Immune modulation in chronic myeloid leukaemia patients treated with nilotinib and interferon-alpha.

The addition of interferon to tyrosine kinase inhibitors (TKIs), to improve deep molecular response (DMR) and potentially treatment-free remission (TFR) rates in chronic-phase chronic myeloid leukaemia (CP-CML) patients is under active investigation. However, the immunobiology of this combination is poorly understood. We performed a comprehensive longitudinal assessment of immunological changes in CML patients treated with nilotinib and interferon-alpha (IFN-α) within the ALLG CML11 trial (n = 12) or nilotinib alone (n = 17). We demonstrate that nilotinib+IFN transiently reduced absolute counts of natural killer (NK) cells, compared with nilotinib alone. Furthermore, CD16+ -cytolytic and CD57+ CD62L- -mature NK cells were transiently reduced during IFN therapy, without affecting NK-cell function. IFN transiently increased cytotoxic T-lymphocyte (CTL) responses to leukaemia-associated antigens (LAAs) proteinase-3, BMI-1 and PRAME; and had no effect on regulatory T cells, or myeloid-derived suppressor cells. Patients on nilotinib+IFN who achieved MR4.5 by 12 months had a significantly higher proportion of NK cells expressing NKp46, NKp30 and NKG2D compared with patients not achieving this milestone. This difference was not observed in the nilotinib-alone group. The addition of IFN to nilotinib drives an increase in NK-activating receptors, CTLs responding to LAAs and results in transient immune modulation, which may influence earlier DMR, and its effect on long-term outcomes warrants further investigation.

Association of TIM-3 checkpoint receptor expression on T cells with treatment-free remission in chronic myeloid leukemia.

Dysregulation of immune-checkpoint receptors has been reported at diagnosis of chronic myeloid leukemia (CML), however, their role in the maintenance of remission after tyrosine kinase inhibitor (TKI) cessation is unclear. We assessed programmed cell death-1 (PD-1), T-cell immunoglobulin, and mucin-domain containing protein-3 (TIM-3), cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), lymphocyte-activation gene-3 (LAG-3), and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) expression on T-cell subsets, regulatory T cells (T-regs), and natural killer (NK) cells at the time of TKI cessation in 44 patients (22 patients sustained treatment-free remission [TFR] and 22 experienced molecular relapse [MolR]). We confirmed our previous finding that absolute numbers of T-regs are increased in patients who experienced MolR compared with those who sustained TFR. The immune-checkpoint receptors PD-1, CTLA-4, LAG-3, and TIGIT on T or NK cells were not differentially expressed between the MolR and TFR groups. However, TIM-3 was consistently upregulated on bulk T cells (CD3+) and T-cell subsets including, CD4+ T cells, CD8+ T cells, and T-regs, in patients who relapsed in comparison with those who maintained TFR after discontinuation. Furthermore, gene expression analysis from publicly available data sets showed increased TIM-3 expression on CML stem cells compared with normal hematopoietic stem cells. These findings suggest that among the targetable immune-checkpoint molecules, TIM-3 blockade may potentially improve effector immune response in patients with CML stopping TKI, while concomitantly targeting leukemic stem cells and could be a promising therapeutic strategy for preventing relapse after cessation of TKI in patients with CML.

Successful treatment-free remission in chronic myeloid leukaemia and its association with reduced immune suppressors and increased natural killer cells.

There is currently no biomarker that reliably predicts treatment-free remission (TFR) in chronic myeloid leukaemia (CML). We characterised effector and suppressor immune responses at the time of tyrosine kinase inhibitor (TKI) cessation in patients from the CML8 and CML10 clinical studies. Natural killer (NK) cells with increased expression of activating NK receptors were higher in patients who achieved TFR. There was no difference in the proportion of CD4+ or CD8+ T cells. Furthermore, we found that FoxP3+ regulatory T cells (T reg) and monocytic myeloid-derived suppressor cells (Mo-MDSCs) were concomitantly decreased in TFR patients, suggesting that the effector and suppressor arms of the immune system work in concert to mediate TFR. A discovery cohort (CML10) was used to generate a predictive model, using logistic regression. Patients classified into the high-risk group were more likely to relapse when compared with the low-risk group (HR 7·4, 95% CI 2·9-19·1). The model was successfully validated on the independent CML8 cohort (HR 8·3, 95% CI 2·2-31·3). Effective prediction of TFR success may be obtained with an effector-suppressor score, calculated using absolute NK cell, T reg, and Mo-MDSC counts, at TKI cessation, reflecting the contribution of both immune suppressors and effectors in the immunobiology underlying successful TFR.

Sex differences in corneal neovascularization in response to superficial corneal cautery in the rat.

Sex-based differences in susceptibility have been reported for a number of neovascular ocular diseases. We quantified corneal neovascularization, induced by superficial silver nitrate cautery, in male and female inbred albino Sprague-Dawley, inbred albino Fischer 344, outbred pigmented Hooded Wistar and inbred pigmented Dark Agouti rats of a range of ages. Corneal neovascular area was quantified on haematoxylin-stained corneal flatmounts by image analysis. Pro-and anti-angiogenic gene expression was measured early in the neovascular response by quantitative real-time polymerase chain reaction. Androgen and estrogen receptor expression was assessed by immunohistochemistry. Male rats from all strains, with or without ocular pigmentation, exhibited significantly greater corneal neovascular area than females: Sprague-Dawley males 43±12% (n = 8), females 25±5% (n = 12), p = 0.001; Fischer 344 males 38±10% (n = 12) females 27±8% (n = 8) p = 0.043; Hooded Wistar males 32±6% (n = 8) females 22±5% (n = 12) p = 0.002; Dark Agouti males 37±11% (n = 9) females 26±7% (n = 9) p = 0.015. Corneal vascular endothelial cells expressed neither androgen nor estrogen receptor. The expression in cornea post-cautery of Cox-2, Vegf-a and Vegf-r2 was significantly higher in males compared with females and Vegf-r1 was significantly lower in the cornea of males compared to females, p<0.001 for each comparison. These data suggest that male corneas are primed for angiogenesis through a signalling nexus involving Cox-2, Vegf-a, and Vegf receptors 1 and 2. Our findings re-enforce that pre-clinical animal models of human diseases should account for sex-based differences in their design and highlight the need for well characterized and reproducible pre-clinical studies that include both male and female animals.

Identification and In Vitro Expansion of Buccal Epithelial Cells.

Ex vivo-expanded buccal mucosal epithelial (BME) cell transplantation has been used to reconstruct the ocular surface. Methods for enrichment and maintenance of BME progenitor cells in ex vivo cultures may improve the outcome of BME cell transplantation. However, the parameter of cell seeding density in this context has largely been neglected. This study investigates how varying cell seeding density influences BME cell proliferation and differentiation on tissue culture polystyrene (TCPS). The highest cell proliferation activity was seen when cells were seeded at 5×104 cells/cm2. Both below and above this density, the cell proliferation rate decreased sharply. Differential immunofluorescence analysis of surface markers associated with the BME progenitor cell population (p63, CK19, and ABCG2), the differentiated cell marker CK10 and connexin 50 (Cx50) revealed that the initial cell seeding density also significantly affected the progenitor cell marker expression profile. Hence, this study demonstrates that seeding density has a profound effect on the proliferation and differentiation of BME stem cells in vitro, and this is relevant to downstream cell therapy applications.

Interleukin-10 Gene Transfer in Rat Limbal Transplantation.

PURPOSE: To evaluate the gene transfer of the interleukin (IL)-10 cytokine as a treatment modality for prolonging limbal allograft survival in a rat model. MATERIALS AND METHODS: Adenoviral (AV) and lentiviral (LV) vectors were produced for ex vivo gene transfer into limbal graft tissue prior to orthotopic transplantation. Experimental groups comprised unmodified isografts, unmodified allografts, allografts transfected with a reporter gene, and allografts transfected with IL-10. The functional effects of the transgenes were determined by clinical assessment and by following donor cell survival in the recipient animal. Group comparisons were made using survival analysis and tested with the log-rank test. Differences in mean rejection times between groups were tested using the Wilcoxon rank-sum test. RESULTS: Isografts survived during the entire observation period of 56 days. Allografts underwent clinical rejection at a mean of 6.7 days (standard deviation 2.0) postoperatively, irrespective of the presence of transgenes (p < 0.001 for difference in rejection times). For both the AV and LV vector systems, Kaplan-Meier analysis showed a statistically significant difference with respect to time-to-graft failure when comparing allografts transfected with IL-10 with allografts transfected with reporter gene alone (p = 0.011 and p < 0.001, respectively). In the isografts, donor cells could be detected during the complete observation period. In all the allograft groups, however, donor cell detection declined after 1 week and was lost after 4 weeks. CONCLUSIONS: Under the conditions tested in the present model, both the AV and the LV vector systems were able to transfect limbal graft tissue ex vivo with biologically active IL-10, leading to delayed rejection compared to the controls.

Oral Mucosal Epithelial Cells Grown on Porous Silicon Membrane for Transfer to the Rat Eye.

Dysfunction of limbal stem cells or their niche can result in painful, potentially sight-threatening ocular surface disease. We examined the utility of surface-modified porous-silicon (pSi) membranes as a scaffold for the transfer of oral mucosal cells to the eye. Male-origin rat oral mucosal epithelial cells were grown on pSi coated with collagen-IV and vitronectin, and characterised by immunocytochemistry. Scaffolds bearing cells were implanted into normal female rats, close to the limbus, for 8 weeks. Histology, immunohistochemistry and a multiplex nested PCR for sry were performed to detect transplanted cells. Oral mucosal epithelial cells expanded on pSi scaffolds expressed the corneal epithelial cell marker CK3/12. A large percentage of cells were p63+, indicative of proliferative potential, and a small proportion expressed ABCG2+, a putative stem cell marker. Cell-bearing scaffolds transferred to the eyes of live rats, were well tolerated, as assessed by endpoint histology. Immunohistochemistry for pan-cytokeratins demonstrated that transplanted epithelial cells were retained on the pSi membranes at 8 weeks post-implant, but were not detectable on the central cornea using PCR for sry. The pSi scaffolds supported and retained transplanted rat oral mucosal epithelial cells in vitro and in vivo and recapitulate some aspects of an artificial stem cell niche.

An Anti-VEGF-B Antibody Fragment Induces Regression of Pre-Existing Blood Vessels in the Rat Cornea.

Purpose: We tested the ability of an antibody fragment with specificity for vascular endothelial growth factor-B (VEGF-B) to regress nascent and established corneal blood vessels in the rat. Methods: A single chain variable antibody fragment (scFv) with specificity for VEGF-B was engineered from the 2H10 hybridoma. Binding to rat, mouse, and human VEGF-B was confirmed by surface plasmon resonance. Activity of the anti-VEGF-B scFv on developing and established corneal blood vessels was assessed following unilateral superficial cautery in male and female outbred Sprague Dawley rats. Groups (untreated, control scFv-treated, or anti-VEGF-B scFv-treated) comprised 6 to 22 rats. Treatment consisted of 5 µL scFv, 1 mg/mL, applied topically five times per day for 14 days, or two subconjunctival injections, 50 µg scFv each, applied 7 days apart, or combined topical and subconjunctival treatment. Corneal vessel area was quantified on hematoxylin-stained corneal flat-mounts, and groups were compared using the Mann-Whitney U test, with post hoc Bonferroni correction. Immunohistochemistry for cleaved caspase-3 was performed. Results: Topical anti-VEGF-B scFv therapy alone did not regress corneal blood vessels significantly (P > 0.05). Subconjunctival injection and combined treatment regressed 14-day established corneal blood vessels (25% reduction in vessel area [P = 0.04] and 37% reduction in vessel area [P < 0.001], respectively, compared to results in untreated controls). Cleaved caspase-3 was identified in vascular endothelial cells of anti-VEGF-B scFv-treated corneas. In scFv-treated rats, corneal endothelial cell function was maintained to 12 weeks after treatment and a normal blink reflex was present. Conclusions: The anti-VEGF-B scFv significantly regressed established but not developing corneal blood vessels in rats.

Gene Therapy and Gene Editing for the Corneal Dystrophies.

Despite ever-increasing understanding of the genetic underpinnings of many corneal dystrophies, gene therapy designed to ameliorate disease has not yet been reported in any human patient. In this review, we explore the likely reasons for this apparent failure of translation. We identify the requirements for success: the genetic defect involved must have been identified and mapped, vision in the affected patient must be significantly impaired or likely to be impaired, no better or equivalently effective treatment must be available, the treatment must be capable of modulating corneal pathology, and delivery of the construct to the appropriate cell must be practicable. We consider which of the corneal dystrophies might be amenable to treatment by genetic manipulations, summarize existing therapeutic options for treatment, and explore gene editing using clustered regularly interspaced short palindromic repeat/Cas and other similar transformative technologies as the way of the future. We then summarize recent laboratory-based advances in gene delivery and the development of in vitro and in vivo models of the corneal dystrophies. Finally, we review recent experimental work that has increased our knowledge of the pathobiology of these conditions.

Species Cross-Reactivity of Antibodies Used to Treat Ophthalmic Conditions.

PURPOSE: The species cross-reactivity of the monoclonal antibodies infliximab, bevacizumab, and an anti-VEGF-B antibody, 2H10, in humans and rodents was determined. METHODS: The binding of infliximab to human, mouse, and rat TNF-α, of bevacizumab to human, mouse, and rat VEGF-A, and of the 2H10 antibody to human, mouse, and rat VEGF-B was evaluated by ELISA. The sequence of human, mouse, and rat TNF-α and VEGF-A at the binding sites for infliximab and bevacizumab were compared. RESULTS: Infliximab bound to human TNF-α, but no binding to mouse or rat TNF-α was detected between 10 pg/mL and 10 µg/ml. Sequence comparison of the binding site revealed four changes in mouse and five in rat TNF-α compared with human. Bevacizumab bound strongly to human VEGF-A, but showed 5-log weaker binding to both mouse and rat VEGF-A. There was a single amino acid substitution in mouse and rat VEGF-A at the bevacizumab binding site. The 2H10 antibody displayed a similar binding profile to human, mouse, and rat VEGF-B. CONCLUSIONS: The species cross-reactivity of monoclonal antibodies should be determined prior to their use in preclinical animal models. The 2H10 antibody binds to human, mouse, and rat VEGF-B making it suitable for testing in rodent models of human disease.

A novel pressed porous silicon-polycaprolactone composite as a dual-purpose implant for the delivery of cells and drugs to the eye.

Dysfunction of corneal epithelial stem cells can result in painful and blinding disease of the ocular surface. In such cases, treatment may involve transfer of growth factor and normal adult stem cells to the ocular surface. Our purpose was to develop an implantable scaffold for the delivery of drugs and cells to the ocular surface. We examined the potential of novel composite biomaterials fabricated from electrospun polycaprolactone (PCL) fibres into which nanostructured porous silicon (pSi) microparticles of varying sizes (150-250 µm or <40 µm) had been pressed. The PCL fabric provided a flexible support for mammalian cells, whereas the embedded pSi provided a substantial surface area for efficient delivery of adsorbed drugs and growth factors. Measurements of tensile strength of these composites revealed that the pSi did not strongly influence the mechanical properties of the polymer microfiber component for the Si loadings evaluated. Human lens epithelial cells (SRA01/04) attached to the composite materials, and exhibited enhanced attachment and growth when the materials were coated with foetal bovine serum. To examine the ability of the materials to deliver a small-drug payload, pSi microparticles were loaded with fluorescein diacetate prior to cell attachment. After 6 hours (h), cells exhibited intracellular fluorescence, indicative of transfer of the fluorescein diacetate into viable cells and its subsequent enzymatic conversion to fluorescein. To investigate loading of large-molecule biologics, murine BALB/c 3T3 cells, responsive to epidermal growth factor, insulin and transferrin, were seeded on composite materials. The cells showed significantly more proliferation at 48 h when seeded on composites loaded with these biologics, than on unloaded composites. No cell proliferation was observed on PCL alone, indicating the biologics had loaded into the pSi microparticles. Drug release, measured by ELISA for insulin, indicated a burst followed by a slower, continuous release over six days. When implanted under the rat conjunctiva, the most promising composite material did not cause significant neovascularization but did elicit a macrophage and mild foreign body response. These novel pressed pSi-PCL materials have potential for delivery of both small and large drugs that can be released in active form, and can support the growth of mammalian cells.

Surface engineering of porous silicon to optimise therapeutic antibody loading and release.

The proinflammatory cytokine, tumor necrosis factor-α (TNF-α), is elevated in several diseases such as uveitis, rheumatoid arthritis and non-healing chronic wounds. Adding Infliximab, a chimeric IgG1 monoclonal antibody raised against TNF-α, to chronic wound fluid can neutralise human TNF-α, thereby providing a potential therapeutic option for chronic wound healing. However, to avoid the need for repeated application in a clinical setting, and to protect the therapeutic antibody from the hostile environment of the wound, suitable delivery vehicles are required. Porous silicon (pSi) is a biodegradable high surface area material commonly employed for drug delivery applications. In this study, the use of pSi microparticles (pSi MPs) for the controlled release of Infliximab to disease environments, such as chronic wounds, is demonstrated. Surface chemistry and pore parameters for Infliximab loading are first optimised in pSi films and loading conditions are transferred to pSi MPs. Loading regimens exceeding 60 µg of Infliximab per mg of pSi are achieved. Infliximab is released with zero-order release kinetics over the course of 8 days. Critically, the released antibody remains functional and is able to sequester TNF-α over a weeklong timeframe; suitable for a clinical application in chronic wound therapy.

Co-expression of a scFv antibody fragment and a reporter protein using lentiviral shuttle plasmid containing a self-processing furin-2A sequence.

It is often desirable to co-express a reporter protein with a potential therapeutic protein, to verify correct targeting of an expression strategy. Vectors containing a viral self-processing 2A sequence have been reported to drive equimolar expression of two or more transgenes from a single promoter. Here, we report on the co-expression of a secreted antibody fragment and an intracellular reporter protein, enhanced yellow fluorescent protein from lentiviral shuttle plasmids by inserting a furin-2A (F2A) sequence between the two cDNAs, in two different orientations, in the expression cassette. We show that the order of these two transgenes relative to the F2A sequence affects expression levels. Reduced expression of each transgene positioned downstream of F2A, compared with upstream of F2A, was observed (p<0.05). Moreover, protein expression from double-cDNA plasmids was significantly lower than from their corresponding single transgene counterparts (p<0.05).

Novel therapeutic approaches for corneal disease.

Congenital and acquired corneal opacities, and diseases of the ocular surface, are blinding conditions that impose physical, psychological, and financial constraints upon the sufferer. In the past, corneal and corneal epithelial stem cell transplantation have been the major treatment for severe corneal and ocular surface disease, respectively, but the sequelae of neovascularization and inflammatory eye disease cause many grafts to undergo irreversible immunological rejection. Furthermore, in the case of corneal dystrophies, the original disease may recur in the graft. New therapeutic options for diseases of the cornea and ocular surface are now being actively explored in experimental animals and in clinical trials. Antibody-based biologics are being tested for their ability to reduce blood and lymphatic vessel ingrowth into the cornea, and to reduce inflammation. Many new biomaterials are being examined for their capacity to transfer drugs and corneal epithelial cell progenitor cells to the ocular surface and anterior segment of the eye. New component-cell corneal transplantation procedures that may reduce the risk of immunological rejection have been developed and are already in clinical practice. Finally, gene therapy is being tested in experimental animals to improve the outcomes of corneal transplantation, and to halt or reverse the pathophysiology of some corneal dystrophies.

Controlled drug delivery from composites of nanostructured porous silicon and poly(L-lactide).

AIMS: Porous silicon (pSi) and poly(L-lactide) (PLLA) both display good biocompatibility and tunable degradation behavior, suggesting that composites of both materials are suitable candidates as biomaterials for localized drug delivery into the human body. The combination of a pliable and soft polymeric material with a hard inorganic porous material of high drug loading capacity may engender improved control over degradation and drug release profiles and be beneficial for the preparation of advanced drug delivery devices and biodegradable implants or scaffolds. MATERIALS & METHODS: In this work, three different pSi and PLLA composite formats were prepared. The first format involved grafting PLLA from pSi films via surface-initiated ring-opening polymerization (pSi-PLLA [grafted]). The second format involved spin coating a PLLA solution onto oxidized pSi films (pSi-PLLA [spin-coated]) and the third format consisted of a melt-cast PLLA monolith containing dispersed pSi microparticles (pSi-PLLA [monoliths]). The surface characterization of these composites was performed via infrared spectroscopy, scanning electron microscopy, atomic force microscopy and water contact angle measurements. The composite materials were loaded with a model cytotoxic drug, camptothecin (CPT). Drug release from the composites was monitored via fluorimetry and the release profiles of CPT showed distinct characteristics for each of the composites studied. RESULTS: In some cases, controlled CPT release was observed for more than 5 days. The PLLA spin coat on pSi and the PLLA monolith containing pSi microparticles both released a CPT payload in accordance with the Higuchi and Ritger-Peppas release models. Composite materials were also brought into contact with human lens epithelial cells to determine the extent of cytotoxicity. CONCLUSION: We observed that all the CPT containing materials were highly efficient at releasing bioactive CPT, based on the cytotoxicity data.

Evaluation of mesoporous silicon/polycaprolactone composites as ophthalmic implants.

The suitability of porous silicon (pSi) encapsulated in microfibers of the biodegradable polymer polycaprolactone (PCL) for ophthalmic applications was evaluated, using both a cell attachment assay with epithelial cells and an in vivo assessment of biocompatibility in rats. Microfibers of PCL containing encapsulated pSi particles at two different concentrations (6 and 20 wt.%) were fabricated as non-woven fabrics. Given the dependence of Si particle dissolution kinetics on pSi surface chemistry, two different types of pSi particles (hydride-terminated and surface-oxidized) were evaluated for each of the two particle concentrations. Significant attachment of a human lens epithelial cell line (SRA 01/04) to all four types of scaffolds within a 24h period was observed. Implantation of Si fabric samples beneath the conjunctiva of rat eyes for 8 weeks demonstrated that the composite materials did not cause visible infection or inflammation, and did not erode the ocular surface. We suggest that these novel composite materials hold considerable promise as scaffolds in tissue engineering with controlled release applications.

Production of scFv antibody fragments from a hybridoma with functional activity against human vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) plays a major role in the development of aberrant neovascularization in ocular diseases such as diabetic retinopathy and age-related macular degeneration (ARMD), and is an important therapeutic target for these diseases. Monoclonal antibodies specific for VEGF are in clinical use for some patients with ARMD, delivered by intraocular injection. We have shown previously that single chain antibody fragments (scFv) penetrate into the eye when applied topically to the ocular surface. Here we describe the production of a scFv from a monoclonal antibody specific for human VEGF and demonstrate its ability to decrease proliferation of human umbilical vein endothelial cells in culture. A suitably formulated anti-VEGF scFv may have potential as a less invasive topical treatment for potentially blinding neovascular diseases of the eye.

PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts.

Single chain antibody fragment genes are commonly created by splicing together the immunoglobulin light chain (VL) and heavy chain variable (VH) genes of a monoclonal antibody produced by a hybridoma. Selective PCR amplification of the functional immunoglobulin variable gene rearrangements can be complicated by the existence of other unproductive immunoglobulin gene rearrangements in the hybridoma. Here we report the detection and preferential amplification of aberrant transcripts from two unproductive VH gene rearrangements derived from the fusion partner of a hybridoma. The functional VH gene of the monoclonal antibody was successfully amplified by selective use of primers to individual JH segments.