Pharmacokinetics and Biodistribution of 89Zr-Miltuximab and Its Antibody Fragments as Glypican-1 Targeting Immuno-PET Agents in Glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain cancer, accounting for about 85% of all primary central nervous system (CNS) tumors. With standard treatment strategies like surgery, radiation, and chemotherapy, the median survival time of patients with GBM is only 12-15 months from diagnosis. The poor prognosis of GBM is due to a very high tumor recurrence rate following initial treatment, indicating a dire need for improved diagnostic and therapeutic alternatives for this disease. Antibody-based immunotheranostics holds great promise in treating GBM, combining the theranostic applications of radioisotopes and target-specificity of antibodies. In this study, we developed and validated antibody-based positron emission tomography (PET) tracers targeting the heparan sulfate proteoglycan, glypican-1 (GPC-1), for noninvasive detection of disease using diagnostic molecular imaging. GPC-1 is overexpressed in multiple solid tumor types, including GBM, and is a promising biomarker for novel immunotheranostics. Here, we investigate zirconium-89 (89Zr)-conjugated Miltuximab (a clinical stage anti-GPC-1 monoclonal antibody developed by GlyTherix, Ltd.) and engineered fragments for their potential as immuno-PET tracers to detect GPC-1positive GBM tumors in preclinical models. We explore the effects of molecular size, avidity, and Fc-domain on the pharmacokinetics and biodistribution in vivo, by comparing in parallel the full-length antibody (Miltuximab), Fab'2, Fab, and single-chain variable fragment (scFv) formats. High radiolabeling efficiency (>95%) was demonstrated by all the formats and the stability post-radiolabeling was higher for larger constructs of Miltuximab and the Fab. Receptor-mediated internalization of all 89Zr-labeled formats was observed in a human GBM cell line in vitro, while full-length Miltuximab demonstrated the highest tumor retention (5.7 ± 0.94% ID/g, day-9 postinjection (p.i.)) and overall better tumor-to-background ratios than the smaller Fc-less formats. Results from in vivo PET image quantification and ex vivo scintillation counting were highly correlated. Altogether, 89Zr-DFO-Miltuximab appears to be an effective immuno-PET imaging agent for detecting GPC-1positive tumors such as GBM and the current results support utility of the Fc containing whole mAb format over smaller antibody fragments for this target.

Glypican-1 as a target for fluorescence molecular imaging of bladder cancer.

OBJECTIVES: To investigate whether anti-glypican-1 antibody Miltuximab conjugated with near-infrared dye IRDye800CW can be used for in vivo fluorescence imaging of urothelial carcinoma. METHODS: The conjugate, Miltuximab-IRDye800CW, was produced and characterized by size exclusion chromatography and flow cytometry with glypican-1-expressing cells. Balb/c nude mice bearing subcutaneous urothelial carcinoma xenografts were intravenously injected with Miltuximab-IRDye800CW or control IgG-IRDye800CW and imaged daily by fluorescence imaging. After 10 days, tumors and major organs were collected for ex vivo study of the conjugate biodistribution, including its accumulation in the tumor. RESULTS: The intravenous injection of Miltuximab-IRDye800CW to tumor-bearing mice showed its specific accumulation in the tumors with the tumor-to-background ratio of 12.7 ± 2.4, which was significantly higher than that in the control group (4.6 ± 0.9, P < 0.005). The ex vivo imaging was consistent with the in vivo findings, with tumors from the mice injected with Miltuximab-IRDye800CW being significantly brighter than the organs or the control tumors. CONCLUSIONS: The highly specific accumulation and retention of Miltuximab-IRDye800CW in glypican-1-expressing tumors in vivo shows its high potential for fluorescence imaging of urothelial carcinoma and warrants its further investigation toward clinical translation.

A bispecific T cell engager targeting Glypican-1 redirects T cell cytolytic activity to kill prostate cancer cells.

BACKGROUND: Glypican-1 is a heparan sulfate proteoglycan that is overexpressed in prostate cancer (PCa), and a variety of solid tumors. Importantly, expression is restricted in normal tissue, making it an ideal tumor targeting antigen. Since there is clinical and preclinical evidence of the efficacy of Bispecific T cell Engager (BiTE) therapy in PCa, we sought to produce and test the efficacy of a GPC-1 targeted BiTE construct based on the Miltuximab® sequence. Miltuximab® is a clinical stage anti-GPC-1 antibody that has proven safe in first in human trials. METHODS: The single chain variable fragment (scFv) of Miltuximab® and the CD3 binding sequence of Blinatumomab were combined in a standard BiTE format. Binding of the construct to immobilised recombinant CD3 and GPC-1 antigens was assessed by ELISA and BiaCore, and binding to cell surface-expressed antigens was measured by flow cytometry. The ability of MIL-38-CD3 to activate T cells was assessed using in vitro co-culture assays with tumour cell lines of varying GPC-1 expression by measurement of CD69 and CD25 expression, before cytolytic activity was assessed in a similar co-culture. The release of inflammatory cytokines from T cells was measured by ELISA and expression of PD-1 on the T cell surface was measured by flow cytometry. RESULTS: Binding activity of MIL-38-CD3 to both cell surface-expressed and immobilised recombinant GPC-1 and CD3 was retained. MIL-38-CD3 was able to mediate the activation of peripheral blood T cells from healthy individuals, resulting in the release of inflammatory cytokines TNF and IFN-g. Activation was reliant on GPC-1 expression as MIL-38-CD3 mediated only low level T cell activation in the presence of C3 cells (constitutively low GPC-1 expression). Activated T cells were redirected to lyse PCa cell lines PC3 and DU-145 (GPC-1 moderate or high expression, respectively) but could not kill GPC-1 negative Raji cells. The expression of PD-1 was up-regulated on the surface of MIL-38-CD3 activated T cells, suggesting potential for synergy with checkpoint inhibition. CONCLUSIONS: This study reports preclinical findings into the efficacy of targeting GPC-1 in PCa with BiTE construct MIL-38-CD3. We show the specificity and efficacy of the construct, supporting its further preclinical development.

The Role of Glypican-1 in the Tumour Microenvironment.

Glypican-1 (GPC-1) is a cell surface heparan sulphate proteoglycan that is critical during normal development, but which is not required for normal homoeostasis in the adult. It is, however, overexpressed in a variety of solid tumours and is known to regulate tumour growth, invasion, metastasis and progression, through modulation of tumour cell biology as well as influence on the tumour microenvironment (TME). The role of GPC-1 in the TME and on the tumour cell is broad, as GPC-1 regulates signalling by several growth factors, including FGF, HGF, TGF-ß, Wnt and Hedgehog (Hh). Signalling via these pathways promotes tumour growth and invasive and metastatic ability (drives epithelial-to-mesenchymal transition (EMT)) and influences angiogenesis, affecting both tumour and stromal cells. Broad modulation of the TME via inhibition of GPC-1 may represent a novel therapeutic strategy for inhibition of tumour progression. Here, we discuss the complex role of GPC-1 in tumour cells and the TME, with discussion of potential therapeutic targeting strategies.

The immune modulatory peptide FhHDM-1 secreted by the helminth Fasciola hepatica prevents NLRP3 inflammasome activation by inhibiting endolysosomal acidification in macrophages.

The NLRP3 inflammasome is a multimeric protein complex that controls the production of IL-1ß, a cytokine that influences the development of both innate and adaptive immune responses. Helminth parasites secrete molecules that interact with innate immune cells, modulating their activity to ultimately determine the phenotype of differentiated T cells, thus creating an immune environment that is conducive to sustaining chronic infection. We show that one of these molecules, FhHDM-1, a cathelicidin-like peptide secreted by the helminth parasite, Fasciola hepatica, inhibits the activation of the NLRP3 inflammasome resulting in reduced secretion of IL-1ß by macrophages. FhHDM-1 had no effect on the synthesis of pro-IL-1ß. Rather, the inhibitory effect was associated with the capacity of the peptide to prevent acidification of the endolysosome. The activation of cathepsin B protease by lysosomal destabilization was prevented in FhHDM-1-treated macrophages. By contrast, peptide derivatives of FhHDM-1 that did not alter the lysosomal pH did not inhibit secretion of IL-1ß. We propose a novel immune modulatory strategy used by F. hepatica, whereby secretion of the FhHDM-1 peptide impairs the activation of NLRP3 by lysosomal cathepsin B protease, which prevents the downstream production of IL-1ß and the development of protective T helper 1 type immune responses that are detrimental to parasite survival.-Alvarado, R., To, J., Lund, M. E., Pinar, A., Mansell, A., Robinson, M. W., O'Brien, B. A., Dalton, J. P., Donnelly, S. The immune modulatory peptide FhHDM-1 secreted by the helminth Fasciola hepatica prevents NLRP3 inflammasome activation by inhibiting endolysosomal acidification in macrophages.

Free Ig light chains interact with sphingomyelin and are found on the surface of myeloma plasma cells in an aggregated form.

Free κ L chains (FκLCs) are expressed on the surface of myeloma cells and are being assessed as a therapeutic target for the treatment of multiple myeloma. Despite its clinical potential, the mechanism by which FκLCs interact with membranes remains unresolved. In this study, we show that FκLCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound FκLCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of FκLCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated FκLCs exposed on the plasma membrane after exocytosis. Although it is well known that protein aggregates bind membranes, this is only the second example of an aggregate being found on the surface of cells that also secrete the protein in its native form. We postulate that many other aggregation-prone proteins may associate with cell membranes by similar mechanisms.

Radioimmunotherapy for solid tumors: spotlight on Glypican-1 as a radioimmunotherapy target.

Radioimmunotherapy (i.e., the use of radiolabeled tumor targeting antibodies) is an emerging approach for the diagnosis, therapy, and monitoring of solid tumors. Often using paired agents, each targeting the same tumor molecule, but labelled with an imaging or therapeutic isotope, radioimmunotherapy has achieved promising clinical results in relatively radio-resistant solid tumors such as prostate. Several approaches to optimize therapeutic efficacy, such as dose fractionation and personalized dosimetry, have seen clinical success. The clinical use and optimization of a radioimmunotherapy approach is, in part, influenced by the targeted tumor antigen, several of which have been proposed for different solid tumors. Glypican-1 (GPC-1) is a heparan sulfate proteoglycan that is expressed in a variety of solid tumors, but whose expression is restricted in normal adult tissue. Here, we discuss the preclinical and clinical evidence for the potential of GPC-1 as a radioimmunotherapy target. We describe the current treatment paradigm for several solid tumors expressing GPC-1 and suggest the potential clinical utility of a GPC-1 directed radioimmunotherapy for these tumors.

Safety and tolerability of Miltuximab® - a first in human study in patients with advanced solid cancers.

OBJECTIVES: Miltuximab® is a chimeric antibody targeting Glypican-1 (GPC-1), a cell surface antigen which is overexpressed in solid cancers. Miltuximab® has shown promising safety and efficacy in radioimmunotherapy models of prostate cancer. This first in human study used Miltuximab® radiolabelled with Gallium-67 ([67Ga]Ga-DOTA-Miltuximab®). The primary study endpoint was to establish safety and tolerability of Miltuximab®. Secondary endpoints were biodistribution, tumour targeting and pharmacokinetic analysis. METHODS: Four cohorts of three patients (9 with advanced prostate cancer, 2 with pancreatic and 1 with bladder cancer) were dosed with 1 mg, ~250 MBq of [67Ga]Ga-DOTA-Miltuximab®. Cohort 1 received [67Ga]Ga-DOTA-Miltuximab® alone, while cohorts 2-4 were pre-infused with increasing doses (3.5, 11.5 and 24 mg, respectively) of unlabelled Miltuximab®-DOTA 1 hour prior to [67Ga]Ga-DOTA-Miltuximab®. Safety and tolerability were assessed by clinical and standard laboratory assessments. Patients underwent whole body gamma-camera scans and SPECT/CT scans up to 144 h post-infusion. Total organ radiation exposure was determined by dosimetry of whole-body gamma scans. RESULTS: The dosing regimen was well tolerated, with no drug-related adverse events observed. Liver and spleen uptake of [67Ga]Ga-DOTA-Miltuximab® was observed. Liver uptake was reduced by pre-infusion of unlabelled Miltuximab®-DOTA. Dosimetry analysis showed a favorable exposure profile. [67Ga]Ga-DOTA-Miltuximab® targeting to tumour sites was observed in two prostate cancer patients who had failed enzalutamide treatment. Higher doses of unlabelled antibody achieved lower liver uptake and increased antibody serum half life. CONCLUSIONS: This study is the first in human for Miltuximab® a first in class antibody targeting GPC-1. The trial met its primary endpoint of safety, demonstrating its potential as a safe and tolerable monoclonal antibody. This safety data, together with targeting to tumour lesions and biodistribution information supports the further clinical development of Miltuximab® as a theranostic agent in a planned Phase I human trial.

The feasibility of Miltuximab®-IRDye700DX-mediated photoimmunotherapy of solid tumors.

BACKGROUND: Photoimmunotherapy (PIT) is an emerging method of cancer treatment based on the use of a photosensitizer near-infrared dye IRDye700DX (IR700) conjugated to a monoclonal antibody. The antibody selectively delivers IR700 to cancer cells, which can then be killed after photoexcitation. Glypican-1 (GPC-1) is a novel target expressed specifically in malignant tumors. We aimed to investigate whether anti-GPC-1 antibody Miltuximab® (Glytherix Ltd., Sydney, Australia) can be conjugated with IR700 for PIT of solid tumors. METHODS: The dye IR700 was conjugated with Miltuximab® and characterized by spectrophotometry and flow cytometry. Miltuximab®-IR700-mediated PIT was tested in prostate (DU-145), bladder (C3 and T-24), brain (U-87 and U-251) and ovarian (SKOV-3) cancer cell lines. After 1 h incubation with Miltuximab®-IR700, the cells were washed by PBS and illuminated using a 690-nm light-emitting diode. The viability of the cells was assessed by a CCK-8 viability kit 24 h later. RESULTS: Miltuximab®-IR700-mediated PIT caused 67.3-92.3% reduction in viability of cells with medium-high GPC-1 expression and did not affect the viability of GPC-1-low cells. Cytotoxicity was attributed to the targeted binding of the conjugate with subsequent photoactivation, as the conjugate or light exposure alone had no effect on the cell viability. Miltuximab®-IR700 did not induce cytotoxicity in cells blocked by unconjugated Miltuximab®. CONCLUSIONS: PIT with Miltuximab®-IR700 appears to be highly specific and effective against GPC-1-expressing cancer cells, indicating that it holds promise for an effective and safe treatment of early stage solid tumors or as adjuvant therapy following surgical resection. These findings necessitate further investigation of PIT with Miltuximab®-IR700 in other GPC-1-expressing cancer cell lines in vitro and in vivo in xenograft tumor models.

Near-Infrared Molecular Imaging of Glioblastoma by Miltuximab®-IRDye800CW as a Potential Tool for Fluorescence-Guided Surgery.

Glioblastoma (GBM) is one of the most aggressive tumors and its 5-year survival is approximately 5%. Fluorescence-guided surgery (FGS) improves the extent of resection and leads to better prognosis. Molecular near-infrared (NIR) imaging appears to outperform conventional FGS, however, novel molecular targets need to be identified in GBM. Proteoglycan glypican-1 (GPC-1) is believed to be such a target as it is highly expressed in GBM and is associated with poor prognosis. We hypothesize that an anti-GPC-1 antibody, Miltuximab®, conjugated with the NIR dye, IRDye800CW (IR800), can specifically accumulate in a GBM xenograft and provide high-contrast in vivo fluorescent imaging in rodents following systemic administration. Miltuximab® was conjugated with IR800 and intravenously administered to BALB/c nude mice bearing a subcutaneous U-87 GBM hind leg xenograft. Specific accumulation of Miltuximab®-IR800 in subcutaneous xenograft tumor was detected 24 h later using an in vivo fluorescence imager. The conjugate did not cause any adverse events in mice and caused strong fluorescence of the tumor with tumor-to-background ratio (TBR) reaching 10.1 ± 2.8. The average TBR over the 10-day period was 5.8 ± 0.6 in mice injected with Miltuximab®-IR800 versus 2.4 ± 0.1 for the control group injected with IgG-IR800 (p = 0.001). Ex vivo assessment of Miltuximab®-IR800 biodistribution confirmed its highly specific accumulation in the tumor. The results of this study confirm that Miltuximab®-IR800 holds promise for intraoperative fluorescence molecular imaging of GBM and warrants further studies.

Detection of glypican-1 (GPC-1) expression in urine cell sediments in prostate cancer.

While measurement of serum prostate specific antigen (PSA) is an important screening tool for prostate cancer, new biomarkers are necessary for better discrimination between presence and absence of disease. The MIL-38 monoclonal antibody is specific for the membrane glycoprotein glypican 1 (GPC-1) and binds to prostate cancer tissue. Urine is known to be a source of cellular material. Thus, we hypothesized that detection of GPC-1 in urine cellular material may identify individuals with prostate cancer. Urine samples from patients with prostate cancer, benign prostatic hyperplasia (BPH), or normal controls were collected and cell sediments prepared. GPC-1-positive cells were detected using a MIL-38 immunofluorescence assay (IFA) and samples were classed positive or negative for GPC-1 expressing cells. Assay sensitivity and specificity, stratified by PSA, was reported. A total of 125 patient samples were analyzed (N = 41 prostate cancer; N = 37 BPH; N = 47 normal controls). The use of MIL-38 to detect GPC-1 by IFA discriminated between prostate cancer and BPH urine specimens with a sensitivity and specificity of 71% and 76%, respectively. Assay specificity increased with increasing PSA, with the highest specificity (89%) for patients with PSA ≥4 ng/ml. At lower PSA (<2 ng/ml) specificity decreased, as evidenced by a greater number of false positives in this concentration range. The odds ratio (OR) and 95% confidence intervals (CIs) for GPC-1-positive cells in patients with prostate cancer, adjusted for PSA, was greatest at the lowest serum PSA (<2 ng/ml; OR = 13.4; 95% CI: 4.0-44.7) compared with no adjustment for PSA (OR = 6.4; 95% CI: 2.8-14.9). The use of MIL-38 for detection of GPC-1 may be a useful tool for detection of prostate cancer.

Development of a reliable assay to measure glypican-1 in plasma and serum reveals circulating glypican-1 as a novel prostate cancer biomarker.

Prostate cancer is responsible for hundreds of thousands of annual deaths worldwide. The current gold standard in early detection of prostate cancer, the prostate specific antigen test, boasts a high sensitivity but low specificity, resulting in many unnecessary prostate biopsies. Thus, emphasis has been placed on identifying new biomarkers to improve prostate cancer detection. Glypican-1 has recently been proposed as one such biomarker, however further exploration into its predictive power has been hindered by a lack of available, dependable glypican-1 immunoassays. Previously, we identified human glypican-1 as the antigenic target of the MIL-38 monoclonal antibody. Additionally, we have now generated another monoclonal antibody, 3G5, that also recognizes human glypican-1. Here we report the development of a reliable, custom Luminex® assay that enables precise quantitation of circulating human glypican-1 in plasma and serum. Using this assay, we show for the first time that circulating glypican-1 levels can differentiate non-cancer (normal and benign prostatic hyperplasia) patients from prostate cancer patients, as well as benign prostatic hyperplasia patients alone from prostate cancer patients. Our findings strongly promote future investigation into the use of glypican-1 for early detection of prostate cancer.

The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus.

The human monocytic cell line, THP-1, is the most widely used model for primary human monocytes/macrophages. This is because, following differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire a macrophage-like phenotype, which mimics, in many respects, primary human macrophages. Despite the widespread use of THP-1 cells in studies elucidating macrophage responses to inflammatory stimuli, as well as the development and screening of potential therapeutics, there is currently no standardised protocol for the reliable differentiation of THP-1 monocytes to a macrophage phenotype using PMA. Consequently, reports using THP-1 cells have demonstrated significant phenotypic and functional differences between resultant THP-1 macrophage populations, which are largely attributable to the varying PMA differentiation methods used. Thus, to guarantee consistency and reproducibility between studies, and to ensure the relevance of THP-1 cells as an appropriate model for primary human macrophages, it is crucial to develop a standardised protocol for the differentiation of THP-1 macrophages. Accordingly, we compared the function and phenotype of THP-1 macrophages generated using the range of published PMA differentiation protocols, specifically in response to the pro-inflammatory stimulus, lipopolysaccharide (LPS). Our results demonstrated that the function of the resultant THP-1 macrophage populations, as determined by tumour necrosis factor (TNF) secretion in response to LPS stimulation, varied significantly, and was dependent upon the concentration of PMA used to stimulate the differentiation of monocytes, and the period of rest following PMA exposure. These data indicate that exposure of monocytic THP-1 cells to 25 nM PMA over 48 h, followed by a recovery period of 24h in culture in the absence of PMA, was the optimal protocol for the differentiation of THP-1 cells.

A parasite-derived 68-mer peptide ameliorates autoimmune disease in murine models of Type 1 diabetes and multiple sclerosis.

Helminth parasites secrete molecules that potently modulate the immune responses of their hosts and, therefore, have potential for the treatment of immune-mediated human diseases. FhHDM-1, a 68-mer peptide secreted by the helminth parasite Fasciola hepatica, ameliorated disease in two different murine models of autoimmunity, type 1 diabetes and relapsing-remitting immune-mediated demyelination. Unexpectedly, FhHDM-1 treatment did not affect the proliferation of auto-antigen specific T cells or their production of cytokines. However, in both conditions, the reduction in clinical symptoms was associated with the absence of immune cell infiltrates in the target organ (islets and the brain tissue). Furthermore, after parenteral administration, the FhHDM-1 peptide interacted with macrophages and reduced their capacity to secrete pro-inflammatory cytokines, such as TNF and IL-6. We propose this inhibition of innate pro-inflammatory immune responses, which are central to the initiation of autoimmunity in both diseases, prevented the trafficking of autoreactive lymphocytes from the periphery to the site of autoimmunity (as opposed to directly modulating their function per se), and thus prevented tissue destruction. The ability of FhHDM-1 to modulate macrophage function, combined with its efficacy in disease prevention in multiple models, suggests that FhHDM-1 has considerable potential as a treatment for autoimmune diseases.

Secreted proteins from the helminth Fasciola hepatica inhibit the initiation of autoreactive T cell responses and prevent diabetes in the NOD mouse.

Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-γ secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFß and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.

Cathelicidin-like helminth defence molecules (HDMs): absence of cytotoxic, anti-microbial and anti-protozoan activities imply a specific adaptation to immune modulation.

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects.