anti-CD6 mAb Research Articles

Surface CD6 regulates T cell adhesion and migration through direct ligand interaction and stabilization of VLA4

Abstract The CD6-ALCAM pathway is an important regulator of T cell activity and trafficking. However, its precise role in modulating T-cell adhesion and migration during inflammation is unclear. To investigate its role in adhesion and transendothelial migration, we used itolizumab, an anti-CD6 mAb that can induce cleavage of CD6 from the T cell surface when in the presence of monocytes. PBMCs were stimulated overnight or left unstimulated prior to treatment with itolizumab to generate CD6low T cells. Cells were then tested in standard adhesion and migration assays. The CD6 low T cells had ~ 50% decreased adherence to HUVEC cells compared to the control T cells (CD6high); similar results were observed by blocking ALCAM on HUVEC. Modulating CD6 on T cells or blocking ALCAM on the HUVEC also translated to a ~3-fold decrease in migration of Teff cells. Loss of CD6 was associated with reduced protein levels of VLA4, but not of other integrins, under both stimulated (~70%) and non-stimulated conditions (~40%). Antibody blockade of CD6 without cleavage did not affect VLA4 levels, but still decreased migration of unstimulated T cells though less so than in stimulated T cells, where VLA4 reduction is required to modulate migration. Our data demonstrate that CD6 contributes to cell adhesion/migration both through direct ligand interactions with ALCAM, and through the stabilization of cell membrane VLA4 and that these functions can be abrogated by modulation of cell surface levels of CD6.

CD6-targeted antibody-drug conjugate as a new therapeutic agent for T cell lymphoma.

T cell lymphomas (TCL) are heterogeneous, aggressive, and have few available targeted therapeutics. In this study, we determined that CD6, an established T cell marker, was expressed at high levels on almost all examined TCL patient specimens, suggesting that CD6 could be a new therapeutic target for this life-threatening blood cancer. We prepared a CD6-targeted antibody-drug conjugate (CD6-ADC) by conjugating monomethyl auristatin E (MMAE), an FDA-approved mitotic toxin, to a high-affinity anti-human CD6 monoclonal antibody (mAb). In contrast to both the unconjugated anti-CD6 mAb, and the non-binding control ADC, CD6-ADC potently and selectively killed TCL cells in vitro in both time- and concentration-dependent manners. It also prevented the development of tumors in vivo in a preclinical model of TCL. More importantly, systemic or local administration of the CD6-ADC or its humanized version, but not the controls, significantly shrank established tumors in the preclinical mouse model of TCL. These results suggest that CD6 is a novel therapeutic target in TCLs and provide a strong rationale for the further development of CD6-ADC as a promising therapy for patients with these potentially fatal lymphoid neoplasms.

Modulating levels of cell surface CD6 is a novel mechanism for regulating T cell activity.

Abstract CD6 is a co-stimulatory receptor expressed on the surface of T cells that promotes immune synapse formation, T cell activation, and migration into tissues by engaging its ligand, ALCAM. CD6high cells have been described to be more pathogenic relative to CD6low cells. Furthermore, T cells in CD6 knockout mice have decreased proliferation and inflammatory responses. Hence, a therapeutic that can modulate surface levels of CD6 may have favorable outcomes for autoimmune/inflammatory conditions. We recently demonstrated that the anti-CD6 mAb, itolizumab, induces proteolytic cleavage of cell surface CD6. The goal of the work shown here is to characterize the mechanism of cleavage and the downstream functional consequences of reduced cell surface CD6. Treatment of PBMCs with itolizumab induces CD6 cleavage from the cell surface and a concomitant increase in the soluble form detected in the supernatant. Itolizumab-induced loss of CD6 was not observed with isolated T cells, however CD6 levels were reduced by 87% when T cells and monocytes were cocultured with itolizumab. The cleavage of CD6 is initiated through rapid cell-to-cell contact between T cells and monocytes via FcγRI. The dose-dependent reduction in surface levels of CD6 positively correlates with decreases in T cell activation markers such as CD25, PD-1, and CD71 (Pearson, p<0.001) and cytokine production including IL-2 (p<0.01) and TNFα (p<0.05). Itolizumab-treated cells are also less alloreactive as shown by a reduction in proliferation and cytokine production of responder cells in a mixed-lymphocyte reaction. This data further supports targeting CD6 as an effective means to inhibit pathogenic T cell activity in the treatment of autoimmune and inflammatory diseases.

A CD6-Targeted Antibody-Drug Conjugate As a Potential Therapy for T Cell-Mediated Disorders

INTRODUCTION Pathogenic T cells cause many diseases including most autoimmune diseases and graft-versus-host disease (GVHD). Selectively targeting these pathogenic T cells while sparing the normal T cells and other tissues is a “holy grail” of therapeutics development in modern clinical immunology. So far, pan-immunosuppressive drugs such as corticosteroids are used to treat these patients, with limited efficacy and severe adverse effects. It is also well-established that these pathogenic T cells, whether auto- or alloreactive, proliferate following antigen recognition to cause tissue damage while the other normal T cells remain quiescent. Selectively targeting the proliferating T cells could be an effective strategy to develop new drugs for diseases mediated by the pathogenic T cells.Antibody-drug conjugates (ADCs) are developed by conjugating a potent toxin onto a monoclonal antibody (mAb) specific for a cancer cell surface antigen. Monomethyl auristatin E (MMAE), a synthetic mitotic toxin, is the payload in several FDA-approved ADCs, and it kills the actively dividing cancer cells by blocking the polymerization of tubulin, rapidly inducing apoptosis. These cancer cells and pathogenic T cells have one feature in common-both of them are actively proliferating, thus, this ADC approach proven successful in cancer treatment could be re-purposed to selectively kill pathogenic T cells for the treatment of T cell-mediated diseases.CD6 is a cell surface glycoprotein that is expressed at high levels on all T cells except Treg cells, a small portion of B cells and many human NK cells. CD6 is not detectable on other tissue cells, making it a highly specific target candidate for T cells. We have generated CD6 knockout mice and CD6 humanized mice, and developed anti-CD6 mAbs that treat mouse models T cell-mediated diseases. A CD6-targeted ADC (CD6-ADC) thus might be effective for treating T cell-mediated diseases by selectively eliminating the proliferating pathogenic T cells.METHODS A CD6-ADC was developed by conjugating a latent form of MMAE onto the high-affinity anti-human CD6 mAb. Its potency of selectively killing pathogenic T cells was evaluated in an antigen-specific T cell recall assay with BrdU-incorporation followed by flow cytometric analyses. To determine the toxicity of the CD6-ADC on normal quiescent T cells, naïve CD6 humanized mice were treated with the CD6-ADC or non-binding control ADC (0.5 mg/kg) by intraperitoneal injection, then numbers of circulating T cells were monitored by flow cytometry daily. To evaluate the efficacy of CD6-ADC in treating T cell-mediated autoimmune diseases, the same dose (0.5 mg/kg) of CD6-ADC, or the parental anti-CD6 mAb, or the control ADC was used to treat a model of autoimmune uveitis, and disease severities were assessed by various ocular imaging techniques including indirect ophthalmoscopy, confocal scanning laser ophthalmoscopy and optical coherence tomography. To examine the potential of the CD6-ADC in treating GVHD, the same dose (0.5mg/kg) of CD6-ADC or control ADC was tested in a model of GVHD induced in NSG mice after adoptive transfer of human PBMC. GVHD severities were assessed by flow cytometric analyses of circulating human T cells and by histopathological analyses of different tissues.RESULTS: The CD6-ADC selectively killed antigen-specific proliferating T cells in vitro. Treating naive CD6-humanized mice with this CD6-ADC (0.5 mg/kg) did not significantly eliminate normal T cells in vivo. Furthermore, systemic delivery of the same dose (0.5 mg/kg) of CD6-ADC, but not the anti-CD6 mAb alone nor the control IgG effectively reduced retinal inflammation in a preclinical model of autoimmune uveitis. The same dose of CD6-ADC, but not the control ADC, also effectively depleted activated xenogeneic T cells and prevented the development of GVHD in NSG mice after the adoptive transfer of human PBMC.CONCLUSION: These data indicate that this CD6-ADC holds promise as a new drug for treating diseases in which T cells are integrally involved in the pathogeneses such as GVHD. [Display omitted] DisclosuresLin: Takeda Pharma: Consultancy, Research Funding.

CD6-Targeted Antibody-Drug Conjugate As a Potential Therapeutic Agent for T Cell Lymphomas

Introduction: Peripheral T-cell lymphomas (PTCLs) comprise a subset of blood cancers that are molecularly heterogeneous and often associated with poor survival. While brentuximab vedotin has shown efficacy in CD30-expressing lymphomas such as anaplastic large cell lymphoma, effective targeted therapies for PTCLs are lacking and this represents an unmet medical need.CD6, an established surface marker for T cells, is a key regulator of T cell responses. CD6 has been suggested as a target for treating T cell-mediated autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis. We previously reported that CD6 knockout mice show reduced pathogenic T-cell responses and thus are protected against several models of autoimmune diseases. We also found that CD6 could be targeted therapeutically by anti-CD6 mAbs in these mouse models of autoimmune diseases. Although the distribution of CD6 and its role in regulating normal, non-malignant T-cells has been established, the expression of this marker on PTCL cells and its potential use as a selective targeted therapeutic agent for PTCLs has not been explored.METHODS: To determine the expression profile of CD6 on the malignant T-cells, different specimens including established T-cell lymphoma/leukemia cell lines HH, MOLT-4, HUT-78 and JurkaT-cells, as well as PTCL patient-derived xenografts (PDX) were analyzed for CD6 expression by flow cytometry using UMCD6, a high-affinity anti-human CD6 mAb. Besides, a custom-made tissue microarray containing biopsy samples from different PTCL patients was also evaluated by immunohistochemical staining using the same mAb.A CD6-targeted antibody-drug conjugate (CD6-ADC) was prepared by conjugating monomethyl auristatin E (MMAE), a potent antimitotic toxin successfully used in other FDA-approved ADCs, to UMCD6. Control non-binding ADC was prepared the same way using purified mouse IgGs. The potency of the CD6-ADC of killing PTCL was first evaluated in vitro using HH and HUT78 cells with trypan blue staining. For in vivo treatment studies, NSG mice were subcutaneously inoculated with HH cells, then treated with CD6-ADC or controls at different time points either systemically by intraperitoneal injections or locally by intratumor injections. Treatment efficacies were evaluated by measuring subcutaneous tumor volumes and detecting metastasized tumors in bone marrows, spleens and livers.RESULTS: Our flow cytometric and immunohistochemical staining studies showed that most, if not all the specimens examined expressed high levels of CD6 on the surface, suggesting that CD6 could be a novel therapeutic target for this molecularly heterogeneous blood cancer. Besides, our anti-CD6 mAb, was quickly internalized by PTCL cells after binding to CD6 on their cell surface in an in vitro assay. In contrast to either the parental anti-CD6 mAb, or the non-binding control ADC, the CD6-ADC potently and selectively killed T lymphoma cells in vitro in a time- and concentration-dependent manner. It further prevented the development of tumors in vivo when given 1 day after PTCL cell inoculations. More importantly, systemic or local administration of CD6-ADC, but not the control ADC, significantly shrank existing tumors, reduced tumor metastases from the already developed tumors, and prolonged overall survival in the preclinical mouse model of PTCL without apparent adverse effects.CONCLUSIONS: These results suggest that CD6 is a novel therapeutic target for PTCLs and provide a strong rationale for the further development of CD6-ADC as a promising therapy for patients with this aggressive blood cancer. [Display omitted] DisclosuresHsi: AbbVie Inc, Eli Lilly: Research Funding. Lin: takeda Pharma: Consultancy, Research Funding.

Itolizumab, a Novel Targeted Anti-CD6 Therapy, in Combination with Corticosteroids, Is Well-Tolerated, with Rapid Pharmacodynamic and Clinical Response in Newly Diagnosed Acute Graft-Versus-Host Disease

Itolizumab, a Novel Targeted Anti-CD6 Therapy, in Combination with Corticosteroids, Is Well-Tolerated, with Rapid Pharmacodynamic and Clinical Response in Newly Diagnosed Acute Graft-Versus-Host Disease

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<0.001) and significantly improved BUN levels (p<0.01) compared to vehicle control mice. Histology also significantly improved with anti-CD6 treatment (p<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 <0.01) inflammatory macrophages (p<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.

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.

Equate, a Phase 1b/2 Study Evaluating the Safety, Tolerability, Pharmacokinetics, Pharmacodynamics, and Clinical Activity of a Novel Targeted Anti-CD6 Therapy, Itolizumab, in Subjects with Newly Diagnosed Acute Graft Versus Host Disease

Equate, a Phase 1b/2 Study Evaluating the Safety, Tolerability, Pharmacokinetics, Pharmacodynamics, and Clinical Activity of a Novel Targeted Anti-CD6 Therapy, Itolizumab, in Subjects with Newly Diagnosed Acute Graft Versus Host Disease

Itolizumab As a Potential Therapeutic for the Prevention and Treatment of Graft Vs Host Disease

Background: 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 presentation 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 inflammation. While effector T cell (Teff) are CD6hi and upregulate expression upon activation, regulatory T cells (Treg) remain CD6lo/-, making this an attractive target to modulate Teff activity while preserving Treg activity. Early studies by Soiffer and colleagues demonstrated using T12, an anti-CD6 monoclonal antibody (mAb) that ex-vivo depletion of CD6+ donor cells prior to transplantation decreased the incidence of both acute and chronic GVHD, highlighting the importance of CD6+ cells in GVHD pathogenesis and validating it as a therapeutic target. However, it remains to be shown whether modulating the CD6-ALCAM pathway in vivo can attenuate GVHD. We investigated the use of itolizumab, a humanized anti-CD6 mAb that has demonstrated clinical efficacy in other autoimmune diseases, as both a preventive and therapeutic treatment for GVHD, using a humanized xenograft mouse model. Methods: Humanized xenograft mice were generated by intravenous transfer of 2x10^7 human PBMCs into 6-8 weeks old NOD/SCID IL2rγ-null (NSG). To investigate the ability of itolizumab to prevent GVHD, mice were dosed with either 60μg or 300μg of itolizumab, 150μg of abatacept (CTLA4-Ig), or vehicle, starting one day prior to PBMC transplantation. To investigate the therapeutic effect of itolizumab, mice were dosed with either 150μg of itolizumab or vehicle, starting at Day 5 post-PBMC transfer, when transplanted T cells are already activated. All treatments were administered IP every other day. Weight and disease scores were monitored throughout the study. At Days 18 and 35, peripheral blood was evaluated by flow cytometry to examine T cell prevalence, and tissues were collected for histological examination of pathology and T cell infiltration. Results: When administered as prevention (Day -1), treatment with either 60μg or 300μg of itolizumab significantly decreased mortality compared to the vehicle control (100% vs. 10%); this decrease was similar to the positive control group treated with abatacept (Figure 1). At 60μg, itolizumab-treated mice demonstrated significant reductions in the prevalence of human T cells in peripheral blood vs. vehicle-treated mice at Day 18 (<0.2% vs. 74.5%; p < 0.001). The reduction in peripheral T cells was accompanied by reductions in tissue-infiltrating T cells in lung (85-fold) and gut (9.5-fold), as well as reductions in disease scores and weight loss. When administered therapeutically, treatment with itolizumab was associated with a survival rate of 50% compared to 10% in the control group (Figure 2). Similarly, peripheral T cell prevalence (34.3% vs. 65.1%; p < 0.001), weight loss, and disease scores were inhibited by itolizumab compared to vehicle control mice. Conclusions: These data suggest that systemic treatment with itolizumab can modulate pathogenic Teff cell activity, establishing this antibody as a potential therapeutic for patents with GvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Ng: Equillium: Employment, Equity Ownership. Ampudia:Equillium: Employment. Soiffer:Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Jazz: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Avrobio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; LifeVault Bio: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Kite Pharma: Research Funding. Connelly:Equillium: Employment, Equity Ownership.

Biocon's first-in-class anti-CD6 mAb reaches the market

Biocon's first-in-class anti-CD6 mAb reaches the market

Long-term outcomes of nonconditioned patients with severe combined immunodeficiency transplanted with HLA-identical or haploidentical bone marrow depleted of T cells with anti-CD6 mAb

Between 1981 and 1995, 20 children with severe combined immunodeficiency (SCID; median age at transplant, 6.5 [range, 0.5-145] mo, 12 with serious infection) were treated with haploidentical T cell-depleted (anti-CD6 antibody) bone marrow (median number of 5.7 [0.8-18.8] x 10(8) nucleated cells/kg) from mismatched related donors (MMRDs), and 5 children with SCID (median age at transplant, 1.8 [0.5-5.0] mo, 1 with serious infection) were given unmanipulated bone marrow from matched related donors (MRDs). No conditioning or graft-versus-host disease (GvHD) prophylaxis was used. To assess the outcomes of patients with SCID who received bone marrow from MMRDs or MRDs. We reviewed the medical records of these 25 consecutive patients with SCID (4 with Omenn syndrome). Of the 20 patients who received bone marrow from MMRDs, 12 engrafted, 10 survived at a median age of 15.2 [10.0-19.1] years, 4 had chronic GvHD (lung, intestine, skin), 5 required intravenous immunoglobulin, and 8 attended school or college. Two of 5 patients who died had chronic GvHD, and 2 developed lymphoproliferative disease. Of the 5 patients who received bone marrow from MRDs, 5 engrafted, 5 survived at a median age of 23.3 [18.5-26] years, 1 had chronic GvHD (lung, skin), 2 required intravenous immunoglobulin, and 4 attended school or college. Treatment of critically ill patients with SCID with anti-CD6 antibody T cell-depleted MMRD marrow resulted in an overall 50% long-term survival of patients (83% survival of those engrafted). The principal barriers to long-term survival were delay in diagnosis, life-threatening infection, failure to engraft, and chronic GvHD. Educational goals were achieved in most of the survivors.

Immunodiagnosis and therapeutic immunosuppression in rheumatoid arthritis with ior t1 (anti-CD6) monoclonal antibody

CD6 antigen is a typeI cell membrane glycoprotein belonging to the scavenger receptor cysteine-rich (SRCR) superfamily groupB, predominantly expressed by T cells and a B-cell subset. CD6 binds activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily (IgSF). ALCAM is expressed on activated T cells, B cells, monocytes, skin fibroblasts, keratinocytes and rheumatoid arthritis synovium, and mediates homophilic and heterophilic adhesion. CD6-ligand interaction has been implicated in cell adhesion, T-cell maturation and regulation of activation, constituting an uncommon type of protein-protein superfamilies interaction. The ior t1 is a murine IgG2a mAb recognizing a different epitope compared to other anti-CD6 mAbs. It is in a phase II clinical trial (PIICT) for cutaneous T-cell lymphomas treatment. Recently, we reported its intravenous therapeutic effect in a Psoriasis vulgaris patient. A PIICT was performed in 18 rheumatoid arthritis patients. Technetium-99m-labeled ior t1 mAb (ior t1-99Tcm) joint uptake and body distribution was assessed by anterior and posterior whole body scans and specific regional imaging. Forty-eight hours apart started a therapeutic dose-finding study based on 7 consecutive daily doses at 0.2 mg/kg, 0.4 mg/kg or 0.8 mg/kg of iort1 mAb intravenous infusion. Clinical evaluation and laboratory analysis were performed weekly. A rapid ior t1-99Tcm/lymphocytes association and a marked radioactivity uptake form inflamed and silent joints were obtained. From biodistribution studies was estimated that more than 0.5% of the ior t1-99Tcm infusion penetrates into hands and feet with inflamed joints. iort1 joint-imaging was superior to MDP-99Tcm, used as standard method. iort1 mAb intravenous infusion induced a dose-dependent therapeutic effect. 0.4 mg/kg was defined as the Optimum Biological Dose, with a long-lasting clinical improvement observed in this group. This treatment reduced the number of tender and swollen joints starting at day 4 of the infusions. Adverse events were dose-depending but controlled by medications. It was not observed deep lymphopenia neither opportunistic infections. This is the first clinical report supporting the relevance of the CD6/CD6-ligand model as a potential target for rheumatoid arthritis immunotherapy. A PI-IICT with a humanized version for iort1 mAb is underway.

Identification of residues in CD6 which are critical for ligand binding.

CD6 is a member of the scavenger receptor cysteine rich protein superfamily (SRCRSF). This family includes many cell surface proteins whose three-dimensional structures and functions are presently not well understood. The extracellular region of CD6 includes 3 SRCR domains. The membrane proximal SRCR domain specifically binds the activated leukocyte cell adhesion molecule (ALCAM), a CD6 ligand belonging to the immunoglobulin superfamily. CD6-ALCAM interactions mediate immune cell adhesion and are implicated in T cell maturation and the regulation of T cell function. On the basis of SRCRSF sequence comparison, a mutagenesis analysis of the membrane proximal SRCR domain of CD6 (CD6D3) has been carried out. Fifteen mutants were characterized. Three CD6 residues were identified in a region of low sequence conservation which, when mutated, abolish ligand binding but not the binding to a panel of conformationally sensitive anti-CD6 mAbs. This study provides the first analysis of residues critical for ligand binding to a member of the SRCRSF.

Evidence for Protein Tyrosine Kinase Involvement in CD6-Induced T Cell Proliferation

Several studies have demonstrated that addition of soluble anti-CD6 mAbs to 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated naive T cells can induce cell proliferation. We showed in the present study that cell proliferation in TPA-treated T cell cultures can be enhanced several fold when the anti-CD6 mAbs are either immobilized or crosslinked with rabbit anti-mouse immunoglobulins (RAM Ig). Using a src family protein tyrosine kinase (PTK) inhibitor, herbimycin A, the cell proliferation induced by the anti-CD6 mAb, IOR-T1, in TPA-treated T cells were effectively abolished. Analysis of the cellular proteins in these cells after crosslinking the CD6 receptor with IOR-T1 (followed by RAM Ig) in the presence of TPA resulted in an increased level of tyrosine phosphorylation. Pretreatment of naive T cells with herbimycin A (0.5 and 1 μg/ml) for 18 hr completely inhibited the tyrosine phosphorylation on cellular substrates in T cell cultures stimulated with IOR-T1/RAM Ig and TPA. Similar concentrations of herbimycin A also inhibited the increase in IL-2 mRNA expression and cell proliferation in T cell cultures after IOR-T1/RAM Ig and TPA treatment. Furthermore, the increase in cytosolic free Ca2+concentration in naive T cells after crosslinking of the CD6 receptor with IOR-T1/RAM Ig was also inhibited by herbimycin A. Taken together, our results suggest that CD6-mediated T cell proliferation is IL-2 dependent, and involves tyrosine kinase activity which is strictly dependent on protein kinase C activation.

Characterization of a CD6 Ligand(s) Expressed on Human- and Murine-Derived Cell Lines and Murine Lymphoid Tissues

CD6, a type I cell surface glycoprotein expressed predominantly by thymocytes and mature T lymphocytes, becomes phosphorylated on tyrosine residues following T cell activation and has been implicated as an accessory molecule in T cell activation. The purpose of this study was to identify cell lines and tissues which express CD6 ligand(s), determine the requirements for CD6 binding, and biochemically characterize the putative CD6 ligand(s). Binding studies with a CD6 immunoglobulin fusion protein, CD6-Rg, allowed the identification of a number of human cell lines which express a CD6 ligand(s). The binding to these cell lines was trypsin sensitive, in part required divalent cations, was blocked by an anti-CD6 mAb, and could be downregulated by tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) and interferon-γ (IFN-γ). Among the cell lines tested, the human breast carcinoma-derived cell line HBL-100 expressed the highest levels of CD6 ligand(s) and was used for immunoprecipitation studies. Following metabolic labeling, CD6-Rg immunoprecipitated glycoproteins of ∼100, ∼90, and ∼45 kDa from HBL-100 cells. Using CD6-Rg we were able to show that murine thymus, lymph nodes, and skin express high levels of CD6 ligand(s) and that CD6-Rg bound to a murine thymic epithelial cell line and to cultured human epidermal keratinocytes.

The Anti-CD6 mAb, IOR-T1, Defined a New Epitope on the Human CD6 Molecule That Induces Greater Responsiveness in T Cell Receptor/CD3-Mediated T Cell Proliferation

The T lymphocyte cell surface molecule, CD6, has been shown in a number of studies to play an important role in T cell activation. Its physiological ligand or function is still unknown. A panel of five anti-CD6 mAbs was used in the present study to investigate the structure-function relationship of this molecule. Cross-blocking assays indicate that three different epitopes were defined on the CD6 molecule by these mAbs. One of these epitopes defined by the mAb, IOR-T1, is insensitive to thiol-reducing agents, such as dithiothreitol and 2-mercaptoethanol. Of the other two epitopes, one was defined by 2H1 and the other was shared by three other mAbs, T12, 6D3, and Dako-CD6. All the CD6 mAbs at optimal concentration exhibit equal potency in enhancing T cell proliferation mediated through the T cell receptor/CD3 complex by optimal concentration of the anti-CD3 mAb, OKT3 (100 ng/ml). Simultaneous cross-linking of both the anti-CD6 mAbs and OKT3 is essential for the synergistic effect. When suboptimal concentrations of OKT3 (1 ng/ml) were used (no detectable cell proliferation), the synergistic effect of the anti-CD6 mAbs was still evident but with a differential effect. The epitope defined by IOR-TI consistently induced greater T cell responsiveness under these conditions. Our results suggest that the CD6 molecule may play an important role in T cell activation, and that signals through an epitope of stable conformation appear to be of importance when antigen levels are low or interacting with low-avidity antigen receptors.

Activation of human T cells through CD6: functional effects of a novel anti-CD6 monoclonal antibody and definition of four epitopes of the CD6 glycoprotein.

The CD6 glycoprotein is expressed primarily on lymphocytes and conveys co-activating signals to T cells, but its exact function and ligand(s) are unknown. A novel mAb, termed UMCD6, was demonstrated to recognize CD6 by immunoprecipitation, Western blotting, and reactivity with COS cells transfected with CD6 cDNA. UMCD6 was mitogenic for T cells and was strongly synergistic with phorbol ester in inducing T cell activation. UMCD6 enhanced the autologous mixed lymphocyte reaction as previously observed with another anti-CD6 mAb, anti-T12. The activating effects of UMCD6 were more striking than those of other anti-CD6 mAbs and encompassed all of the diverse stimulatory properties previously reported for other anti-CD6 reagents. However, neither UMCD6 nor other anti-CD6 antibodies alone or in combination with phorbol ester or IL-2 were able to induce thymocytes to proliferate. Stimulation by UMCD6 is dependent on accessory cell function in a manner not accounted for simply by antibody cross-linking. UMCD6 did not induce an increase in cytoplasmic free Ca2+, but the CD6 activation pathway appears to involve protein kinase C. UMCD6 and a panel of seven other anti-CD6 mAbs were used in a series of experiments to define four discrete epitopes of CD6 using the criteria of antibody cross-blocking, reactivity on reduced Western blots, and resistance to controlled V8 protease digestion. The functional mAbs UMCD6, 2H1, and anti-T12 each recognized a different epitope. Taken together, the results of these studies strongly reinforce the hypothesis that CD6 plays a significant and distinct role in T cell activation, and suggest that multiple regions of CD6 may be functionally active.

Phosphorylation-dephosphorylation of the CD6 glycoprotein renders two isoforms of 130 and 105 kilodaltons. Effect of serum and protein kinase C activators.

The molecular nature of the structural changes on the T cell-CD6 glycoprotein upon cell activation has been investigated. Cell surface 125I labeling and immunoprecipitation studies from PBMC revealed that after stimulation by different activators of protein kinase C, or after exposure to either human or FCS, the anti-CD6 mAb precipitated an additional protein of 130 kDa, together with the 105-kDa protein present in resting cells. Cell surface expression of this 130-kDa CD6 protein form could be detected as early as 15 min after PKC activation, without requiring de novo protein synthesis. Pulse and chase activation experiments of radioiodinated cells suggested that the 130-kDa molecule is the result of a posttranslational modification of the 105-kDa protein and that this conversion is a reversible process. Studies of 32P-cell labeling and immunoprecipitation by anti-CD6 mAb revealed that only the 130-kDa form was phosphorylated, whereas the 105-kDa protein was unphosphorylated both in resting and activated cells. Moreover, the removal of phosphate groups from the 130-kDa CD6-form by enzymatic treatment with alkaline phosphatase resulted in its conversion to the 105-kDa form. Taken together, these results demonstrate the existence of two CD6 molecular forms that are in a dynamic equilibrium and differ only at their degree of phosphorylation: a 105-kDa unphosphorylated form present in resting T cells that changes very rapidly to a 130-kDa phosphorylated form by exposure of cells either to serum or to activators of PKC.