The molecular mechanism of CD81 antibody inhibition of metastasis.

Metastases are reduced in CD81KO mice. In addition, a unique anti-CD81 antibody, 5A6, inhibits metastasis in vivo and invasion and migration in vitro. Here, we probed the structural components of CD81 required for the antimetastatic activity induced by 5A6. We found that the removal of either cholesterol or the intracellular domains of CD81 did not affect inhibition by the antibody. We show that the uniqueness of 5A6 is due not to increased affinity but rather to its recognition of a specific epitope on the large extracellular loop of CD81. Finally, we present a number of CD81 membrane-associated partners that may play a role in mediating the 5A6 antimetastatic attributes, including integrins and transferrin receptors.

Targeting the tetraspanin CD81 reduces cancer invasion and metastasis.

Tetraspanins are an evolutionary conserved family of proteins involved in multiple aspects of cell physiology, including proliferation, migration and invasion, protein trafficking, and signal transduction; yet their detailed mechanism of action is unknown. Tetraspanins have no known natural ligands, but their engagement by antibodies has begun to reveal their role in cell biology. Studies of tetraspanin knockout mice and of germline mutations in humans have highlighted their role under normal and pathological conditions. Previously, we have shown that mice deficient in the tetraspanin CD81 developed fewer breast cancer metastases compared to their wild-type (WT) counterparts. Here, we show that a unique anti-human CD81 antibody (5A6) effectively halts invasion of triple-negative breast cancer (TNBC) cell lines. We demonstrate that 5A6 induces CD81 clustering at the cell membrane and we implicate JAM-A protein in the ability of this antibody to inhibit tumor cell invasion and migration. Furthermore, in a series of in vivo studies we demonstrate that this antibody inhibits metastases in xenograft models, as well as in syngeneic mice bearing a mouse tumor into which we knocked in the human CD81 epitope recognized by the 5A6 antibody.

CD81 as a tumor target.

CD81 participates in a variety of important cellular processes such as membrane organization, protein trafficking, cellular fusion and cell-cell interactions. In the immune system, CD81 regulates immune synapse, receptor clustering and signaling; it also mediates adaptive and innate immune suppression. CD81 is a gateway in hepatocytes for pathogens such as hepatitis C virus and Plasmodium; it also confers susceptibility to Listeria infection. These diverse biological roles are due to the tendency of CD81 to associate with other tetraspanins and with cell-specific partner proteins, which provide the cells with a signaling platform. CD81 has also been shown to regulate cell migration and invasion, and has therefore been implicated in cancer progression. Indeed, we have recently shown that CD81 contributes to tumor growth and metastasis. CD81 is expressed in most types of cancer, including breast, lung, prostate, melanoma, brain cancer and lymphoma, and the overexpression or down-regulation of this molecule has been correlated with either good or bad prognosis. Here, we discuss the role of CD81 in cancer and its potential therapeutic use as a tumor target.

B-cell receptors expressed by lymphomas of hepatitis C virus (HCV)-infected patients rarely react with the viral proteins.

Chronic hepatitis C virus (HCV) infection has been implicated in the induction and maintenance of B-cell lymphomas. The strongest evidence for this derives from clinical observations of tumor regressions upon antiviral treatments. Here we used multiple methods to test the hypothesis that the expansion of HCV-specific B cells gives rise to lymphomas. We obtained lymphoma tissues from HCV-infected lymphoma patients, including some that later regressed upon antiviral treatments. We expressed the lymphoma B-cell receptors as soluble immunoglobulin Gs and membrane IgMs, and analyzed their reactivity with HCV proteins and with HCV virions. We confirmed previous reports that HCV-associated lymphomas use a restricted immunoglobulin variable region gene repertoire. However, we found no evidence for their binding to the HCV antigens. We conclude that most lymphomas of HCV-infected patients do not arise from B cells aimed at eliminating the virus.

A mutation in the human tetraspanin CD81 gene is expressed as a truncated protein but does not enable CD19 maturation and cell surface expression.

A homozygous mutation in a splice site of the CD81 gene was identified previously in a patient, as the cause in a case of common variable immune deficiency (CVID). CD19 expression is reduced in mice that lack CD81; however, B cells in this patient lacked completely CD19 surface expression. The mutation led to an absence of the CD81 protein on the cell surface and it was assumed that the CD81 protein was not produced. Here we demonstrate that a truncated human CD81 mutant (CD81mut) was actually produced, but retained intracellularly. We also demonstrate that the truncated CD81mut protein is in close proximity to the intracellularly sequestered CD19. However, this interaction does not enable normal CD19 maturation and surface expression. In addition, we show that specific domains of CD81 enable retrieval and trafficking of human CD19 to the cell surface. Finally, we demonstrate that surface expression of CD19 requires CD81, even in non-B cells.

Role of an arginine-lysine rich motif in maturation and trafficking of CD19.

Normal expression of CD19 on the surface of B cells requires the presence of the tetraspanin molecule CD81. Previous studies have shown that surface expression of CD19 is highly reduced in CD81-deficient mouse B cells and that it is completely absent in an antibody deficient human patient with a mutation in the CD81 gene. The current study explored the contribution of an arginine-lysine rich motif, present in the membrane-proximal cytoplasmic domain of CD19, for the maturation and trafficking of this molecule. We demonstrate that this motif plays a role in the maturation and recycling of CD19 but in a CD81-independent manner.

Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin.

CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of B-lymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin-binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. After engagement of CD81, it colocalized with ezrin and F-actin, and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This mechanism might explain the pleiotropic effects induced in response to stimulation of cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

Cell-free production of Gaussia princeps luciferase--antibody fragment bioconjugates for ex vivo detection of tumor cells.

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Building of the tetraspanin web: distinct structural domains of CD81 function in different cellular compartments.

The tetraspanin web is composed of a network of tetraspanins and their partner proteins that facilitate cellular interactions and fusion events by an unknown mechanism. Our aim was to unravel the web partnership between the tetraspanin CD81 and CD19, a cell surface signaling molecule in B lymphocytes. We found that CD81 plays multiple roles in the processing, intracellular trafficking, and membrane functions of CD19. Surprisingly, these different roles are embodied in distinct CD81 domains, which function in the different cellular compartments: the N-terminal tail of CD81 has an effect on the glycosylation of CD19; the first transmembrane domain of CD81 is sufficient to support the exit of CD19 from the endoplasmic reticulum, although the large extracellular loop (LEL) of CD81 associates physically with CD19 early during biosynthesis; and finally, the TM2 and TM3 domains of CD81 play a role in the transmission of signals initiated upon engagement of the LEL. The participation of distinct CD81 domains in varied functions may explain the pleiotropic effects of CD81 within the tetraspanin web.

CD81 is a novel immunotherapeutic target for B cell lymphoma.

The tetraspanin CD81 was initially discovered by screening mAbs elicited against a human B cell lymphoma for their direct antiproliferative effects. We now show that 5A6, one of the mAbs that target CD81, has therapeutic potential. This antibody inhibits the growth of B cell lymphoma in a xenograft model as effectively as rituximab, which is a standard treatment for B cell lymphoma. Importantly, unlike rituximab, which depletes normal as well as malignant B cells, 5A6 selectively kills human lymphoma cells from fresh biopsy specimens while sparing the normal lymphoid cells in the tumor microenvironment. The 5A6 antibody showed a good safety profile when administered to a mouse transgenic for human CD81. Taken together, these data provide the rationale for the development of the 5A6 mAb and its humanized derivatives as a novel treatment against B cell lymphoma.

Tetraspanin CD81, a modulator of immune suppression in cancer and metastasis.

Cancer cells can escape the antitumor immune response by recruiting immune suppressor cells. However, although innate myeloid-derived suppressor cells (MDSCs) and T regulatory (Treg) cells accumulate normally in tumor-bearing CD81-deficient mice, both populations are impaired in their ability to suppress the antitumor immune response.

Tetraspanin CD81 promotes tumor growth and metastasis by modulating the functions of T regulatory and myeloid-derived suppressor cells.

Tumor cells counteract innate and adaptive antitumor immune responses by recruiting regulatory T cells (Treg) and innate myeloid-derived suppressor cells (MDSC), which facilitate immune escape and metastatic dissemination. Here we report a role in these recruitment processes for CD81, a member of the tetraspanin family of proteins that have been implicated previously in cancer progression. We found that genetic deficiency in CD81 reduced tumor growth and metastasis in two genetic mouse backgrounds and multiple tumor models. Mechanistic investigations revealed that CD81 was not required for normal development of Treg and MDSC but was essential for immunosuppressive functions. Notably, adoptive transfer of wild-type Treg into CD81-deficient mice was sufficient to promote tumor growth and metastasis. Our findings suggested that CD81 modulates adaptive and innate immune responses, warranting further investigation of CD81 in immunomodulation in cancer and its progression.

CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency.

Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans.

CD81-dependent binding of hepatitis C virus E1E2 heterodimers.

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV is also the major cause of mixed cryoglobulinemia, a B-lymphocyte proliferative disorder. Direct experimentation with native viral proteins is not feasible. Truncated versions of recombinant E2 envelope proteins, used as surrogates for viral particles, were shown to bind specifically to human CD81. However, truncated E2 may not fully mimic the surface of HCV virions because the virus encodes two envelope glycoproteins that associate with each other as E1E2 heterodimers. Here we show that E1E2 complexes efficiently bind to CD81 whereas truncated E2 is a weak binder, suggesting that truncated E2 is probably not the best tool with which to study cellular interactions. To gain better insight into virus-cell interactions, we developed a method by which to isolate E1E2 complexes that are properly folded. We demonstrate that purified E1E2 heterodimers bind to cells in a CD81-dependent manner. Furthermore, engagement of B cells by purified E1E2 heterodimers results in their aggregation and in protein tyrosine phosphorylation, a hallmark of B-cell activation. These studies provide a possible clue to the etiology of HCV-associated B-cell lymphoproliferative diseases. They also delineate a method by which to isolate biologically functional E1E2 complexes for the study of virus-host cell interaction in other cell types.