Selective haematological cancer eradication with preserved haematopoiesis.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs2-5. Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies.

Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant.

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) require conditioning to ablate the recipient's hematopoietic stem cells (HSCs) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities and resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. In vitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof of concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSCs with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late effects of these procedures, which would greatly increase their applicability.

Aviation Decompression Sickness in Aerospace and Hyperbaric Medicine.

INTRODUCTION: The U.S. Navy experienced a series of physiological events in aircrew involving primarily the F/A-18 airframe related to rapid decompression of cabin pressures, of which aviation decompression sickness (DCS) was felt to contribute. The underlying pathophysiology of aviation DCS is the same as that of diving-related. However, based on the innate multifactorial circumstances surrounding hypobaric DCS, in clinical practice it continues to be unpredictable and less familiar as it falls at the intersect of aerospace and hyperbaric medicine. This retrospective study aimed to review the case series diagnosed as aviation DCS in a collaborative effort between aerospace specialists and hyperbaricists to increase appropriate identification and treatment of hypobaric DCS.METHODS: We identified 18 cases involving high-performance aircraft emergently treated as aviation DCS at a civilian hyperbaric chamber. Four reviewers with dual training in aviation and hyperbaric medicine retrospectively reviewed cases and categorized presentations as "DCS" or "Alternative Diagnosis".RESULTS: Reviewers identified over half of presenting cases could be attributed to an alternative diagnosis. In events that occurred at flight altitudes below 17,000 ft (5182 m) or with rapid decompression pressure changes under 0.3 atm, DCS was less likely to be the etiology of the presenting symptoms.CONCLUSIONS: Aviation physiological events continue to be difficult to diagnose. This study aimed to better understand this phenomenon and provide additional insight and key characteristics for both flight physicians and hyperbaric physicians. As human exploration continues to challenge the limits of sustainable physiology, the incidence of aerospace DCS may increase and underscores our need to recognize and appropriately treat it.Kutz CJ, Kirby IJ, Grover IR, Tanaka HL. Aviation decompression sickness in aerospace and hyperbaric medicine. Aerosp Med Hum Perform. 2023; 94(1):11-17.

An Antibody Targeting ICOS Increases Intratumoral Cytotoxic to Regulatory T-cell Ratio and Induces Tumor Regression.

The immunosuppressive tumor microenvironment constitutes a significant hurdle to immune checkpoint inhibitor responses. Both soluble factors and specialized immune cells, such as regulatory T cells (Treg), are key components of active intratumoral immunosuppression. Inducible costimulatory receptor (ICOS) can be highly expressed in the tumor microenvironment, especially on immunosuppressive Treg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we performed immune profiling of samples from tumor-bearing mice and patients with cancer to demonstrate differential expression of ICOS in immune T-cell subsets in different tissues. ICOS expression was higher on intratumoral Treg than on effector CD8 T cells. In addition, by immunizing an Icos knockout transgenic mouse line expressing antibodies with human variable domains, we selected a fully human IgG1 antibody called KY1044 that bound ICOS from different species. We showed that KY1044 induced sustained depletion of ICOShigh T cells but was also associated with increased secretion of proinflammatory cytokines from ICOSlow effector T cells (Teff). In syngeneic mouse tumor models, KY1044 depleted ICOShigh Treg and increased the intratumoral TEff:Treg ratio, resulting in increased secretion of IFNγ and TNFα by TEff cells. KY1044 demonstrated monotherapy antitumor efficacy and improved anti-PD-L1 efficacy. In summary, we demonstrated that using KY1044, one can exploit the differential expression of ICOS on T-cell subtypes to improve the intratumoral immune contexture and restore an antitumor immune response.

Bioaccumulation and effects of dietary exposure to the alternative flame retardant, bis(2-ethylhexyl) tetrabromophthalate (TBPH), in the Atlantic killifish, Fundulus heteroclitus.

Bis(2-ethylhexyl) tetrabromophthalate (TBPH), a high production volume flame retardant chemical used as a replacement for banned flame retardants, has been detected in media and human and wildlife tissues globally. We describe bioaccumulation and biological effects from dietary exposure of TBPH to an estuarine fish, Atlantic killifish, Fundulus heteroclitus. Briefly, adult fish were fed carrier control or chemically amended diets for 28 d, followed by 14 d of control diet feeding. Diets were amended with TBPH (TBPH_LO diet, 139 µg/g dry wt, or TBPH_HI diet, 4360 µg/g dry wt) or a polychlorinated biphenyl congener (PCB153 diet, 13 µg/g dry wt), which was included as a positive control for bioaccumulation. Although bioaccumulation of either chemical correlated with fish size, only a small proportion of the TBPH offered (<0.5% total TBPH) had bioaccumulated into TBPH-treated fish by 28 d. In contrast, 24.5% of the PCB153 offered was accounted for in 28-d PCB-treated fish. Although 28-d bioaccumulated concentrations of TBPH differed by sex and treatment, sexes did not differ in their rates of TBPH bioaccumulation, and the time to achieve 50% of 28 d concentration (T1/2 ) was estimated to be 13 d. Depuration rates of TBPH did not differ by sex or treatment, and the time after exposure to achieve T1/2 was estimated to be 22 d. Independent of treatment, male fish grew faster than female fish, but for both sexes reproductive condition (gonadal somatic index) declined unexpectedly over the experimental period. Across treatments, only the TBPH_LO treatment affected growth, reducing male but increasing female growth rates by small amounts relative to respective controls. In summary, our study used very high concentrations of dietary TBPH to contaminate fish tissues above the highest levels reported to date in wild biota, yet we observed few adverse biological effects. Environ Toxicol Chem 2018;37:2350-2360. © 2018 SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Combined OX40L and mTOR blockade controls effector T cell activation while preserving Treg reconstitution after transplant.

A critical question facing the field of transplantation is how to control effector T cell (Teff) activation while preserving regulatory T cell (Treg) function. Standard calcineurin inhibitor-based strategies can partially control Teffs, but breakthrough activation still occurs, and these agents are antagonistic to Treg function. Conversely, mechanistic target of rapamycin (mTOR) inhibition with sirolimus is more Treg-compatible but is inadequate to fully control Teff activation. In contrast, blockade of OX40L signaling has the capacity to partially control Teff activation despite maintaining Treg function. We used the nonhuman primate graft-versus-host disease (GVHD) model to probe the efficacy of combinatorial immunomodulation with sirolimus and the OX40L-blocking antibody KY1005. Our results demonstrate significant biologic activity of KY1005 alone (prolonging median GVHD-free survival from 8 to 19.5 days), as well as marked, synergistic control of GVHD with KY1005 + sirolimus (median survival time, >100 days; P < 0.01 compared to all other regimens), which was associated with potent control of both TH/TC1 (T helper cell 1/cytotoxic T cell 1) and TH/TC17 activation. Combined administration also maintained Treg reconstitution [resulting in an enhanced Treg/Teff ratio (40% over baseline) in the KY1005/sirolimus cohort compared to a 2.9-fold decrease in the unprophylaxed GVHD cohort]. This unique immunologic signature resulted in transplant recipients that were able to control GVHD for the length of analysis and to down-regulate donor/recipient alloreactivity despite maintaining anti-third-party responses. These data indicate that combined OX40L blockade and sirolimus represents a promising strategy to induce immune balance after transplant and is an important candidate regimen for clinical translation.

Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery.

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.

Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor.

PCSK9 (proprotein convertase subtilisin/kexin type 9) promotes degradation of the LDLR [LDL (low-density lipoprotein) receptor] through an as-yet-undefined mechanism, leading to a reduction in cellular LDLc (LDL-cholesterol) and a concomitant increase in serum LDLc. Central to the function of PCSK9 is a direct protein-protein interaction formed with the LDLR. In the present study, we investigated a strategy to modulate LDL uptake by blocking this interaction using specific antibodies directed against PCSK9. Studies using surface plasmon resonance demonstrated that direct binding of PCSK9 to the LDLR could be abolished with three different anti-PCSK9 antibodies. Two of these antibodies were raised against peptide epitopes in a region of the catalytic domain of PCSK9 that is involved in the interaction with the LDLR. Such antibodies restored LDL uptake in HepG2 cells treated with exogenous PCSK9 and in HepG2 cells engineered to overexpress recombinant PCSK9. This latter observation indicates that antibodies blocking the PCSK9-LDLR interaction can inhibit the action of PCSK9 produced endogenously in a cell-based system. These antibodies also disrupted the higher-affinity interaction between the natural gain-of-function mutant of PCSK9, D374Y, and the LDLR in both the cell-free and cell-based assays. These data indicate that antibodies targeting PCSK9 can reverse the PCSK9-mediated modulation of cell-surface LDLRs.

Analysis of the interaction between RGD-expressing adenovirus type 5 fiber knob domains and alphavbeta3 integrin reveals distinct binding profiles and intracellular trafficking.

Adenovirus (Ad) vectors are used widely for experimental and therapeutic gene transfer. Ad-mediated gene delivery is often inefficient and, thus, there is considerable interest in developing Ad vectors that overcome biological barriers to efficient virus uptake. For this strategy to succeed, it is imperative that the interaction between such Ad vectors and their novel receptors is well understood. In this study, three surface-exposed loops (HI, CD and IJ loops) on the Ad5 fiber knob domain were selected as sites for insertion of an alphavbeta3 integrin-binding RGD sequence. Three RGD-containing Ad5 fiber knob-domain mutants were produced as recombinant proteins and all were shown to interact with soluble alphavbeta3 integrin by using biomolecular cell-free assays. Cell adsorption and subsequent internalization and intracellular trafficking of each of these proteins were assessed by confocal microscopy. Whilst the Ad5 fiber knob domain expressing the RGD sequence in the HI and CD loops bound with similar association and dissociation profiles, the fiber knob domain expressing the RGD sequence in the IJ loop bound with slower association and faster dissociation rates. By using molecular modelling, it was shown that the Ad5 fiber knob domain in which the RGD peptide was expressed in the IJ loop was only capable of binding to one alphavbeta3 integrin molecule per trimer. In contrast, fiber knob domains in which the RGD peptide was expressed in the HI and CD loops were capable of binding to one integrin molecule per monomer. These differences in the interactions between each mutant and alphavbeta3 may explain our observation that the three RGD-bearing Ad5 fiber knob domains demonstrated similar internalization rates, but distinct patterns of endosomal transport and escape.

Adenovirus type-5 entry and disassembly followed in living cells by FRET, fluorescence anisotropy, and FLIM.

We have used fluorescence resonance energy transfer (FRET) to follow the process of capsid disassembly for adenovirus (Ad) serotype 5 (Ad5) in living CHO-CAR cells. Ad5 were weakly labeled on their capsid proteins with FRET donor and acceptor fluorophores. A progressive decrease in FRET efficiency recorded during Ad5 uptake revealed that the time course of Ad5 capsid disassembly has two sequential protein dissociation rates with half-times of 3 and 60 min. Fluorescence anisotropy measurements of the segmental motions of fluorophores on Ad5 indicate that the first rate is linked to the detachment from the capsid of the protruding, flexible fiber proteins. The second rate was shown to report on the combined dissociation of protein IX, penton base, and hexons, which form the rigid icosahedral capsid shell. Fluorescence lifetime imaging microscopy measurements using a pH-sensitive probe provided information on the pH of the microenvironment of Ad5 particles during intracellular trafficking, and confirmed that the fast fiber dissociation step occurred at the onset of endocytosis. The slower dissociation phase was shown to coincide with the escape of Ad5 from endocytic compartments into the cytosol, and its arrival at the nuclear membrane. These results demonstrate a rapid, quantitative live-cell assay for the investigation of virus-cell interactions and capsid disassembly.