Activated Leukocyte Cell Adhesion Molecule Research Articles

Itolizumab-induced antigenic modulation of CD6 inhibits T cell activity

Abstract CD6 is an immunomodulatory receptor on T cells that promotes immune synapse formation, T-cell activation, and cell migration into tissues by binding activated leukocyte cell adhesion molecule (ALCAM). Excessive activation through the CD6-ALCAM pathway has been implicated in the pathogenesis of multiple autoimmune and inflammatory diseases. Hence, the ability to modulate the level of activation is beneficial to disease resolution. Itolizumab is a humanized anti-CD6 monoclonal antibody that is specific for the membrane distal domain 1 of CD6. Previously, itolizumab was believed to sterically hinder the CD6-ALCAM interaction, thereby blocking T-cell co-stimulation. Here we describe antigenic modulation as an additional mechanism whereby binding of itolizumab to CD6 induces proteolytic cleavage of the extracellular portion of CD6. Upon treatment with itolizumab, surface levels of CD6 decreased in a dose- and time-dependent manner, as monitored by flow cytometry using a noncompetitive anti-CD6 monoclonal antibody. This loss was inhibited in the presence of protease inhibitors and the decrease in surface levels of CD6 was accompanied by an increase in levels of soluble CD6 in the supernatant. Furthermore, we assessed the effect of surface levels of CD6 on the response of cells to T cell stimulation in the presence of ALCAM. CD6low cells showed reduced T-cell activity compared to CD6highcells as measured by cell surface activation makers, including CD25, CD69 and PD-1, and cytokine production. These findings demonstrate that CD6 is an important regulator of T-cell activity and that modulating surface levels of CD6 is an effective method for fine-tuning the activity of T-cells.

Blockade of the CD6-ALCAM pathway modulates effector T cell function

Abstract CD6 is a T-cell costimulatory receptor predominantly expressed on T cells and promotes immune synapse formation, T-cell activation, and T-cell migration via interaction with activated leukocyte cell adhesion molecule (ALCAM). The CD6-ALCAM pathway has been linked to the pathogenesis of multiple autoimmune and inflammatory diseases. While the contribution of CD6 to T cell activation has been well described, less is known regarding the expression levels and role of CD6 on effector and memory T cells which contribute to ongoing disease. Consequently, the aim of this study was to determine the role of CD6 specifically on Th1, Th2 and Th17 effector T cells (Teff). Blockade of the CD6 pathway using itolizumab, a humanized mAb currently being tested in the clinic, during restimulation of fully differentiated Th1, Th2 or Th17 Teff cells in the presence of ALCAM inhibited multiple effector functions, including proliferation and changes in cell size. An average 40% decrease in proliferation was observed across multiple donors. Furthermore, treatment of Teff cells with itolizumab resulted in a significant decrease in relevant cytokines such as IFNγ and TNFα for Th1, IL-4 and IL-13 for Th2, TNFα and IL-17 for Th17, as well as in expression levels of T cell markers of activation and exhaustion such as CD69, CD25, and PD-1. This effect was exclusively observed in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. This study is the first to characterize the CD6-ALCAM pathway as a key regulator of effector Teff cell function. As these are the cells that drive disease pathogenicity, these data support targeting the CD6-ALCAM pathway to inhibit effector Teff cell populations in autoimmune and inflammatory diseases.

Abstract PS17-48: Potential role of activated leukocyte cell adhesion molecule (ALCAM) in hepatocyte growth factor (HGF) signalling in vascular endothelial cells and implications in breast cancer

Abstract Background. Activated Leukocyte Cell Adhesion Molecule, ALCAM (also known as CD166) is a member of the immunoglobulin superfamily and a cell surface adhesion molecule involved in heterophilic and homophilic interactions during cell-cell adhesion in epithelial cells, cancer cells and endothelial cells. ALCAM appears to be linked to tumour progression in a number of cancers including breast cancer, though there are conflicting reports as to its precise prognostic implications and significance. We have previously reported that ALCAM has a tumour suppressive role in breast cancer and is inversely correlated with clinical outcome (1) and interestingly bone metastasis of breast cancer (2). We have also reported that ALCAM has a diverse role in other cell types including keratinocytes and endothelial cells. In the present study, we investigated the association between ALCAM and hepatocyte growth factor (HGF)/cMET, a signalling pathway established as a key promoter of cancer progression, influencing both the aggressive nature of cancer cells and acting as an angiogenic and lymphangiogenic factor, in breast cancer and endothelial cells. Methods. The expression pattern and correlation of ALCAM, HGF and cMET in human breast cancer were deduced from a clinical breast cancer cohort. ALCAM manipulated HECV cell lines, previously established in our laboratories, were used in conjunction with recombinant human HGF and cMET inhibitors to assess cellular functions and the implications of this relationship at a cellular level. Results. We compared the expression of ALCAM with that of HGF and cMET within the breast cancer cohort and found that ALCAM/CD166 showed a highly significant negative correlation with the HGF receptor cMET (r=-0.17, p<0.0001) and a weak negative correlation with HGF, although this was not significant. ALCAM was not found to correlate with its heterophilic partner CD6. In previously generated HECV models, ALCAM manipulation showed a marked influence on cellular function and responsiveness to HGF treatment. Discussion. ALCAM’s role in breast cancer progression and related mechanisms is complex. Our current data suggests this may involve an interaction with the cMET/HGF signalling pathway and may also impact the endothelial component within the tumour microenvironment to influence disease progression. Reference1 King JA, Ofori-Acquah SF, Stevens T, et al. Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator. Breast Cancer Res. 2004;6:R478-87. Reference2 Davies SR, Dent C, Watkins G et al. Expression of the cell to cell adhesion molecule, ALCAM, in breast cancer patients and the potential link with skeletal metastasis. Oncol Rep. 2008;19:555-61. Citation Format: Andrew James Sanders, David Guo Jiang, Jianyuan Zeng, Robert Edward Mansel, Eleri Davies, Amber Xinyu Li, Keith Gordon Harding, Wen Guo Jiang. Potential role of activated leukocyte cell adhesion molecule (ALCAM) in hepatocyte growth factor (HGF) signalling in vascular endothelial cells and implications in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-48.

Abstract OT-03-08: A Phase 2, open-label study to evaluate the safety and efficacy of the probody therapeutic (Pb-Tx) CX-2009 in metastatic HR-Positive/HER2-negative breast cancer (mHR+/HER2− BC) and of CX-2009 as monotherapy and in combination therapy with CX-072 in metastatic triple-negative breast cancer (TNBC)

Abstract Background: Probody® therapeutics (Pb-Tx) are masked antibodies designed to be selectively activated in the tumor microenvironment by tumor-associated proteases, while remaining largely inactive in normal tissue and in circulation. This allows Pb-Tx to address previously undruggable targets that are highly expressed in both tumor and normal tissue, such as the activated leukocyte cell adhesion molecule (ALCAM) CD166. CX-2009 is an investigational Probody drug conjugate consisting of an anti-CD166 monoclonal antibody conjugated to the microtubule inhibitor DM4. A Phase 1 study demonstrated safety and durable clinical activity in patients with mHR+/HER2− BC and in mTNBC patients who had received a median of 7 prior regimens. In the efficacy evaluable subgroup receiving doses of at least 4 mg/kg Q3W, the overall response rate (ORR; includes confirmed and unconfirmed responses) was 11% (HR+/HER2−; n=18) and 38% (TNBC; n=8), with a clinical benefit rate at 24 weeks (CBR24) of 35% in all breast cancer patients. Gr ≥3 DM4-associated ocular toxicities related to CX-2009 ≤7mg/kg were reported in 2%. In this Phase 2 study, CX-2009 monotherapy will be evaluated in patients with mHR+/HER2− BC and mTNBC. In addition, CX-2009 will be combined with CX-072, a Probody therapeutic directed against PD-L1, to evaluate the safety of the combination and whether the cytotoxic activity of the DM4 payload is additive with PD-L1 blockade via CX-072 in patients with mTNBC. CD166 expression in patients with HR+/HER2− BC was high (>90%) in the Phase 1 study; this cohort will enroll without screening for CD166. CD166 expression in patients with TNBC was variable and, as such, patients will undergo screening for CD166 positivity. Methods: This Phase 2, open-label noncomparative study with 3 parallel arms (n≈40/arm) is investigating the safety and activity of CX-2009 monotherapy (7 mg/kg Q3W) and the combination of CX-2009 (7 mg/kg Q3W) + CX-072 (1200 mg Q3W) in patients with previously treated locally advanced or metastatic HER2− BC. All patients will be adults with an ECOG status of 0-1, acceptable end-organ function, measurable disease, and willingness to receive ocular prophylaxis for DM4 related toxicities. Tumor tissue is required for CD166 expression analysis. Key eligibility criteria for the mHR+/HER2− BC cohort include 2-4 prior regimens (not including single-agent hormonal therapy). Patients with mTNBC must be CD166 positive by IHC and have received 1-3 regimens. For mTNBC patients who receive the doublet, key exclusion criteria include known PD-L1-negative tumor status, history of or active autoimmune disease, and progression within 120 days of first dose of an IO agent. Patients with active or chronic corneal disorders will be excluded from the study. All treatments will be given until disease progression or unacceptable toxicity. Radiology assessments will be done Q6W. The primary endpoint will be ORR assessed by an independent radiology committee per RECIST v1.1. Secondary endpoints will include ORR, DoR, CBR16 and CBR24, and PFS by investigator, OS will also be assessed. This study will also evaluate tolerability, pharmacokinetics, and antidrug antibodies with CX-2009 as monotherapy and in combination with CX-072. Exploratory endpoints will include potential predictive markers of response or toxicity. This trial is actively enrolling. Email address for questions or comments: gpaton@cytomx.com Citation Format: Kathy D. Miller, Leisha A. Emens, Sara Tolaney, Sara A. Hurvitz, Erika Hamilton, Virginia Paton, Alison Hannah, Valentina Boni. A Phase 2, open-label study to evaluate the safety and efficacy of the probody therapeutic (Pb-Tx) CX-2009 in metastatic HR-Positive/HER2-negative breast cancer (mHR+/HER2− BC) and of CX-2009 as monotherapy and in combination therapy with CX-072 in metastatic triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-03-08.

Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion.

Cluster of differentiation (CD) 166 or activated leukocyte cell adhesion molecule (ALCAM) is a transmembrane molecule known to be an intercellular adhesion factor. The expression and function of ALCAM in medulloblastoma (MB), a pediatric brain tumor with highly advanced molecular genetics, remains unclear. Therefore, this study aimed to clarify the significance and functional role of ALCAM expression in MB. ALCAM expression in 45 patients with MB was evaluated by immunohistochemical analysis of formalin-fixed paraffin-embedded clinical specimens and the relationship between ALCAM expression and pathological type/molecular subgroup, such as WNT, SHH, Group 3, and Group 4, was examined. Eight ALCAM positive (18%), seven partially positive (16%), and 30 negative (67%) cases were detected. All seven cases of the WNT molecular subgroup were ALCAM positive and ALCAM expression strongly correlated with this subgroup (P < 0.0001). In addition, functional studies using MB cell lines revealed ALCAM expression affected proliferation and migration as a positive regulator in vitro. However, ALCAM silencing did not affect survival or the formation of leptomeningeal dissemination in an orthotopic mouse model, but did induce a malignant phenotype with increased tumor cell invasion at the dissemination sites (P = 0.0029). In conclusion, our results revealed that ALCAM exhibited highly specific expression in the WNT subgroup of MB. Furthermore, we demonstrated that the cell kinetics of MB cell lines can be altered by the expression of ALCAM.

A novel HDGF-ALCAM axis promotes the metastasis of Ewing sarcoma via regulating the GTPases signaling pathway.

Ewing sarcoma (ES) is a type of highly aggressive pediatric tumor in bones and soft tissues and its metastatic spread remains the most powerful predictor of poor outcome. We previously identified that the transcription factor hepatoma-derived growth factor (HDGF) promotes ES tumorigenesis. However, the mechanisms underlying ES metastasis remain unclear. Here, we show that HDGF drives ES metastasis in vitro and in vivo, and HDGF reduces metastasis-free survival (MFS) in two independent large cohorts of human ES patients. Integrative analyses of HDGF ChIP-seq and gene expression profiling in ES cells reveal that HDGF regulates multiple metastasis-associated genes, among which activated leukocyte cell adhesion molecule (ALCAM) emerges as a major HDGF target and a novel metastasis-suppressor in ES. HDGF down-regulates ALCAM, induces expression and activation of the downstream effectors Rho-GTPase Rac1 and Cdc42, and promotes actin cytoskeleton remodeling and cell-matrix adhesion. In addition, repression of ALCAM and activation of Rac1 and Cdc42 are required for the pro-metastatic functions of HDGF in vitro. Moreover, analyses in murine models with ES tumor orthotopic implantation and experimental metastasis, as well as in human ES samples, demonstrate the associations among HDGF, ALCAM, and GTPases expression levels. Furthermore, high HDGF/low ALCAM expression define a subgroup of patients harboring the worst MFS. These findings suggest that the HDGF/ALCAM/GTPases axis represents a promising therapeutic target for limiting ES metastasis.

A novel molecular magnetic resonance imaging agent targeting activated leukocyte cell adhesion molecule as demonstrated in mouse brain metastasis models

Molecular magnetic resonance imaging (MRI) allows visualization of biological processes at the molecular level. Upregulation of endothelial ALCAM (activated leukocyte cell adhesion molecule) is a key element for leukocyte recruitment in neurological disease. The aim of this study, therefore, was to develop a novel molecular MRI contrast agent, by conjugating anti-ALCAM antibodies to microparticles of iron oxide (MPIO), for detection of endothelial ALCAM expression in vivo. Binding specificity of ALCAM-MPIO was demonstrated in vitro under static and flow conditions. Subsequently, in a proof-of-concept study, mouse models of brain metastasis were induced by intracardial injection of brain-tropic human breast carcinoma, lung adenocarcinoma or melanoma cells to upregulate endothelial ALCAM. At selected time-points, mice were injected intravenously with ALCAM-MPIO, and ALCAM-MPIO induced hypointensities were observed on T2*-weighted images in all three models. Post-gadolinium MRI confirmed an intact blood-brain barrier, indicating endoluminal binding. Correlation between endothelial ALCAM expression and ALCAM-MPIO binding was confirmed histologically. Statistical analysis indicated high sensitivity (80–90%) and specificity (79–83%) for detection of endothelial ALCAM in vivo with ALCAM-MPIO. Given reports of endothelial ALCAM upregulation in numerous neurological diseases, this advance in our ability to image ALCAM in vivo may yield substantial improvements for both diagnosis and targeted therapy.

Early Reconstitution of CD6+ T Cells after Hematopoietic Cell Transplantation Identifies a Suitable Target for Acute Graft Versus Host Disease Treatment Using Anti-CD6 Monoclonal Antibody Itolizumab

Early Reconstitution of CD6+ T Cells after Hematopoietic Cell Transplantation Identifies a Suitable Target for Acute Graft Versus Host Disease Treatment Using Anti-CD6 Monoclonal Antibody Itolizumab

Impact of Itolizumab on the Expression of CD6 and the Function of Effector T Cells

Introduction: CD6 is a T-cell costimulatory receptor that has been implicated in the pathogenesis of multiple autoimmune and inflammatory (AI) diseases. In GVHD, CD6 is expressed on reconstituting T cells soon after transplant (Rambaldi et al., 2019), including Th1 and Th17 cells which are both implicated in the induction and pathogenesis of acute GVHD (aGVHD). CD6 is highly expressed on these cells and promotes immune synapse formation, T-cell activation, and T-cell migration via interaction with its ligand activated leukocyte cell adhesion molecule (ALCAM). Furthermore, studies have demonstrated that ex-vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998), highlighting the importance of CD6. While the contribution of CD6 to T cell activation has been well described, less is known regarding the expression levels and role of CD6 on effector and memory T cells (Teff) which are prominent in all diseases including aGVHD. Consequently, the aim of this study was to determine the role of CD6 specifically on effector T cells, and further illuminate the mechanism of itolizumab, an anti-CD6 monoclonal antibody. Methods: Naïve T cells were enriched from frozen PBMCs via a naïve T cell magnetic separation kit (Stemcell). Naïve T cells were polarized towards a Th1 phenotype for 6 days with a Th1 differentiation cocktail (Stemcell) and CD3/CD28 T cell activator (Stemcell) and rested overnight prior to entering restimulation conditions. Naïve T cells were polarized towards a Th17 phenotype for 8 days with IL-6, IL-1β, TGF-β, IL-23, anti-IL-4 and IFN-γ, the CD3/CD28 T cell activator was used to activate the T cells. To re-stimulate differentiated T cells, anti-CD3 mAb and ALCAM-Fc or anti-CD3 mAb alone were coated on 48-well plates overnight at 4oC. Th1 or Th17 T cells were labeled with CFSE and seeded with isotype control or itolizumab for 72hrs. Cells were collected for flow cytometry analysis and supernatant collected for relevant cytokine detection. To assess surface levels of CD6, cryopreserved PBMCs were thawed and incubated with itolizumab or isotype at 37oC for specific timepoints. Following incubation, cells were washed and stored at 4oC for subsequent staining. All samples were stained at the same time and surface levels of CD6 was detected using a monoclonal anti-CD6 antibody that does not compete with itolizumab. Results: Blockade of the CD6 pathway, using itolizumab during restimulation of differentiated Teff cells in the presence of ALCAM, inhibited multiple effector functions including proliferation and changes in cell size. An average of a 40% decrease in CFSE proliferation was observed across multiple donors. Furthermore, treatment of Teff cells with itolizumab resulted in a significant decrease in expression level of T cell markers of activation and exhaustion such as CD25, PD-1 and Tim3. This effect was exclusively in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. When levels of CD6 were assessed, CD45RO+ (Teff/mem) cells expressed higher levels than CD45RA+CD45RO- (Tnaive). Itolizumab treatment of in vitro generated CD45RO+ T cells inhibited this stimulation-induced increase in CD6 in a dose-dependent manner, suggesting that the drug may modulate surface CD6 expression as a mechanism separate from physical blockade of CD6. Conclusions: These findings are the first to characterize the CD6-ALCAM pathway as a key regulator of differentiated effector T-cell function. Modulation of activation markers and CD6 itself by itolizumab, suggest modulation of Teff activity by both direct and indirect inhibition of CD6 signaling. These data further support targeting the CD6-ALCAM pathway to inhibit both naïve and effector T cell populations in aGVHD. Disclosures Ampudia: Equillium: Current Employment, Current equity holder in publicly-traded company. Chu:Equillium: Current Employment, Current equity holder in publicly-traded company. Doherty:Equillium Inc.: Research Funding. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company.

Erratum: Yao et al., "Cocaine Hijacks σ1 Receptor to Initiate Induction of Activated Leukocyte Cell Adhesion Molecule: Implication for Increased Monocyte Adhesion and Migration in the CNS".

Erratum: Yao et al., "Cocaine Hijacks σ1 Receptor to Initiate Induction of Activated Leukocyte Cell Adhesion Molecule: Implication for Increased Monocyte Adhesion and Migration in the CNS".

Activated leukocyte cell adhesion molecule as a biomarker for disease severity and efficacy of sublingual immunotherapy in allergic rhinitis

BackgroundActivated leukocyte cell adhesion molecule (ALCAM) plays an important role in T cell activation and immune response, but the role of ALCAM in allergic rhinitis (AR) remains unclear. The objective of the current study was to validate serum ALCAM as a biomarker in assessing disease severity and predicting the efficacy of sublingual immunotherapy (SLIT) in AR patients. MethodsWe recruited 40 healthy controls (HC group), 38 mild AR patients (MAR group) and 80 moderate-severe AR patients (MSAR group) in this study. Serum levels of ALCAM were determined by ELISA, and the association between ALCAM levels and disease severity was evaluated. In the MSAR group, 68 patients underwent and finished 3-years of SLIT, and were divided into effective group and ineffective group, the relationship between ALCAM levels and efficacy of SLIT was exampled. ResultsALCAM levels were elevated in the serum of AR patients in comparison with HC. Moreover, serum ALCAM concentrations were higher in MSAR group than in MAR group and HC group, and levels of ALCAM significantly correlated with AR total nasal symptom score (TNSS) (r = 0.330, P < 0.001), visual analogue scale (VAS) (r = 0.387, P < 0.001) and serum total IgE levels (r = 0.442, P < 0.001). In the effective group, the ALCAM levels were significantly lower than in the ineffective group. Receiver operating characteristic (ROC) curve exhibited good accuracy for predicting clinical efficacy of SLIT (area under the curve = 0.805, P < 0.001). ConclusionsThe serum ALCAM maybe a novel biomarker for assessing disease severity and predicting clinical efficacy of SLIT in AR patients.

Abstract IA08: Incorporating proteomics in bladder cancer molecular diagnosis

Abstract Proteins are directly linked to cell phenotype, hence accrue interest as biomarkers and therapeutic targets. Investigating the urine proteome as a source of biomarkers entails multiple technical challenges, with the main being the protein concentration vast dynamic range and presence of proteins in multiple modified forms. As a result, protein enrichment strategies in combination to different mass spectrometry platforms are regularly employed as main discovery tools, providing complementary experimental outputs. Verification and further biomarker validation regularly involve application of ELISA and, more recently, multiplex mass spectrometry (MS)-based assays (Multiple Reaction Monitoring–MS). In the course of this presentation, an overview of urine proteomics, as applied in bladder cancer research, will be provided. In brief, using the armamentarium of existing methodologic approaches, multiple urinary proteins have been associated with bladder cancer phenotypes and/or attributed prognostic or predictive value, including the Nuclear Matrix Protein 22 (NMP22) and Bladder Tumor Antigen (BTA; corresponding to complement factor H-related protein), approved by the FDA for use in disease diagnosis and/or surveillance. Additional proteins frequently reported in the literature as associated with bladder cancer detection and/or staging include various forms of apolipoproteins, proteins of the extracellular matrix (such as collagen and fibrinogen fragments), matrix metalloproteinases, cytokeratin fragments as well as proteins involved in apoptosis (such as survivin) or inflammation (including multiple cytokines). Reports also support potential of some of these proteins when evaluated in the form of biomarker panels, as predictors of BCG response in high-risk non-muscle invasive bladder cancer. Along these lines, simultaneous analysis of multiple markers through ELISA or mass spectrometry-based platforms has provided increased accuracies in comparison to single biomarkers. Hematuria is a frequent confounding variable in urinary protein measurements, rendering establishment of signal specificity via optimized ELISA assays or mass spectrometry platforms a current technological target. Direct links of urinary protein changes to bladder tissue events have been frequently reported (such as the cases of profilin 1 or activated leukocyte cell adhesion molecule). Even though disproportionate to the numerous reported findings (frequently compromised by suboptimal experimental design), collectively, promising urinary protein findings in association to bladder cancer phenotypes and contexts of use are existent, awaiting proper validation in large-scale prospective trials. Main research paths that now open up include: refinement of existing bladder cancer molecular subtypes, with the addition of information at the protein level, and placing urinary markers in this context; and, in parallel, design and running of proper validation studies as well as clinical sample sharing, to define optimal marker combinations per clinically relevant bladder cancer context of use. In the latter, special emphasis should be placed on prediction of treatment response, in order to maximize benefit from available treatment modalities. Citation Format: Antonia Vlahou. Incorporating proteomics in bladder cancer molecular diagnosis [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr IA08.

Negatively charged amino acids in the stalk region of membrane proteins reduce ectodomain shedding

Ectodomain shedding is a post-translational modification mechanism by which the entire extracellular domain of membrane proteins is liberated through juxtamembrane processing. Because shedding rapidly and irreversibly alters the characteristics of cells, this process is properly regulated. However, the molecular mechanisms governing the propensity of membrane proteins to shedding are largely unknown. Here, we present evidence that negatively charged amino acids within the stalk region, an unstructured juxtamembrane region at which shedding occurs, contribute to shedding susceptibility. We show that two activated leukocyte cell adhesion molecule (ALCAM) protein variants produced by alternative splicing have different susceptibilities to ADAM metallopeptidase domain 17 (ADAM17)-mediated shedding. Of note, the inclusion of a stalk region encoded by a 39-bp-long alternative exon conferred shedding resistance. We found that this alternative exon encodes a large proportion of negatively charged amino acids, which we demonstrate are indispensable for conferring the shedding resistance. We also show that the introduction of negatively charged amino acids into the stalk region of shedding-susceptible ALCAM variant protein attenuates its shedding. Furthermore, we observed that negatively charged amino acids residing in the stalk region of Erb-B2 receptor tyrosine kinase 4 (ERBB4) are indispensable for its shedding resistance. Collectively, our results indicate that negatively charged amino acids within the stalk region interfere with the shedding of multiple membrane proteins. We conclude that the composition of the stalk region determines the shedding susceptibility of membrane proteins.

MicroRNA expression profiling and the role of ALCAM modulating tumor growth and metastasis in benzo[a]pyrene-transformed 16HBE cells

Benzo[a]pyrene (BaP) is a potent carcinogen and microRNAs (miRNAs) may play an important role in carcinogenesis. Activated leukocyte cell adhesion molecule (ALCAM) was up-regulated in BaP-transformed 16HBE cell line (THBEc1), and may be a key molecule for THBEc1 cells to gain and maintain the malignant phenotype. Here we screened the differentially expressed miRNAs which resulted in up-regulation of ALCAM in THBEc1 cells by comparing miRNA expression profiles between THBEc1 and 16HBE (HBE) cells. Results showed that a total of 555 miRNAs differentially expressed between THBEc1 and HBE cells, of which 351 miRNAs were down-regulated and 204 miRNAs were up-regulated in THBEc1 cells. MiR-152-3p, miR-142-5p and miR-211-5p down-regulated in THBEc1 cells were demonstrated to participate in the regulation of ALCAM. With THBEc1 as a tumor cell model, we determined the role of ALCAM in tumor growth and metastasis employing two ALCAM knockout THBEc1 cell lines via CRISPR/Cas9 technology. Results showed that ALCAM knockout inhibited colony formation and tumor growth, but enhanced cell migration and lung metastasis of THBEc1 cells. In conclusion, miR-152-3p/ALCAM, miR-142-5p/ALCAM and miR-211-5p/ALCAM axes may be involved in BaP-induced carcinogenesis. BaP might induce up-regulation of ALCAM via inhibiting miR-152-3p, miR-142-5p and miR-211-5p, which in turn allows ALCAM to exert its role promoting cell proliferation and tumor growth, and suppressing cell migration and metastasis.

MO007A PHASE 1B MULTIPLE ASCENDING-DOSE STUDY OF A CD6 TARGETED THERAPY, ITOLIZUMAB, IN SUBJECTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS WITH OR WITHOUT ACTIVE PROLIFERATIVE LUPUS NEPHRITIS

Abstract Background and Aims Lupus nephritis (LN) is a leading cause of morbidity and mortality in systemic lupus erythematosus (SLE) patients. T cells are believed to play a central role in the pathogenesis of both SLE and LN. CD6 is a co-stimulatory receptor, predominantly expressed on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation, differentiation and trafficking, and is central to immune mediated inflammation. Itolizumab (EQ001) is a humanized IgG1 monoclonal antibody that binds CD6, blocks the interaction between CD6 and ALCAM, and inhibits both the activation and trafficking of T cells. Inhibiting the CD6-ALCAM pathway with itolizumab potentially represents a promising therapeutic approach for the treatment of LN. The aim of this study is to assess the safety and tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of subcutaneously administered itolizumab in patients with SLE with and without active proliferative lupus nephritis (apLN). Method This cohort-based dose escalation study includes two types of patients: The Type A cohort will enroll ∼24 patients with SLE without apLN (all treated with itolizumab) and the Type B cohort will randomize in a blinded manner ∼36 patients (3:1, itolizumab:placebo) with biopsy-proven ISN/RPS class III or IV (+ V) apLN who have had inadequate response to induction and/or post-induction maintenance treatment, exhibiting urine protein to creatinine ratio [UPCR] ≥1 g/g and active serology. Within both the Type A and Type B cohorts, up to 4 dose groups will be tested (Figure). Background treatments for SLE or LN are allowed. Following 4 weeks of treatment in a new higher dose Type A cohort and recommendation by an independent safety data review committee (DRC), the dose studied in the Type A cohort may then be studied in a Type B Cohort for a 12-week treatment duration (Figure). The primary endpoint is the safety and tolerability of itolizumab. Efficacy endpoints (in the Type B cohorts) include UPCR, estimated glomerular filtration rate, prednisone dose requirements, renal response, Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K), FACIT Fatigue Scale, serologic markers, and other patient reported outcomes. In Type A cohort patients, clinical responses and pharmacologic activity will be assessed based on changes in serologic markers, SLEDAI-2K, FACIT Fatigue Scale. Pharmacodynamic markers, including markers that may allow future risk stratification, urinary ALCAM and CD6, will be examined in both cohort types. Results The study is ongoing. Six patients have been enrolled in Type A Cohort 1 (0.4 mg/kg dose) and completed both treatment and 4 weeks of post-treatment follow-up. The mean age was 59.5 (12.9) years, 100% were female; 67% were Hispanic/Latino; and 50% were White, and 50% were Black. Duration of SLE ranged from 3 years to 31 years. Concomitant medications for lupus included prednisone (83%, dose range 2.5 mg – 10 mg), methotrexate (33%), and anti-malarials (33%). Baseline SLEDAI-2K (mean 7.5 [2.2]) was based on findings of alopecia (83%); arthritis (67%); mucosal ulcers and rash (50% each); fever, increased dsDNA, and low complement (17% each). There were no adverse events. Additional data from this ongoing study will be presented. Conclusion Itolizumab, a monoclonal antibody blocking the CD6-ALCAM pathway, is a novel experimental treatment for LN. This is the first trial of itolizumab in patients with SLE and apLN. Data from the first cohort of patients suggest that the drug is safe and well-tolerated at a dose of 0.4 mg/kg over a 4-week treatment period. Additional cohorts of patients with SLE and apLN are currently being enrolled.

Urinary activated leukocyte cell adhesion molecule as a novel biomarker of lupus nephritis histology

BackgroundLupus nephritis (LN) is one of the most severe complications of SLE patients. We aim to validate urinary ALCAM as a biomarker in predicting renal disease histpathology in a Chinese lupus cohort.MethodsIn this cross-sectional study, a total of 256 patients and controls were recruited. Urinary levels of ALCAM were determined by ELISA. Renal histopathology was reviewed by an experienced renal pathologist.ResultsUrinary ALCAM levels were significantly increased in active LN patients when compared to active SLE patients without renal involvement (p < 0.001), inactive LN patients (p = 0.023), inactive SLE patients without renal involvement (p < 0.001), and healthy controls (p < 0.001). Correlation analysis revealed a positive correlation between urinary ALCAM and general disease activity—SLEDAI score (r = 0.487, p < 0.001), as well as renal disease activity—rSLEDAI (r = 0.552, p < 0.001) and SLICC RAS (r = 0.584, p < 0.001). Urinary ALCAM also correlated with lab parameters including 24-h urine protein, hemoglobin, and complement 3. Moreover, urinary ALCAM levels were significantly increased in class III and IV (proliferative) LN as compared to those in class V (membranous) LN. It outperformed conventional biomarkers (anti-dsDNA antibody, C3, C4, proteinuria) in discriminating the two groups of LN. On renal histopathology, urinary ALCAM levels correlated positively with activity index (r = 0.405, p < 0.001) but not chronicity index (r = 0.079, p = 0.448).ConclusionUrinary ALCAM is a potential biomarker for predicting renal pathology activity in LN and may serve as a valuable surrogate marker of renal histopathology.

Amelioration of skin and kidney disease in a spontaneous murine lupus model via CD6 modulation

Abstract Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that can affect multiple organ systems, including the kidneys, skin, and brain. T cells are an important mediator in this end organ damage. CD6 is a co-stimulatory receptor, predominantly expressed on T cells, which binds with activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. This signaling pathway is vital for T cell activation, proliferation, differentiation and trafficking. We found increased expression of both CD6 and ALCAM in the kidneys of MRL/lpr mice (a spontaneous model of SLE) versus healthy control B6 mice. In a separate experiment, female MRL/lpr mice were aged to 9–10 weeks of age, at which point we began treating with either anti-CD6 antibody (60 ug/dose, intraperitoneally twice per week), isotype control (60 ug/dose, twice per week), or cyclophosphamide (25 mg/kg, once per week). We also included a no treatment group and a group of MRL/MpJ mice, a congenic healthy control strain. Mice treated with anti-CD6 show lower levels of proteinuria and BUN (p&amp;lt;0.05), improved survival rates, and decreased renal pathology compared to isotype control mice. Flow cytometry revealed decreased numbers of activated and effector T cells within the kidneys of anti-CD6 treated mice compared to isotype control mice. While there was no difference in anti-DNA levels, anti-CD6 treatment significant improved the spontaneous skin lesions associated with disease progression. Overall, these results indicate that targeting CD6-ALCAM interactions may have promising therapeutic potential within the context of different end organ pathologies within lupus.

CD6-ALCAM signaling regulates multiple effector/memory T cell functions

Abstract CD6 is a T-cell costimulatory receptor that has been implicated in the pathogenesis of multiple autoimmune and inflammatory diseases. Primarily expressed on CD4 T cells, CD6 promotes immune synapse formation, T-cell activation and T-cell migration via interaction with its ligand activated leukocyte cell adhesion molecule (ALCAM). While the contribution of CD6 to T cell activation has been well described, less is known regarding the role of CD6 on effector and memory T cells (Teff). Thus, to better characterize this, we examined phosphorylation signaling patterns during CD6 co-stimulation together with the impact of CD6 on differentiated Teff functions. Profiling of ~100 phosphorylation targets associated with T-cell receptor signaling revealed that CD6 co-stimulation on T cells activates factors in pathways involved in actin polymerization, motility, integrin activation and T-cell activation. Comparison of Tnaive cells vs. Teff demonstrated differing levels of phosphorylation in response to CD6 stimulation. Furthermore, CD6 signaling on Teff sustained phosphorylation of these pathways at later timepoints compared to CD28 stimulation. Blockade of the CD6 pathway, using the clinically tested anti-CD6 mAb itolizumab during re-stimulation of CD4 Teff cells in the presence of ALCAM, inhibited multiple effector functions including proliferation and changes in blast size. This effect was observed exclusively in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. These findings demonstrate that the CD6-ALCAM pathway is a key regulator of effector T-cell functions and further support targeting this pathway to directly inhibit both naïve and effector T cell populations.

CD6 modulation ameliorates immune complex-mediated glomerulonephritis

Abstract Lupus nephritis (LN) is a serious end organ complication of systemic lupus erythematosus (SLE) in which T cells are thought to play an essential role. CD6 is a co-stimulatory receptor on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation and trafficking, and increased levels of CD6 are associated with pathogenic T cell responses. To assess the role of the CD6-ALCAM pathway in LN pathogenesis, we tested a monoclonal antibody against CD6 in a short-term, validated, inducible murine model of lupus nephritis known as nephrotoxic serum nephritis (NTN). NTN mice were treated 3× per week with an anti-CD6 mAb (10D12, 60ug/dose, n=23) or with vehicle control (n=23). Healthy mice were also included as a control (n=12). Mice treated with the anti-CD6 mAb displayed decreased levels of proteinuria (p&amp;lt;0.001) and significantly improved BUN levels (p&amp;lt;0.01) compared to vehicle control mice. Histology also significantly improved with anti-CD6 treatment (p&amp;lt;0.05). RT-PCR revealed significantly decreased levels of VCAM and RANTES in the kidneys of treated mice, while anti-inflammatory IL-10 was increased, compared to vehicle control mice. Flow cytometry analysis indicated decreased accumulation of both renal-infiltrating activated T cells (CD4+CD25+CD69+, p &amp;lt;0.01) inflammatory macrophages (p&amp;lt;0.05). Overall, these results indicate that the CD6-ALCAM pathway is an important driver of inflammation and pathology in LN and, thus, a promising therapeutic option that is more selective than the immunosuppressive therapies currently offered.

Genetic variations in the host dependency factors ALCAM and TPST2 impact HIV-1 disease progression.

Recently, the activated leukocyte cell adhesion molecule (ALCAM) and tyrosylprotein sulfotransferase 2 (TPST2) have been identified as important host dependency factors (HDFs) for in-vitro HIV-1 replication. To determine whether these genes play a role in HIV-1 pathogenesis, we analysed whether naturally occurring genetic variations were associated with the clinical course of infection. Single nucleotide polymorphisms (SNPs) in ALCAM and TPST2 were analysed in a cohort of 304 HIV-1-infected men who have sex with men and survival analysis was used to determine their effect on the outcome of untreated HIV-1 infection. Flowcytometry was used to determine the effect of SNPs on CD4 T-cell activation prior to HIV-1 infection and 1 and 5 years after infection. In-vitro HIV-1 infections were performed to analyse the effect of the SNPs on HIV-1 replication. We observed that the minor allele of rs1344861 in ALCAM was associated with accelerated disease progression, whereas the minor allele of rs9613199 in TPST2 was associated with delayed disease progression. In-vitro infection assays did not demonstrate any differences in HIV-1 replication associated with rs9613199. However, the increase in CD4 T-cell immune activation levels during HIV-1 infection was less pronounced in infected individuals homozygous for rs9613199, which is in agreement with delayed disease progression. Our data demonstrate that ALCAM and TPST2 play a role in HIV-1 pathogenesis. SNPs in these genes, without known functional implications, had a major effect on disease progression, and therefore, these HDFs may be attractive and effective targets for new treatment strategies.