silent mutations in surface loops of the viral major capsid protein. The temporal appearance and disappearance of these mutations highlights the intra-patient evolution of BKV. Some of the observed mutations conferred resistance to antibody-mediated neutralization. The mutations also modified the spectrum of receptor glycans engaged by BKV during host cell entry. Intriguingly, all observed mutations were consistent with DNA damage caused by antiviral APOBEC3 cytosine deaminases. Moreover, APOBEC3 expression was evident upon immunohistochemical analysis of renal biopsies from KTRs. These results provide a snapshot of in-host BKV evolution and suggest that APOBEC3 may drive BKV mutagenesis in vivo.

Infectious Entry and Neutralization of Pathogenic JC Polyomaviruses.

Progressive multifocal leukoencephalopathy (PML) is a lethal brain disease caused by uncontrolled replication of JC polyomavirus (JCV). JCV strains recovered from the brains of PML patients carry mutations that prevent the engagement of sialylated glycans, which are thought to serve as receptors for the infectious entry of wild-type JCV. In this report, we show that non-sialylated glycosaminoglycans (GAGs) can serve as alternative attachment receptors for the infectious entry of both wild-type and PML mutant JCV strains. After GAG-mediated attachment, PML mutant strains engage non-sialylated non-GAG co-receptor glycans, such as asialo-GM1. JCV-neutralizing monoclonal antibodies isolated from patients who recovered from PML appear to block infection by preventing the docking of post-attachment co-receptor glycans in an apical pocket of the JCV major capsid protein. Identification of the GAG-dependent/sialylated glycan-independent alternative entry pathway should facilitate the development of infection inhibitors, including recombinant neutralizing antibodies.

Identification of a Second Raccoon-Associated Polyomavirus.

Raccoon polyomavirus 1 (RacPyV1) is the suspected cause of an outbreak of fatal brain tumors among raccoons (Procyon lotor) in the western United States. Spleen samples from Georgia raccoons were screened for polyomaviruses. Although RacPyV1 was not detected, a previously unknown polyomavirus, which we designate RacPyV2, was identified and sequenced.

The Ancient Evolutionary History of Polyomaviruses.

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.

Antiviral activity of a single-domain antibody immunotoxin binding to glycoprotein D of herpes simplex virus 2.

Despite years of research dedicated to preventing the sexual transmission of herpes simplex virus 2 (HSV-2), there is still no protective vaccine or microbicide against one of the most common sexually transmitted infections in the world. Using a phage display library constructed from a llama immunized with recombinant HSV-2 glycoprotein D, we identified a single-domain antibody VHH, R33, which binds to the viral surface glycoprotein D. Although R33 does not demonstrate any HSV-2 neutralization activity in vitro, when expressed with the cytotoxic domain of exotoxin A, the resulting immunotoxin (R33ExoA) specifically and potently kills HSV-2-infected cells, with a 50% neutralizing dilution (IC50) of 6.7 nM. We propose that R33ExoA could be used clinically to prevent transmission of HSV-2 through killing of virus-producing epithelial cells during virus reactivation. R33 could also potentially be used to deliver other cytotoxic effectors to HSV-2-infected cells.