CD2 Subset Research Articles

Advances in Understanding the Roles of CD244 (SLAMF4) in Immune Regulation and Associated Diseases.

Proteins in the signaling lymphocytic activating molecule (SLAM) family play crucial roles in regulating the immune system. CD244 (SLAMF4) is a protein in this family, and is also a member of the CD2 subset of the immunoglobulin (Ig) superfamily. CD244 is a cell surface protein expressed by NK cells, T cells, monocytes, eosinophils, myeloid-derived suppressor cells, and dendritic cells. CD244 binds to the ligand CD48 on adjacent cells and transmits stimulatory or inhibitory signals that regulate immune function. In-depth studies reported that CD244 functions in many immune-related diseases, such as autoimmune diseases, infectious diseases, and cancers, and its action is essential for the onset and progression of these diseases. The discovery of these essential roles of CD244 suggests it has potential as a prognostic indicator or therapeutic target. This review describes the molecular structure and function of CD244 and its roles in various immune cells and immune-related diseases.

Expression of T-Bet, Eomesodermin, and GATA-3 Correlates With Distinct Phenotypes and Functional Properties in Porcine γδ T Cells.

Unlike mice and humans, porcine γδ T cells represent a prominent subset of T cells in blood and secondary lymphatic organs. GATA-3, T-bet and Eomesodermin (Eomes) are transcription factors with crucial functions in T-cell development and functional differentiation, but their expression has not been investigated in porcine γδ T cells so far. We analyzed the expression of these transcription factors in γδ thymocytes, mature γδ T cells from blood, spleen, lymph nodes, and lung tissue as well as in vitro stimulated γδ T cells on the protein level by flow cytometry. GATA-3 was present in more than 80% of all γδ-thymocytes. Extra-thymic CD2− γδ T cells expressed high levels of GATA-3 in all investigated organs and had a CD8α−/dimCD27+perforin− phenotype. T-bet expression was mainly found in a subset of CD2+ γδ T cells with an opposing CD8αhighCD27dim/−perforin+ phenotype. Eomes+ γδ T cells were also found within CD2+ γδ T cells but were heterogeneous in regard to expression of CD8α, CD27, and perforin. Eomes+ γδ T cells frequently co-expressed T-bet and dominated in the spleen. During aging, CD2−GATA-3+ γδ T cells strongly prevailed in young pigs up to an age of about 2 years but declined in older animals where CD2+T-bet+ γδ T cells became more prominent. Despite high GATA-3 expression levels, IL-4 production could not be found in γδ T cells by intracellular cytokine staining. Experiments with sorted and ConA + IL-2 + IL-12 + IL-18-stimulated CD2− γδ T cells showed that proliferating cells start expressing CD2 and T-bet, produce IFN-γ, but retain GATA-3 expression. In summary, our data suggest a role for GATA-3 in the development of γδ-thymocytes and in the function of peripheral CD2−CD8α−/dimCD27+perforin− γδ T cells. In contrast, T-bet expression appears to be restricted to terminal differentiation stages of CD2+ γδ T cells, frequently coinciding with perforin expression. The functional relevance of high GATA-3 expression levels in extra-thymic CD2− γδ T cells awaits further clarification. However, their unique phenotype suggests that they represent a thymus-derived separate lineage of γδ T cells in the pig for which currently no direct counterpart in rodents or humans has been described.

Characterization of leukocyte subsets in buffalo (Bubalus bubalis) with cross-reactive monoclonal antibodies specific for bovine MHC class I and class II molecules and leukocyte differentiation molecules

Although buffaloes (Bubalus bubalis) are a major component of the livestock industry worldwide, limited progress has been made in the study of the mechanisms regulating the immune response to pathogens and parasites affecting their health and productivity. This has been, in part, attributable to the limited availability of reagents to study immune responses in buffalo. As reported here, a set of cross-reactive monoclonal antibodies (mAbs), developed against bovine, ovine and caprine leukocyte differentiation molecules (LDM) and major histocompatibility complex (MHC) molecules, were identified and used to compare expression of LDM in Italian and Egyptian buffalo. The results show most of the epitopes identified with the mAbs are conserved on LDM and MHC I and II molecules in both lineages of buffalo. Comparison of the composition of lymphocyte subsets between buffalo and cattle revealed they are similar except for expression of CD2 and CD8 on workshop cluster one (WC1) positive γδ T cells. In cattle, CD8 is expressed on a subset of CD2+/WC1- γδ T cells that are present in low frequency in blood of young and old animals, whereas, CD8-/CD2-/WC1+ γδ T cells are present in high frequency in young animals, decreasing with age. In the buffalo, CD2 is expressed on a subset of WC1+ γδ T cells and CD8 is expressed on all WC1+ γδ T cells. The availability of this extensive set of mAbs provides opportunities to study the immunopathogenesis of pathogens and parasites affecting the health of buffalo.

Identification and Validation of HLA-A24 XBP1us, XBP1sp, CD138, and CS1 Peptides to Generate Antigens Specific-Cytotoxic T Lymphocytes: Preclinical Basis for Vaccine Therapy in HLA-A24 Patients with Multiple Myeloma and Other Cancers

XBP1 (X-box binding protein 1), CD138 (Syndecan-1), and CS1 (CD2 subset 1, CRACC, SLAMF7, CD319) are highly expressed antigens associated with tumor pathogenesis in multiple myeloma cells and various solid tumor cells. We have previously reported on the use of HLA-A2 peptides specific to these antigens to generate antigen-specific and HLA-2-restricted cytotoxic T cells (CTL) in preclinical studies and phase I/II clinical trials in patients with smoldering MM. These studies extend this concept to HLA-A24 patients. Immunogenic HLA-A24 peptides specific to both the spliced and unspliced isoforms of XBP1, CD138, and CS1 were identified, synthesized, and evaluated in vitro for their ability to generate the antigen-specific cytotoxic T lymphocytes (CTL) targeting tumor cells. The specific epitopes were selected based on their binding affinity to HLA-A24, extended half-time disassociation rates, proteasomal C terminal cleavage, and TAP transport. We report here on novel highly immunogenic HLA-A24-specific peptides XBP1 US185 -193 (I S P W I L A V L), XBP1 SP223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F), and CS1240-248 (L F V L G L F L W), which induce antigen-specific CD3+CD8+ CTL against MM and various solid tumors. Each of the single peptide-specific CTL displayed immune functional activities including IFN-g/IL-2 cytokine production and cytotoxic activity against HLA-A24+ MM cells. Furthermore, our studies demonstrated that a cocktail of the four HLA-A24-specific peptides induced multipeptide-specific CD3+CD8+ CTL with functional anti-tumor properties against myeloma cells. Phenotypically, the multipeptide-specific CTL were central memory (CM; CCR7+CD45RO+/CD3+CD8+), effector memory (EM; CCR7-CD45RO+/CD3+CD8+) and terminal effector (TE; CCR7-CD45RO-/CD3+CD8+) cells expressing high levels of cell activation markers CD69, CD38, and CD40L. The multipeptide-specific effector and memory CTL subsets demonstrated HLA-A24-restricted and peptide-specific anti-tumor activities, and the highest activities were detected within the CM CTL subset. Treatment of the multipeptide-specific CTL or tumor target cells with different types of checkpoint inhibitors (a-PD1, a-TIM3, a-PDL-1, a-Galectin-9) or the immune modulator drug, lenalidomide, enhanced their anti-myeloma activities and antigen-specific CD3+CD8+ T cell proliferation to HLA-A24+ tumor cells. Therefore, a cocktail of the four immunogenic HLA-A24 epitopes provides the framework for development of a cancer vaccine based targeted immunotherapy. In conclusion, we report here on novel immunogenic HLA-A24-specific XBP1 unspliced, XBP1 spliced, CD138, and CS1 peptides that elicit CTL with anti-myeloma activities. These peptides will allow us to extend our immunotherapeutic vaccine approach beyond the current HLA-A2-specific peptides to HLA-A24 patients with smoldering myeloma, multiple myeloma, other plasma cell disorders and potentially with solid tumors. These preclinical studies also demonstrate that incorporation of immunomodulatory agents and/or checkpoint inhibitors can amplify and maintain antigen-specific memory CTL responses against tumor. DisclosuresBae:OncoPep Inc.: Consultancy, Equity Ownership. Raje:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Chauhan:Stemline Therapeutics: Consultancy. Munshi:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Anderson:Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

The comparative profile of lymphoid cells and the T and B cell spectratype of germ-free piglets infected with viruses SIV, PRRSV or PCV2.

Lymphocyte subsets isolated from germ-free piglets experimentally infected with swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) were studied and the profile of these subsets among these three infections was monitored. Germ-free piglets were used since their response could be directly correlated to the viral infection. Because SIV infections are resolved even by colostrum-deprived neonates whereas PRRSV and PCV2 infections are not, SIV was used as a benchmark for an effectively resolved viral infection. PRRSV caused a large increase in the proportion of lymphocytes at the site of infection and rapid differentiation of B cells leading to a high level of Ig-producing cells but a severe reduction in CD2—CD21+ primed B cells. Unlike SIV and PCV2, PRRSV also caused an increase in terminally differentiated subset of CD2+CD8α+ γδ cells and polyclonal expansion of major Vβ families suggesting that non-specific helper T cells drive swift B cell activation. Distinct from infections with SIV and PRRSV, PCV2 infection led to the: (a) prevalence of MHC-II+ T cytotoxic cells, (b) restriction of the T helper compartment in the respiratory tract, (c) generation of a high proportion of FoxP3+ T cells in the blood and (d) selective expansion of IgA and IgE suggesting this virus elicits a mucosal immune response. Our findings suggest that PRRSV and PCV2 may negatively modulate the host immune system by different mechanisms which may explain their persistence.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0091-x) contains supplementary material, which is available to authorized users.

CS1-Directed Monoclonal Antibody Therapy for Multiple Myeloma

Therapeutic monoclonal antibodies (mAbs) have revolutionized treatment options for many cancers. However, the extension of effective mAb-mediated therapy for multiple myeloma (MM) has been limited thus far by lack of an optimal target. Results of experimental agents targeting a number of potential candidate molecules expressed on the surface of MM tumor cells have been reported (including CD20, CD40, CD56, CD74, CD138, CD200, epidermal growth factor receptor, and beta-2 microglobulin), as have those of mAbs targeting other proteins involved in the MM immunologic microenvironment (interleukin-6, vascular endothelial growth factor, and killer immunoglobulin-like inhibitory receptors). Despite these efforts, the benefit of mAb-based therapy directed at these targets in MM remains incompletely articulated. The most promising target for mAb-mediated MM therapy to date seems to be CS1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), a human membrane glycoprotein and member of the immunoglobulin superfamily. CS1 cell surface expression seems nearly ubiquitous in both primary MM tumor cells as well as virtually all MM cell lines. Soluble CS1 is also detectable in serum of patients with MM and correlates with both disease stage and need for therapy. Furthermore, CS1 is expressed in some leukocyte subsets (eg, natural killer [NK] cells and some T-cell subsets) but not in the majority of healthy tissues or other forms of malignancy. Relatively little is known regarding the function of CS1 in MM cells. The CS1 gene resides on the long arm of chromosome 1 (1q23.11q24.1), and gains of chromosome 1q are frequent alterations in MM tumor cells; thus CS1 overexpression may contribute to the pathobiology of the disease. CS1 has been shown to colocalize with CD138 in uropod membranes on the surface of polarized MM cells, which may promote cell-cell adhesion. Moreover, a direct role for CS1 in MM cell adhesion to, and interaction with, bone marrow stromal cells (BMSCs) has been conclusively demonstrated. This is important for MM pathobiology, because the protective effect of BMSCs comprises a substantial component of therapeutic resistance in this tumor type. Much of what is known about human CS1 signaling, however, is derived from studies in other lymphocyte subsets. In NK cells, for example, CS1 is a self ligand that promotes activation through selective expression of the adaptor protein EAT-2. In fact, CS1 may serve an inhibitory role in T cells that lack EAT-2. CS1 ligation independent of Fc R ligation in NK cells results in signal transduction events that activate several pathways including phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and phospholipase C gamma. Whether EAT-2 is expressed in MM cells remains unknown; similarly, the signal transduction events consequent to CS1 ligation in MM cells are a matter of ongoing investigation. Elotuzumab (formerly HuLuc63), a humanized immunoglobulin G1 anti-CS1 mAb, has been shown to impair MM cell adhesion and exert direct (albeit modest) effects on primary MM cell survival in vitro, either alone or in the presence of BMSCs. Its principal mechanism of action, however, is thought to be antibody-dependent cellular cytotoxicity (ADCC). Here, ADCC is mediated by NK cells through interaction of their Fc RIIIa receptor with the Fc binding region of the therapeutic antibody. Preliminary data suggest that elotuzumab may also directly activate NK cells through direct CS1 ligation. Interestingly, elotuzumab-associated NK-cell cytotoxicity via ADCC is directed specifically against CS1-bearing MM tumor cells and not fraternal CS1-bearing NK cells, as demonstrated in both in vitro and correlative studies performed during clinical investigation. Figure 1 summarizes the known functions of CS1 in MM and apparent

Is It Relevant to Determine Routinely Levels of Circulating NK and NK Like Cells in Myelodyplastic Syndroms?.

Abstract 2786Poster Board II-762 Purpose:Myelodysplastic syndromes (MDS) are characterized by an ineffective haematopoiesis due to different pathways: Intrinsic factors as cytogenetic and molecular abnormalities and extrinsic factors as disturbance in cellular immunity. An excess or abnormal natural killer (NK) cells cytolytic function have been previously described in MDS. A sub-group of MDS characterized by bone marrow hypoplasia, an excess of NK, HLA-DR15 and trisomy 8 may respond to an immune-suppressive therapy by antithymocyte globuline (ATG) or Cyclosporin. Recent advances in immunophenotyping of hematopoietic dysplastic cells in blood and bone marrow pointed to a useful role for multiparameter flow cytometry (FCM) in the diagnosis of MDS. This prospective study reports the rates of NK and NK like circulating cells in MDS patients (pts) assessed by routine FCM. Methods:This study concern a cohort of pts with MDS, in a university hospital, proved by morphological criteria on blood samples and bone marrow aspirate or trephine biopsy. Pts were classified according to 2001 WHO classification. IPSS score has been determined for pts according to cytopenia, blasts percent and cytogenetic results. We performed between 02/2005 and 09/2008 a prospective analysis of immunophenotyping data using a double platform method that allowed the enumeration of NK cells according to two different antibodies combinations (CD45+, CD3-, CD16 or CD56+, so called “total” NK cells vs CD45+ CD2+ CD3- so called 2+3- NK cells) and NK like cells (CD3+, CD16+ or CD56+). The statistical analysis was made with STATVIEW software. Results:The cohort counts 58 pts, median age 75 years (50-86), sex ratio 0,93. MDS subtype were distributed as 34.5% RAEB-1 (n=20), 22.4% RAEB-2 (n=13), 15.5 RA (n=9), 13.7% CMML (n=8), 8.6% RARS (n=5), 1.7% RA/RCMD (n=1), 1.7% 5q- syndrome (n=1), 1.7% inclassifiable (n=1). Cytogenetic analysis could be performed in 96.5% of cases (56/58) and revealed 78.5% of favourable prognosis (44/56) with 60.7% normal, 7.1% del (5q), 7.1% -Y, 3.6% del (20q), intermediate prognosis 12.5% (7/56) with 10.7% trisomy 8 and 1.8% inv(9), 5.4% of unfavourable prognosis (3/56) with 3.6% complex and 1.8% del(7q) and 3.6% failure (2/56). IPSS was calculated for 93% of the files (54/58) with 68.5% of low risk (18.5% low IPSS (n=10), 50% int-1 IPSS (n=27)) and 31.5% of high risk (25.9% int-2 IPSS (n=14), 5.6% high IPSS (n=3). Median rate of 2+3- NK cells, obtained on 96.5% of pts, was 79 cells/μl (6.7-798) (normal values : 130-430). This rate is decreased for 69.6% of pts (n=39), normal for 25% (n=14) and increased for 3.4% (n=3). The classification of these 3 pts was RARS, RAEB-2 and CMML. All had a normal cytogenetic profile. Median rate of “total” NK cells (determined on 60% of pts) was 119.7 cells/μl (17.6-473.1) (normal values 130-430). This rate is decreased for 57.1% (n=20), normal 40% (n=14) and increased 2.9% (n=1). This pts was suffering CMML with absence of cytopenia, 11% blast excess, normal cytogenetic and IPSS Int-2. Individually, the two approaches for NK numeration gives, as expected, close values with superior values for “total” NK cells that comprised the subset of CD2- NK cells. Median rate of NK like cells (60% of pts) was 58.5 cell/μl (7-1264) (normal values: 5-95). This rate is normal for 71.4% of pts (n=25) and increased in 28.6% (n=10). The classification of these 10 pts was 10% of RARS (n=1), 40% RAEB-1 (n=4), 20% RAEB-2 (n=2) and 30% CMML (n=3). Cytogenetic was normal 60% (n=6), trisomy 8 20% (n=2), inv 9 10% (n=1) and –Y 10% (n=1). IPSS was low 10% (n=1), Int-1 50% (n=5), Int-2 40% (n=4). Conclusion:European Leukemia Net recommendation proposes to use FCM for diagnosis and prognostication of MDS, based abnormal phenotype of myeloid lineages. The interest of lymphocytes immunophenotyping is discussed. Our study shows that cases with elevated circulating NK cells are rare, in addition without correlation with IPSS score and cytogenetic data. By contrast, an increase in circulating NK like cells is more frequent, and seems correlated with a blast excess. Such data could be of interest regarding to the potential therapeutic issues using immunosupressors. Disclosures:No relevant conflicts of interest to declare.

Identification of CS1 Peptides for Induction of Antigen-Specific CTLs in Multiple Myeloma.

Despite progress in the treatment of multiple myeloma (MM), the disease remains incurable. Therefore novel therapeutic approaches are required to achieve better outcome in patients. Development of peptide-based immunotherapy against tumor specific or associated antigens offers an attractive approach that may lead to tumor-targeted cellular therapy in MM patients. Recently, CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family, was found to be highly expressed by the tumor cells of the majority of MM patients but not other normal tissues. Importantly, a novel anti-CS1 HuLuc63 mAb induced significant anti-myeloma killing in vitro and in vivo (Abstract #950059). In addition, CS1 may play a role in MM cell survival (Abstract #953659). In this study, we asked whether CS1 is a suitable myeloma-associated antigen for cellular therapy in MM. We wanted to identify potential immunogenic peptides derived from CS1 and tested whether these peptides evoke MM-specific cytotoxic T lymphocytes (CTLs). First, we predicted the potential peptide sequences of CS1 that could bind to HLA-A*0201 molecule using three databases (RANKPEP, BIMAS and NetMHC). A total of four peptides were selected and synthesized according to the high binding scores among all the databases. Next, peptide-T2 cells binding assay confirmed these peptides with high binding affinity to HLA-A*0201 molecule (Fluorescence index: 2.19, 3.28, 3.44 and 3.49). To generate the peptide-specific CTLs, HLA-A*0201-positive normal human CD8+ T lymphocytes were stimulated with autologuous dendritic cells (DCs) or T2 cells pulsed with candidate CS1-peptides. The CTL cell lines were established after several rounds restimulation. We have evaluated the immunogenicity of the expanded CTLs by the stimulation with peptide-pulsed or unpulsed T2 cells. Our data demonstrated that one peptide-induced CTLs (CS1-CTLs) possessed high antigen-specific immune responses with the HLA-A*0201-restriction relative to a certain level of immune responses displayed by another three peptide-induced CTLs. We further evaluated the proliferative activity of CS1-CTLs by 3H-TdR incorporation assay. Upon stimulation by peptide-pulsed T2 cells, the cell proliferation of CS1-specific CTLs increased approximately 55-fold, compared with unstimulated cells (pulsed vs. unpulsed: 22894 vs. 423 cpm). The activation of CS1-CTLs was monitored by the expression of CD25 (IL-2Rα chain) on peptide-stimulated CS1-CTLs (%CD25+ cells: pulsed vs. unpulsed: 98% vs. 13%). CS1-CTLs significantly secreted 14-fold higher levels of IFN- γ in response to peptide-pulsed T2 cells (pulsed vs. unpulsed: 615 vs. 46 pg/ml). Importantly, the expanded CS1-CTLs generated high cytotoxicity (93%) in response to peptide pulsed T2 cells. Furthermore, more than 30% of killing activity by CS1-CTLs against HLA-A*0201+CS1+ MCCAR cells was observed at the effector: target ratio of 20:1. In contrast, these CS1-CLTs did not kill HLA-A*0201+/CS1− U266 and HLA-A*0201−/CS1+ MM1S target cells. These results support CS1 as an antigenic target for the induction of peptide-specific CTLs against MM cells. Further functional studies are underway to confirm potential CS1-specific cellular therapy to treat MM.

HuLuc63 in Combination Regimens with Conventional and Targeted Therapies Has Additive and Synergistic Anti-Tumor Activity in Pre-Clinical Models of Myeloma.

Introduction: HuLuc63 is a humanized monoclonal antibody that targets CS1 (CD2 subset 1, CRACC, SLAMF7, CD319), a cell surface glycoprotein that is highly and universally expressed on myeloma cells. In preclinical studies, we have shown that HuLuc63 treatment of mice with multiple myeloma (MM) xenograft tumors resulted in significant in vivo anti-tumor activity that is mediated at least in part by an antibody-dependent cellular cytotoxicity (ADCC) mechanism of action. The purpose of this study was to examine whether using HuLuc63 in combination with a panel of drugs having distinct modes of action (dexamethasone, thalidomide, bevacizumab (Avastin®), bortezomib (Velcade®)) could result in additional therapeutic benefit and provide a rationale for the design of future clinical trials.Methods: HuLuc63 in combination with other agents was tested in vivo for anti-tumor activity using the human L363 and OPM2 xenograft models. SCID mice were implanted subcutaneously with myeloma cells and randomized into different groups (10–15 mice per treatment group) when the average tumor volume reached ∼100 mm3. HuLuc63 was administered via intra-peritoneal injection twice per week at doses of 1–10 mg/kg. Dosing for dexamethasone was 10 mg/kg twice weekly, thalidomide 50 mg/kg daily, bevacizumab twice weekly at 0.5 mg/kg, and bortezomib 1 mg/kg for two dosing cycles, each cycle consisting of twice weekly dosing for 2 weeks followed by a week of rest.Results: The combination of dexamethasone with HuLuc63 showed a statistically significant increase in anti-tumor activity over either agent alone (p < 0.04). Combination with thalidomide only showed a slight enhancement of tumor inhibition when dosed in combination with HuLuc63 but its anti-tumor activity did not reach a statistically significant increase in over that of HuLuc63 alone. Co-treatment of the anti-VEGF anti-angiogenic monoclonal antibody bevacizumab with HuLuc63 resulted in a significant increase in tumor inhibition (p < 0.05) over that observed with either antibody when used as a single agent. The strongest anti-myeloma activity was observed when HuLuc63 was combined with bortezomib, which appeared to result in a synergistic inhibition of tumor cell growth. None of the agents tested changed the CS1 expression level on the myeloma cells or diminished the anti-myeloma activity of HuLuc63.Conclusions: These results suggest that HuLuc63 may be combined with different classes of drugs to enhance its anti-myeloma effects. In particular, agents that may induce apoptosis of myeloma cells (dexamethasone and bortezomib) and anti-angiogenics (bevacizumab) may be of particular interest for future clinical testing. Further preclinical studies using HuLuc63 in combination with other agents are in progress. HuLuc63 is currently being evaluated in a phase I clinical study as monotherapy for the treatment of relapsed/refractory multiple myeloma.

Human natural killer cell receptor 2B4 (CD244) down-regulates its own expression by reduced promoter activity at an Ets element

2B4 (CD244), a member of the CD2 subset of the immunoglobulin superfamily, is important for stimulating human natural killer (NK) cell cytotoxicity and cytokine production. It is expressed on all NK cells, a subpopulation of T cells, monocytes, basophils and eosinophils. 2B4 interaction with its ligand CD48 regulates NK, T and B lymphocyte functions and thus plays a central role in various immune responses. Previous study indicated a role for AP-1 and Ets in the transcription of the 2B4 gene. In this study we report that stimulation of NK cells through surface 2B4 down-regulates its own expression due to a reduction in the promoter activity at the Ets element. The down-regulation of 2B4 could be a mechanism to attenuate the co-stimulatory signal from 2B4--CD48 interactions.

Killing of Drug-Sensitive and Resistant Myeloma Cells and Disruption of Their Bone Marrow Stromal Interaction by HuLuc63, a Novel Humanized Anti-CS1 Monoclonal Antibody.

Introduction: Current monoclonal antibody (mAb) therapies for multiple myeloma (MM) have had limited success due to narrow target expression across MM patient samples. A preferred strategy would be to develop cytotoxic human mAbs against novel antigens that are highly expressed in MM cells yet have limited expression in other cell types. CS1 (CD2 subset 1, CRACC, SLAMF7), a member of the CD2 family of cell surface glycoproteins, was found to be highly expressed in myeloma cells. In this study, we investigated the anti-myeloma activity of HuLuc63, a novel humanized anti-CS1 mAb.Methods: Microarray expression profiling was used to determine the CS1 mRNA levels in CD138-expressing myeloma cells from 101 MM patient samples. For detection of CS1 protein, flow cytometry was performed using the anti-CS1 mAb HuLuc63. Functional characterization of HuLuc63 was performed by assessing antibody-dependent cellular cytotoxicity (ADCC) and by assessing MM and bone marrow stromal cell (BMSC) interactions.Results: CS1 mRNA was expressed in CD138 cells from more than 96% (97/101) of MM patients. Flow cytometric analysis confirmed that protein expression mirrors the mRNA profile. Importantly, CS1 is also present in 12 MM cell lines that are either drug-sensitive or resistant. HuLuc63, but not an isotype control antibody, induced ADCC in a CS1-specific, dose-dependent manner against CD138-expressing MM lines and patient MM cells including dexamethasone (dex)-sensitive MM1S and dex-resistant MM1R cells. Significantly, HuLuc63 triggered autologous ADCC against CS1-expressing CD138-purified tumor cells from 11 MM patients resistant to conventional or novel therapies such as bortezomib (Velcade®) and an HSP90 inhibitor. Since CS1 may regulate cell adhesion, we next studied whether HuLuc63 alters MM cell adhesion to BMSCs. HuLuc63 inhibited MM cell adhesion to BMSCs in a dose-dependent manner, whereas human control IgG did not. However, the presence of BMSC appeared to reduce HuLuc63-induced cell lysis against MM1S and MM1R cells. Since the immunomodulatory drug lenalidomide (Revlimid®) enhances NK cell function, we further tested whether HuLuc63-induced ADCC against MM cells is augmented by lenalidomide. Pretreatment with lenalidomide markedly enhanced NK-cell-mediated lysis of autologous patient MM cells triggered by HuLuc63.Conclusions: We show that the new MM antigen, CS1, is expressed in myeloma cells from more than 96% of MM patients. The novel humanized anti-CS1 mAb, HuLuc63, induced significant cytotoxicity against MM cells including drug-resistant cells, and inhibited their interaction with BMSCs. These data suggest that HuLuc63 may have clinical utility in a spectrum of MM patients including those newly diagnosed with the disease as well as patients with late stage refractory disease.

Eradication of Tumors in Pre-Clinical Models of Multiple Myeloma by Anti-CS1 Monoclonal Antibody HuLuc63: Mechanism of Action Studies.

Introduction: We have recently shown that CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family, is uniformly expressed on myeloma cells from multiple myeloma (MM) patients. Based on its high expression in MM and limited expression in normal cells, we propose CS1 as a novel and specific antibody target for the treatment of MM.Methods: A panel of monoclonal anti-CS1 antibodies (mAbs) was generated to identify a potential therapeutic candidate. MAb clones MuLuc63 and MuLuc90 were selected for testing in CS1 positive MM xenograft models in vivo in severe combined immunodeficient mice. HuLuc63, a humanized IgG1 version of MuLuc63, was generated as the potential therapeutic candidate for the treatment of MM. HuLuc63 and Fc-modified versions of HuLuc63 were tested for anti-tumor activity in mouse models vivo. In vitro antibody-dependent cellular cytotoxicity (ADCC) assays were performed to define the potential mechanism of action for HuLuc63.Results: Both MuLuc63 and MuLuc90 exhibited significant in vivo anti-tumor activity compared to isotype control antibodies in the L363 MM xenograft model. MuLuc63 was significantly more potent, resulting in rapid tumor eradication in most of the animals for the length of the study (~4 months). Based on these results, MuLuc63 was humanized to generate HuLuc63, which exhibited similar affinity for CS1 when compared to the mouse parent antibody. In two different MM xenograft models, L363 and OPM2, HuLuc63 exhibited significant anti-tumor activity resulting in tumor eradication in a high proportion of animals. To investigate the mechanism of action, two modified versions of HuLuc63 were tested in xenograft models. One version, HuLuc63-Ala,Ala, exhibits a mutation in the Fc region that decreases the ability to interact with the Fc receptor on natural killer (NK) cells. The second version, HuLuc63-LF, exhibits low levels of fucosylation in the Fc region that would result in increased binding to the Fc receptor. Compared to HuLuc63, the LF version exhibited significantly better in vivo anti-tumor activity towards, while the Ala,Ala mutant exhibited no anti-tumor activity. These data indicate that the Fc region of HuLuc63 is critical for its anti-tumor activity, and suggest ADCC as a possible mechanism of action. In vitro, HuLuc63 exhibits substantial ADCC towards L363 and OPM2 cells. The activity was dose-dependent, with increasing cytotoxicity being observed with concentrations ranging from 0.01μg/mL to10 μg/mL.Conclusions: These pre-clinical data support HuLuc63 as a new therapeutic for the treatment of MM and suggest that ADCC is part of the mechanism of action. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM).Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays.Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner.Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

CS1 Is Expressed on Myeloma Cells from Early Stage, Late Stage, and Drug-Treated Multiple Myeloma Patients, and Is Selectively Targeted by the HuLuc63 Antibody.

Introduction: One-third of multiple myeloma (MM) patients exhibit high-risk features such as abnormal cytogenetics, high LDH, amplification of CKS1-B or spiked expression of MAF, MAF-B or FGFR3. While not affecting complete response rates, median durations of event-free and overall survival, even with high-dose melphalan-based tandem autotransplants of such patients, do not exceed 24 mo and 36 mo, compared to 60 mo and ≥ 90 mo for the remainder. Monoclonal antibody (mAb)-mediated therapy may target a chemotherapy-resistant myeloma cell pool. CS1 (CD2 subset 1, CRACC, SLAMF7), a member of the CD2 family of cell surface glycoproteins, exhibits high-level expression on primary myeloma cells, indicating that CS1 is a potential target for treatment in MM.Methods: Gene expression was assessed using an Affymetrix GeneChip array. Protein expression was measured by flow cytometry, and immunohistochemistry (IHC), using HuLuc63, a novel humanized anti-CS1 mAb. HuLuc63-mediated lysis of myeloma cells via antibody dependent cellular cytotoxicity (ADCC) was measured by 51Cr-release.Results: CS1 mRNA was detected in >95% of CD138+ purified plasma cells from >95% of healthy donors, newly diagnosed myeloma patients, and those with relapsed myeloma (Fig. 1a). CS1 remained highly expressed in patients following VDTPACE treatment, albeit at a reduced level. CS1 expression was also high following bortezomib (Velcade®) treatment, with a subset of patients showing increased expression post-treatment. CS1 protein expression on primary myeloma cells was confirmed by flow cytometry, while IHC analysis of normal tissues revealed anti-CS1 staining primarily on CD138+ tissue plasma cells. Finally, we determined that HuLuc63 could induce killing of myeloma cells using purified allogeneic NK cells (Fig. 1b). Blocking the Fc receptor greatly reduced this activity indicating an ADCC mechanism. Killing of myeloma targets was also observed in autologous systems suggesting that HuLuc63 can overcome KIR-mediated NK cell inhibition of autologous NK cells. In summary, we observed high mRNA and protein expression of CS1 in myeloma from early stage, late stage, and treated patients, and showed enhanced lysis of myeloma cells in vitro with HuLuc63. Our data support the potential clinical utility of CS1-targeted therapy. HuLuc63 will be entering a phase I clinical trial for advanced myeloma patients. [Display omitted] [Display omitted]

The co‐expression of 2B4 (CD244) and CD160 delineates a subpopulation of human CD8+ T cells with a potent CD160‐mediated cytolytic effector function

Within human CD8+ T lymphocytes, the CD27-CD45RAhigh or CD56+ phenotypes contribute to precisely define the cells with CTL effector function. Novel markers were demonstrated to correlate with CTL properties, such as the 2B4 (CD244) receptor, a member of the CD2 subset of the immunoglobulin superfamily or the glycosylphosphatidylinositol-anchored CD160 receptor. We performed a study of these markers to further define the population of effectors with CTL functions. Here we show that cytotoxic subpopulations defined by surface markers CD160, CD56 and CD57 are mostly contained in the 2B4+CD8+ T cell population. Expression of CD160 identifies two populations in the 2B4+ population. The 2B4+CD160+ subset expresses a bona fide CTL phenotype. The co-expression of 2B4 and CD160 defines T cells containing high amounts of perforin and granzyme B. During CTL ontogeny, an up-regulation of 2B4 and CD160 is observed from a naive to a terminally differentiated phenotype. Finally, we demonstrated that CD160 triggering failed to induce cytotoxicity per se, but costimulated CD3-redirected killing. We conclude that the co-expression of 2B4 and CD160 defines a CD8+ T lymphocyte subpopulation with high CTL activity.

Crystal Structure and Binding Properties of the CD2 and CD244 (2B4)-binding Protein, CD48

The structural analysis of surface proteins belonging to the CD2 subset of the immunoglobulin superfamily has yielded important insights into transient cellular interactions. In mice and rats, CD2 and CD244 (2B4), which are expressed predominantly on T cells and natural killer cells, respectively, bind the same, broadly expressed ligand, CD48. Structures of CD2 and CD244 have been solved previously, and we now present the structure of the receptor-binding domain of rat CD48. The receptor-binding surface of CD48 is unusually flat, as in the case of rat CD2, and shares a high degree of electrostatic complementarity with the equivalent surface of CD2. The relatively simple arrangement of charged residues and this flat topology explain why CD48 cross-reacts with CD2 and CD244 and, in rats, with the CD244-related protein, 2B4R. Comparisons of modeled complexes of CD2 and CD48 with the complex of human CD2 and CD58 are suggestive of there being substantial plasticity in the topology of ligand binding by CD2. Thermodynamic analysis of the native CD48-CD2 interaction indicates that binding is driven by equivalent, weak enthalpic and entropic effects, in contrast to the human CD2-CD58 interaction, for which there is a large entropic barrier. Overall, the structural and biophysical comparisons of the CD2 homologues suggest that the evolutionary diversification of interacting cell surface proteins is rapid and constrained only by the requirement that binding remains weak and specific.

Requirement of homotypic NK-cell interactions through 2B4(CD244)/CD48 in the generation of NK effector functions

Abstract2B4 belongs to the CD2 subset of the IgG family of receptors. Members in this family have been shown to function as coreceptors via homophilic or heterophilic interactions. Both 2B4 and CD2 bind to CD48, another member of this family. Because all 3 molecules are expressed on natural killer (NK) cells, it raises a possibility that the binding of 2B4 and CD2 to CD48 among NK cells may have functional consequences. Using specific monoclonal antibodies and gene-deficient NK cells, we found that 2B4/CD48, but not CD2/CD48, interaction is essential for IL-2–driven expansion and activation of murine NK cells. In the absence of 2B4/CD48 interaction, NK cytotoxicity and IFN-γ secretion on tumor target exposure is severely impaired. Impaired activation of NK cells in 2B4-deficient mice was also demonstrated by poor NK-mediated clearance of syngeneic tumor cells in these mice. Functional impairment of NK cells in the absence of 2B4/CD48 interactions was accompanied by defective calcium signaling, suggesting that the early signaling pathway of NK receptors is inhibited. Finally, homotypic interactions among NK cells through 2B4/CD48 was visualized by specific localization of GFP-tagged 2B4 onto NK-NK conjugation sites. Thus, these data identify a novel mechanism whereby NK effector function is regulated via homotypic 2B4/CD48 interactions.

2B4/CD48-Mediated Regulation of Lymphocyte Activation and Function

2B4 (CD244) is a member of the CD2 subset of the Ig superfamily. This molecule is expressed on innate immune cells, including NK cells, and on subsets of T cells. The 2B4 molecule interacts with CD48, which is widely expressed on hemopoietic cells. Although earlier reports demonstrated a role for 2B4 as an activating receptor in both mice and humans, recent studies of 2B4-deficient mice have suggested that 2B4 functions predominantly as an inhibitory receptor in mice. In addition, 2B4 may also act as a costimulatory ligand for cells expressing CD48. Thus, the 2B4 molecule is more multifunctional than previously understood. In this study, we delineate the current view of 2B4-CD48 interactions among lymphocytes and other cells.

Mutational Analysis of the Human 2B4 (CD244)/CD48 Interaction: Lys68 and Glu70 in the V Domain of 2B4 Are Critical for CD48 Binding and Functional Activation of NK Cells

Interaction between receptors and ligands plays a critical role in the generation of immune responses. The 2B4 (CD244), a member of the CD2 subset of the Ig superfamily, is the high affinity ligand for CD48. It is expressed on NK cells, T cells, monocytes, and basophils. Recent data indicate that 2B4/CD48 interactions regulate NK and T lymphocyte functions. In human NK cells, 2B4/CD48 interaction induces activation signals, whereas in murine NK cells it sends inhibitory signals. To determine the structural basis for 2B4/CD48 interaction, selected amino acid residues in the V domain of the human 2B4 (h2B4) were mutated to alanine by site-directed mutagenesis. Following transient expression of these mutants in B16F10 melanoma cells, their interaction with soluble CD48-Fc fusion protein was assessed by flow cytometry. We identified amino acid residues in the extracellular domain of h2B4 that are involved in interacting with CD48. Binding of CD48-Fc fusion protein to RNK-16 cells stably transfected with wild-type and a double-mutant Lys(68)Ala-Glu(70)Ala h2B4 further demonstrated that Lys(68) and Glu(70) in the V domain of h2B4 are essential for 2B4/CD48 interaction. Functional analysis indicated that Lys(68) and Glu(70) in the extracellular domain of h2B4 play a key role in the activation of human NK cells through 2B4/CD48 interaction.

Functional requirements for interactions between CD84 and Src homology 2 domain-containing proteins and their contribution to human T cell activation.

Cell surface receptors belonging to the CD2 subset of the Ig superfamily of molecules include CD2, CD48, CD58, 2B4, signaling lymphocytic activation molecule (SLAM), Ly9, CD84, and the recently identified molecules NTB-A/Ly108/SLAM family (SF) 2000, CD84H-1/SF2001, B lymphocyte activator macrophage expressed (BLAME), and CRACC (CD2-like receptor-activating cytotoxic cells)/CS-1. Some of these receptors, such as CD2, SLAM, 2B4, CRACC, and NTB-A, contribute to the activation and effector function of T cells and NK cells. Signaling pathways elicited via some of these receptors are believed to involve the Src homology 2 (SH2) domain-containing cytoplasmic adaptor protein SLAM-associated protein (SAP), as it is recruited to SLAM, 2B4, CD84, NTB-A, and Ly-9. Importantly, mutations in SAP cause the inherited human immunodeficiency X-linked lymphoproliferative syndrome (XLP), suggesting that XLP may result from perturbed signaling via one or more of these SAP-associating receptors. We have now studied the requirements for SAP recruitment to CD84 and lymphocyte activation elicited following ligation of CD84 on primary and transformed human T cells. CD84 was found to be rapidly tyrosine phosphorylated following receptor ligation on activated T cells, an event that involved the Src kinase Lck. Phosphorylation of CD84 was indispensable for the recruitment of SAP, which was mediated by Y(262) within the cytoplasmic domain of CD84 and by R(32) within the SH2 domain of SAP. Furthermore, ligating CD84 enhanced the proliferation of anti-CD3 mAb-stimulated human T cells. Strikingly, this effect was also apparent in SAP-deficient T cells obtained from patients with XLP. These results reveal a novel function of CD84 on human lymphocytes and suggest that CD84 can activate human T cells via a SAP-independent mechanism.