Targeting CTLA-4 Research Articles

Optimal approach for melanoma

The incidence of cutaneous melanoma is rising faster than that of any other malignancy, and in some parts of Europe it is now the commonest cancer outside of “the big four” common malignancies: i.e. breast, lung, colorectal and prostate cancers. There is little doubt that the major factors in the development of melanoma are skin type, racial origin and sun exposure. Short sharp bursts of sunlight leading to sunburn are dangerous, especially when they occur in children and adolescents. Ten percent of melanomas are familial in origin, and as with other cancers, the biology associated with such tumours has helped us to develop an understanding of the molecular genetics of sporadic melanoma. A number of mutations have been described, including those found in CDKN2A, CDK4, RB1, p14ARF, NRAS and particularly BRAF. BRAF mutations are found in approximately 50% of patients with cutaneous melanoma, and the development of targeted agents against mutations in BRAF has been responsible for one of the most dramatic examples of molecular medicine in oncology. Adjuvant therapy for patients at high risk of relapse following treatment for primary melanoma or locoregional disease remains an area of uncertainty. The use of adjuvant interferon, at various doses and schedules, has been the subject of many randomised trials over 25 years. It is of note that some large trial groups such as those in Europe still feel that there is enough uncertainty as to the efficacy of interferon that randomised trials of adjuvant therapy should still be performed with a no-treatment control arm. The one indisputable fact about adjuvant interferon is that it is associated with a relapse-free survival benefit, but some argue that, unlike treatment in the metastatic setting, the purpose of adjuvant therapy is to improve overall survival. A number of meta-analyses of the randomised trials involving interferon have been published, and it appears that the maximum absolute benefit to overall survival is in the order of 2–3% and again, some argue that this is below the threshold of useful clinical utility. Randomised trials of the newer melanoma therapies are now being brought into the adjuvant arena. Vemurafenib is a BRAF inhibitor, and the results of the first randomised trial of vemurafenib against standard of care – namely, dacarbazine (DTIC) – were dramatic in terms of response rates, progression-free survival and overall survival. The hazard ratio for overall survival at the time of the first analysis was unprecedented in solid tumour oncology. The therapeutic momentum has continued with the development of MEK inhibitors and their combination with BRAF inhibitors. Other targeted therapies are being developed for uveal and acral melanomas, e.g. against c-KIT mutations in the latter. The most important recent development in immunotherapy has been the targeting of inhibitors of the immune system, e.g. CTLA-4, PD-1 and its ligand PD-L1. Ipilimumab targets CTLA-4 and is the first immunotherapy to have shown an overall survival benefit in melanoma within the context of a randomised trial. The magnitude of benefit can be very great in some patients with prolonged complete remissions; however, it is only a minority of patients that benefit. Early results targeting PD-1 and PD-L1 are particularly exciting because they appear to challenge the dogma that immunotherapy only impacts a minority of patients. Early results suggest that the majority of patients show some benefit without necessarily achieving a complete remission. The new immunotherapeutic landscape means that our previous view of follow-up needs to change rapidly. We now know that there is an important immunotherapy that is associated with a survival advantage, but that, as with most immunotherapies, it can take some time before the host response becomes effective. This time-frame may be 2–4 months, and therefore it is completely illogical to wait for a patient to become symptomatic from their metastatic disease before investigating and treating them. Patients must have their metastatic disease diagnosed early, otherwise there is little prospect of a successful outcome to immunotherapy, and therefore patients at high risk of relapse need regular imaging, and treatment should be instituted before high volumes of disease are seen.

Immunomodulatory Monoclonal Antibodies Combined with Peptide Vaccination Provide Potent Immunotherapy in an Aggressive Murine Neuroblastoma Model

Neuroblastoma is one of the commonest extracranial tumors of childhood. The majority of patients present with metastatic disease for which outcome remains poor. Immunotherapy is an attractive therapeutic approach for this disease, and a number of neuroblastoma tumor antigens have been identified. Here, we examine the therapeutic potential of combining immunomodulatory monoclonal antibodies (mAb) with peptide vaccination in murine neuroblastoma models. Neuroblastoma-bearing mice were treated with mAb targeting 4-1BB, CD40, and CTLA-4 alone, or in combination with a peptide derived from the tumor antigen survivin (GWEDPPNDI). Survivin-specific immune response and therapeutic efficacy were assessed. In the Neuro2a model, treatment of established tumor with anti-4-1BB, anti-CD40, or anti-CTLA-4 mAb results in tumor regression and long-term survival in 40% to 60% of mice. This is dependent on natural killer (NK) and CD8(+) T cells and is associated with tumor CD8(+) lymphocyte infiltrate. Successful therapy is achieved only if mAb is given to mice once tumors are established, suggesting dependence on sufficient tumor to provide antigen. In the more aggressive AgN2a and NXS2 models, single-agent mAb therapy provides ineffective therapy. However, if mAb (anti-CTLA-4) is given in conjunction with survivin peptide vaccination, then 60% long-term survival is achieved. This is associated with the generation of survivin-specific T-cell immunity, which again is only shown in the presence of tumor antigen. These data suggest that the combination of antigen and costimulatory mAb may provide effective immunotherapy against neuroblastoma and may be of particular use in the minimal residual disease setting.

Blood mRNA signature to predict survival in patients with metastatic melanoma treated with tremelimumab.

9080 Background: Tremelimumab (Ticilimumumab, Pfizer), a monoclonal antibody targeting CTLA-4, a T cell inhibitory molecule, has shown activity in metastatic melanoma. Ipilimumab (Yervoy, BMS), another antibody targettingCTLA-4, improves survival relative to a peptide vaccine and is now FDA approved. A minority of patients will achieve durable tumor control with CTLA-4 blockade and biomarkers are urgently needed to identify those patients. Methods: 170 inflammatory, melanoma-specific and CTLA4-pathway related mRNA transcripts were measured using RT-PCR in pre-treatment peripheral blood samples from 218 patients with refractory melanoma receiving tremelimumab in a multi-center phase II study. A 2-class latent model yielded a risk score based on 4-genes that was highly predictive of survival (p<0.001), and was used to categorize patients into low, medium and high-risk groups. An independent cohort of 260 treatment naïve melanoma patients receiving tremelimumab as part of a multi-center phase III study was then used to validate the risk score as well as the 3 risk groups defined using the pre-specified cut-points. Results: There was no significant difference between the two cohorts in terms of age, gender, stage of disease or ECOG status. Median time of follow up was 297 days for the training cohort and 386 days for the validation cohort. 67% of patients in the training cohort and 70% of patients in the validation died during time of follow-up. Collectively, the ability of the 170 genes to predict survival exhibited a high degree of consistency across the cohorts (p < 0.001). A 4-gene model including cathepsin D (CTSD), Phopholipase A2 group VII (PLA2G7), Thioredoxin reductase 1 (TXNRD-1) and Interleukin 1 receptor associated kinase 3 (IRAK3) predicted survival in the validation cohort (p=0.001 by log rank test). Multivariable cox analysis showed that the 4-gene model added to the predictive value of clinical predictors (p<0.0001). Conclusions: Expression levels of CTSD, PLA2G7, TXNRD1, and IRAK3 in peripheral blood are predictive of survival in melanoma patients treated with ticilimumab (αCTLA-4). Blood mRNA signatures should be further explored to define patient subsets likely to benefit from immunotherapy.

The role of miR-155 in regulatory T cells and rheumatoid arthritis

Recently, various micro(mi)RNAs have been found deregulated in the setting of rheumatoid arthritis (RA), but their role in the pathogenesis of this disease remains a matter of debate. In the meanwhile, increasing evidence indicates a defective function of regulatory T cells (Tregs) in RA. This review discusses relevant studies addressing the function of Tregs and Cytotoxic T-Lymphocyte Antigen 4 in RA, provides recent data on the role of miRNAs for Tregs homeostasis, and focuses on the role of miR-155 in Tregs. In a final perspective section we discuss the potential impact of therapeutic miR-155 modulation in RA.

Intracellular CTLA4 and Regulatory T Cells in Patients with Laryngeal Squamous Cell Carcinoma

To explore if the increased percentages of Regulatory T (Treg) cells, as well as, overexpression of Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) are involved in laryngeal-squamous cell carcinoma (SCC), 45 patients with laryngeal-SCC and 27 healthy controls were enrolled. Flow cytometry was performed to investigate, in the peripheral blood, the prevalence of CD4+CD25+FoxP3+ Treg cells, as well as, surface and intracellular expression of CTLA-4 by the main lymphocyte subsets (CD4+, CD8+ and CD19+). The results indicated intracellular (In)CTLA4 with considerable higher expression in the CD8+ lymphocytes among patients with laryngeal-SCC compared with the control group (8.2 ± 8.7 versus 2.3 ± 3.5, P = 0.001). The mean percentage of InCTLA4+CD4+ and InCTLA4+CD19+ lymphocytes was also significantly higher in patients (8.7 ± 7.8 versus 4.4 ± 4.2, P = 0.018 and 0.6 ± 0.8 versus 0.2 ± 0.2, P = 0.024, respectively). With respect to surface (Sur)CTLA4, the difference between patients and controls was, however, significant only in the case of CD8+ lymphocytes (0.7 ± 0.6 versus 0.3 ± 0.3, P = 0.003, respectively). The percentage of Treg cells was observed to be significantly higher in patients (7.5 ± 6.3 and 3.2 ± 1.9, P < 0.0001). Furthermore, association analysis revealed the association of Treg cell increase with the higher tumor-size and lymphnode stage (P < 0.005). These data collectively suggest that patients with laryngeal-SCC may benefit from immunotherapy targeting CTLA4 and Treg cells.

Impact of Treatment On CD4+CTLA-4+ T Cell Subset in Brazilian Patients with Classical Hodgkin Lymphoma

AbstractAbstract 4778Cytotoxic T lymphocyte antigen-4 (CTLA-4) is one of the basic antigens involved in immune responses regulation associated with autoimmune diseases and cancer. Its key role in regulating the immune system has made CTLA-4 an attractive target for cancer. Augmentation of the immune response via blockade of CTLA-4 has shown an improvement in survival for patients with metastatic melanoma, which prompted the Food and Drug Administration (FDA) approval of the CTLA-4 function blocking antibody Ipilimumab for this disease. Objective:The aim of the study was to evaluate the surface expression of CTLA-4 on CD4+ T cells in peripheral blood mononuclear cells (PBMC) of patients with classical Hodgkin lymphoma (cHL) at diagnosis and post-treatment and correlate these findings with clinical and epidemiological aspects. Material and Methods:This is an open study and, so far, we included 35 patients from December 2009 to December 2011. Blood was drawn at diagnosis and post-treatment (1 to 4 months after completion of therapy). The T cell phenotype was evaluated by flow cytometry using CD3, CD4, CD8, CTLA-4 and correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. Eighteen healthy blood donors volunteers were recruited as controls. In this study, only cHL patients whose histology could be confirmed and Epstein-Barr (EBV) association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. Three patients relapsed, and blood was also drawn at this time. Results:From the 35 cHL patients, 17 were EBV related and 18 EBV non-related. The percentage of CD4+ T cells with CTLA-4 surface expression was significantly increased in patients with cHL at diagnosis compared with healthy controls (median 7.36 vs 2.73; P<0.001). Additionally, CD4+CTLA-4+ T lymphocytes significantly decreased following treatment and complete response (7.36 vs 4.53; p=0.008), with values similar to healthy controls (4.53 vs 2.73; p=0.07). Interestingly, CD4+CTLA-4+ T lymphocytes on relapse were significantly different from post-treatment values and similar to pre treatment. There was no difference on CD4+CTLA-4+ T lymphocytes in the EBV related and non-related cHL patients. Regarding patient’s baseline characteristics, CD4+CTLA-4+ T lymphocytes strongly correlated with erythrocyte sedimentation rate (ESR) values (r=0.67; p=0.002). Conclusions:We showed that CD4+CTLA-4+ T lymphocytes are increased in Brazilian cHL patients at diagnosis compared with post-treatment values and healthy controls. These results suggest a role of CTLA-4 on Hodgkin lymphomagenesis, possibly negatively regulating host anti-tumor immune response. The promising immunotherapy regimen targeting CTLA-4 might be beneficial in classical Hodgkin lymphoma. Disclosures:No relevant conflicts of interest to declare.

Phase I trial of tremelimumab in combination with short-term androgen deprivation in patients with PSA-recurrent prostate cancer

CTLA-4 blockade has demonstrated antitumor efficacy in human clinical trials. The antitumor mechanism is presumably mediated in part by the expansion of tumor-specific T cells. Androgen deprivation, the cornerstone of treatment for patients with metastatic prostate cancer, has been shown to elicit prostate tissue apoptosis and lymphocytic inflammation. We hypothesized that treatment with androgen deprivation, followed by an anti-CTLA-4 antibody, could augment a tumor-specific immune response elicited by androgen deprivation. We report here the results of a phase I trial evaluating a humanized monoclonal antibody targeting CTLA-4, CP-675,206 (tremelimumab), in combination with androgen deprivation using an antiandrogen. Eligible patients were those with PSA-recurrent prostate cancer after primary surgery and/or radiation therapy, not previously treated with androgen deprivation, and without radiographic evidence of metastatic disease. Subjects were treated in two cycles, 3 months apart, in which they received bicalutamide 150 mg daily days 1-28 and tremelimumab on day 29. The primary endpoint of the trial was safety. Secondary endpoints included measures of PSA kinetics and identification of a maximum tolerated dose. Eleven patients were enrolled and completed at least 1 year of follow-up. Dose-limiting toxicities included grade 3 diarrhea and skin rash. No favorable changes in PSA doubling time were observed in a period shortly after completing treatment; however, three patients experienced a prolongation in PSA doubling time detectable several months after completing treatment. The identification of delayed, prolonged favorable changes in serum PSA suggests that future studies could explore this combination in studies evaluating time to disease progression.

Anti-CTLA-4 Antibody Is Not Able to Trigger Apoptosis in CTLA-4 Expressing Myeloid Tumor Cells

Abstract 5054Cytoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a co-inhibitory molecule normally expressed on activated effector T cells and a subset of regulatory T cells. However, it has been reported also to be expressed on acute myeloid leukemia (AML) cells, CD34+ hematopoietic progenitor cells after stimulation as well as several solid tumor cell types. Furthermore, CTLA-4 engagement with recombinant CD80 and CD86 ligands has been shown to induce apoptosis in AML cells (Laurent et al. Br J Haematol 2007). In this study, we investigated CTLA-4 expression on different cell populations in bone marrow aspirates of myelodysplastic syndrome (MDS) patients, and explored the possibility of targeting CTLA-4 for apoptosis induction using the CTLA-4 antibody tremelimumab. Flow cytometry analysis showed CTLA-4 surface expression on immature CD34+, CD117+ and CD33+ myeloid cells, as well as CD14+ monocytic cells from MDS patients (n=11). In addition, CTLA-4 was expressed by both CD4+ and CD8+ T cells in bone marrow of both low and high risk MDS patients. In order to address whether anti-CTLA-4 antibody is able to induce apoptosis in myeloid leukemic progenitor cells, we incubated the CTLA-4-expressing myeloid cell lines HL-60 and KG1a in vitro for various time points (24h, 48h, 72h) with 25 microgram/ml tremelimumab. However, anti-CTLA-4 antibody alone as well as cross linking with a secondary antibody was unable to induce apoptosis, while serum-free conditions and irradiation induced high numbers of Annexin V and/or 7AAD positive cells. These data indicate that the anti-CTLA-4 antibody tremelimumab is unable to induce an apoptotic signal into CTLA-4-expressing myeloid tumor cells. Disclosures:No relevant conflicts of interest to declare.

Targeting ctla-4 directly on melanoma cells: A possible novel perspective in the immunotherapy of cutaneous melanoma

e22138 Background: CTLA-4 (CD152) is an omodimeric membrane glycoprotein which behaves as the major negative regulator of T cell-mediated immune response. CTLA-4 inhibitory function in T cells mainly occurs upon engagement with the B7 ligands expressed on antigen presenting cells, resulting in inhibition of cytokine production and T cell proliferation. CTLA-4 expression has also been documented in established human melanoma cell lines and primary melanoma tumours. The aim of this study was to investigate the expression and functional role of CTLA-4 in primary melanoma cultures derived from biopsies of metastatic melanoma patients. Methods: Seven primary cultures were derived from melanoma samples by mechanical tissue dissociation, enzymatic digestion and filtration of single cell suspensions. Flow cytometry (FACS) with an anti-melanoma (MCSP) mAb was used to assess the growth of melanoma cells. CTLA-4 expression analysis was performed by FACS and real-time PCR. Cytokine secretion and apoptotic cells were evaluated by Elisa and Annexin V/PI staining after treatment with an agonistic anti-CTLA-4 mAb (3D5). The angiogenic property of melanoma culture supernatants was evaluated on mesenchimal stromal cells (msc) isolated from human thoracic aortas. All the study was approved by our Institutional Ethics Committee and patients gave written informed consent. Results: Constitutive CTLA-4 expression was found on all melanoma cultures with variable intensity at both the protein and transcript levels. CTLA-4 expressed by these cells is functional as its ligation, with an anti-CTLA-4 agonistic mAb, is able to induce inhibition of cell proliferation, due to apoptosis induction, and of IL-8, TNF-alpha and VEGF cytokine secretion. Moreover, CTLA-4 ligation down-modulates the ability of melanoma culture supernatants to induce angiogenic differentiation of msc as detected by the acquisition of their endothelial phenotype. Conclusions: Given the physiologic inhibitory function of CTLA-4, our results suggest its possible role in the functional biology of melanoma and open up the possibility of an anti-melanoma immunotherapy based on targeting CTLA-4 directly on tumour cells as this might allow inhibition of tumour cell growth and angiogenesis. No significant financial relationships to disclose.

Overcoming Immunologic Tolerance to Melanoma: Targeting CTLA-4 with Tremelimumab (CP-675,206)

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade therapies have been evaluated in clinical trials and have shown promise as possible options for treating patients with cancer. One agent under investigation is tremelimumab (CP-675,206), a monoclonal antibody that has been demonstrated to be a safe and efficacious treatment in patients with malignant melanoma. Results of a phase I clinical trial suggested that a dose of 15 mg/kg of tremelimumab would be the maximum-tolerated dose, with the most common grade 3-4 toxicities being diarrhea and rash. Pharmacokinetic studies showed that the postinfusion plasma concentration and area under the plasma disposition curve both increased in an approximately proportional manner with dose. Studies also showed that tremelimumab has a low clearance (0.132 ml/h x kg), a small volume of distribution (81.2 ml/kg), and a long terminal-phase half-life (22.1 days). A pivotal phase II clinical trial assessing single-agent tremelimumab as second-line therapy in metastatic melanoma has completed accrual, with response rate as the primary endpoint. A pivotal phase III trial has also completed accrual; that study compared the overall survival of previously untreated patients receiving single-agent tremelimumab versus dacarbazine or temozolomide.

Overcoming Immunologic Tolerance to Melanoma: Targeting CTLA-4 with Ipilimumab (MDX-010)

Targeted biologic therapies such as anti-cytotoxic T lymphocyte antigen (CTLA-4) monoclonal antibodies, either as monotherapy or in combination with chemotherapy or vaccines, have shown great promise in late-stage melanoma, which has a very poor prognosis. Melanoma is relatively resistant to both chemotherapy and radiotherapy. Blockade of CTLA-4, which inhibits T-cell proliferation, promotes stimulation of adaptive immunity and T-cell activation, resulting in eradication of tumor cells. Two human monoclonal antibodies are under investigation in melanoma. Phase II and III clinical trials are currently evaluating the efficacy and safety of ipilimumab (MDX-010, Medarex, Inc., Princeton, NJ, and Bristol-Myers Squibb, Princeton, NJ) and tremelimumab (CP-675,206; Pfizer Pharmaceuticals, New York) in melanoma. Data are available on ipilimumab, which has been explored as monotherapy and in combination with vaccines, other immunotherapies such as interleukin-2, and chemotherapies such as dacarbazine. Overall response rates range from 13% with ipilimumab plus vaccine in patients with stage IV disease to 17% and 22% with ipilimumab plus dacarbazine or interleukin-2, respectively, in patients with metastatic disease. Responses have been durable, and among those experiencing grade 3 or 4 autoimmune toxicities, even higher response rates have been seen--up to 36%. While the optimal dose of ipilimumab has yet to be established, studies also indicate that higher doses may be more effective. Importantly, the lack of an initial clinical response may not predict ultimate treatment failure, because the onset of a response may follow progressive disease or stable disease. Pending results from registration studies with ipilimumab and lessons learned from registration studies conducted with tremelimumab will help to define the role of anti-CTLA-4 blockade in the treatment of metastatic melanoma.

Clinical Trial of Therapeutic Blockade of CTLA4 with Ipilimumab in Patients with Relapse of Malignancy Following Allogeneic Hematopoietic Transplantation.

Relapse/progression of malignancy (RM) is an important cause of treatment failure and death following allogeneic hematopoietic stem cell transplantation (allo-HCT). CTLA-4 is an important negative regulator of effector T-cell activation. CTLA-4 inhibition has demonstrated potent anti-cancer effects in animal models, and in patients with some solid tumors. CTLA-4 blockade following allo-HCT may potentially augment graft-versus-malignancy but GVHD may also possibly be increased. We report the safety and preliminary efficacy of a dose-escalation study of a neutralizing human monoclonal antibody targeting CTLA-4 (ipilimumab) in patients with RM following allo-HCT. Eligibility criteria included allo-HCT ≥90 days previously, > 50% donor T-cell chimerism, no prior grade 3/4 GVHD, no prophylaxis/therapy for GVHD for ≥ 6 weeks. Patients received a single dose of ipilimumab over 90 min. DLI at a dose of 5 × 10e6 CD3 cells/kg was allowed 8 weeks following ipilimumab if no GVHD occurred and RM was present. A total of 29 patients were treated at four centers (23M, 6F; median age 43 (21–65); Hodgkins disease [HD] =14 Myeloma [MM]=6, CML=2, CLL=2, AML=2, NHL=1, Renal Ca =1, Breast Ca=1; 19 related donors, 10 unrelated; 6 myeloablative, 23 RICT).(4 at dose-level 1 [DL1] 0.1 mg/kg, 3 at 0.33 mg/kg [DL2], 4 at 0.66 mg/kg [DL3], 3 at 1.0 mg/kg [DL4] and 15 at 3.0 mg/kg [DL5]). Eight patients had failed prior DLI. Median time between BMT and ipilimumab was 366 d (125–2368). Dose-limiting toxicity was not encountered. No patient developed clinically significant GVHD within 90 days following ipilimumab alone. Three possible immune adverse events were documented: grade 3 polyarthropathy 14 weeks following ipilimumab, and 6 weeks post DLI, which resolved with corticosteroid therapy, (AML, DL1); grade 1 hyperthyroidism with thyroid-stimulating antibody 6 weeks post ipilimumab (CLL, DL3). Grade 2 pneumonitis responsive to corticosteroids (HD, DL5). Ten patients received DLI after ipilimumab. Three patients developed objective evidence of disease response after ipilimumab alone: PR in a patient with mantle cell NHL lasting 3m [DL4]; ongoing CR in a two patients with HD [DL5]. Two of these patients had failed prior DLI. Three additional patients demonstrated possible anti-cancer effects (MR in a HD patient, reduction of PB and BM blasts in AML, DL1; maintenance of molecular remission in a CML patient given ipilimumab alone for 3.5 yrs despite stopping imatinib, DL1). This study shows that doses of ipilimumab (up to 3.0 mg/kg) can safely be administered to patients with RM following allo-HCT without inducing/exacerbating GVHD while inducing regressions of malignancy including durable CR in some patients.

Molecular Determinants of Inverse Agonist Activity of Biologicals Targeting CTLA-4

Ligation of CD28 or CTLA-4 with some biologicals can activate T cells due to an unexpected superagonist or inverse agonist activity, respectively. The risk of such an outcome limits the therapeutic development of these reagents. Thus, identifying the molecular determinants of superagonist/inverse agonist properties for biologicals targeting costimulatory/inhibitory receptors has not only fundamental value but also important therapeutic implications. In this study, we show that ligation of CTLA-4 with either soluble B7.1 Ig (but not B7.2 Ig) or with a recombinant bispecific in-tandem single chain Fv known as 24:26 induces TCR-independent, T cell activation. Such an inverse agonist activity requires CD28 expression and high CTLA-4 expression and is not seen when CTLA-4 is ligated by membrane-bound B7.1 or B7.2. At the molecular level, the inverse agonist activity of B7.1 Ig or 24:26 correlates with their ability to induce the formation of unique dimer-based, CTLA-4 oligomers on the T cell surface and involves CTLA-4 signaling through its cytoplasmic domain. Our results provide a potential mechanism to explain and to predict inverse agonist activity for CTLA-4 ligands.

183 Targeting CTLA-4 by its recombinant ligands CD80/CD86: Perspectives towards the development of a novel anti-tumor therapeutic approach

183 Targeting CTLA-4 by its recombinant ligands CD80/CD86: Perspectives towards the development of a novel anti-tumor therapeutic approach

TCR signal quality determines trafficking of CTLA-4 to immunological synapse

CD28 and its homolog CTLA-4 share the ability to bind B7 family members and generate crucial regulatory signals for T-cell activation. However, CD28 is expressed on the T-cell surface and enhances T-cell activation, whereas CTLA-4 is not detectable at the protein level in naive cells. Instead, it is upregulated upon T-cell activation and functions to inhibit T-cell proliferation. Furthermore, an intracellular localization motif targets CTLA-4 to the endosomal compartment. The balance between these activating and inhibitory signals can determine the outcome of a T cell's interaction with an antigen-presenting cell (APC). A recent paper has now investigated the trafficking characteristics of CTLA-4 and CD28 to gain insight into how these molecules exert their regulatory function on T-cell activation.

Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model.

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies' growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.

Interaction of CTLA-4 with the clathrin-associated protein AP50 results in ligand-independent endocytosis that limits cell surface expression.

CTLA-4 is a lymphocyte cell surface receptor expressed by activated T cells that functions to down-regulate T cell responses induced by TCR and CD28 stimulation. Since CTLA-4 competes with CD28 for binding to the common ligands B7-1 and B7-2, the level of CTLA-4 surface expression is likely to play an important role in its ability to inhibit CD28-dependent T cell activation. The factors that regulate these levels are poorly understood. Recent studies have revealed that following T cell activation, the majority of CTLA-4 is localized intracellularly rather than on the cell surface, and surface CTLA-4 is rapidly reinternalized. In this study, we investigate the molecular mechanism underlying the rapid clearance of CTLA-4 from the cell surface. The data demonstrate that cell surface CTLA-4 is endocytosed into clathrin-coated vesicles even in the absence of ligand. The targeting of CTLA-4 to clathrin-coated vesicles is mediated by the clathrin-associated adaptor complex AP-2. The cytoplasmic domain of CTLA-4 was found to specifically bind to AP50, the medium chain subunit of AP-2 in both yeast two-hybrid and coimmunoprecipitation assays. The interaction requires the peptide sequence 199-GVYVKM-204 in the cytoplasmic tail of CTLA-4. Mutation of the CTLA-4 amino acid residue Y201 abrogates the interaction with AP50, resulting in the accumulation of CTLA-4 at the cell surface. Together these data suggest that the interaction of CTLA-4 with AP50 plays an important role in regulating the cell surface expression of CTLA-4.