Humanized Chimeric Receptors in the Therapy of Multiple Sclerosis

The role of autoreactive, antigen-specific T-cells in the development of autoimmunity has long been documented. T-cells expressing chimeric receptors are specifically redirected against such cells and have been proven to suppress autoimmune encephalomyelitis, the murine model of multiple sclerosis. We here demonstrate the ability of humanized chimeric receptors to suppress experimental autoimmune encephalomyelitis (EAE) in a humanized mouse model by redirecting T lymphocytes against autoreactive T-cells. The receptors were synthesized by linking the 84-102 epitope of human myelin basic protein (MBP) to the extracellular and transmembrane domains of the beta chain of human major histocompatibility complex (MHC) class II molecule and the cytoplasmic zeta chain of T cell receptor and pairing it to the alpha chain linked to zeta. CD8+ receptor-modified T-cells (RMTC) were able to recognize the cognate TCR receptor of antigen-specific cells and induce cytokine secretion, proliferation, and cytolysis upon engagement. Most importantly, the RMTC were able to specifically kill antigen-specific cells both in vitro and in vivo and prevent EAE disease. We hypothesize that the humanized chimeric receptors could be used as a therapeutic approach for multiple sclerosis in the future

Inhibition of 2A-mediated ‘cleavage’ of certain artificial polyproteins bearing N-terminal signal sequences

Where 2A oligopeptide sequences occur within ORFs, the formation of the glycyl-prolyl peptide bond at the C-terminus of (each) 2A does not occur. This property can be used to concatenate sequences encoding several proteins into a single ORF: each component of such an artificial polyprotein is generated as a discrete translation product. 2A and ‘2A-like’ sequences have become widely utilised in biotechnology and biomedicine. Individual proteins may also be co- and post-translationally targeted to a variety of sub-cellular sites. In the case of polyproteins bearing N-terminal signal sequences we observed, however, that the protein downstream of 2A (no signal) was translocated into the endoplasmic reticulum (ER). We interpreted these data as a form of ‘slipstream’ translocation: downstream proteins, without signals, were translocated through a translocon pore already formed by the signal sequence at the N-terminus of the polyprotein. Here we show this effect is, in fact, due to inhibition of the 2A reaction (formation of fusion protein) by the C-terminal region (immediately upstream of 2A) of some proteins when translocated into the ER. Solutions to this problem include the use of longer 2As (with a favourable upstream context) or modifying the order of proteins comprising polyproteins

The Plasma Concentration of the B Cell Activating Factor Is Increased in Children With Acute Malaria

Malaria-specific antibody responses in children often appear to be short-lived but the mechanisms underlying this phenomenon are not well understood. In this study, we investigated the relationship between the B-cell activating factor (BAFF) and its receptors expressed on B cells with antibody responses during and after acute malaria in children. Our results demonstrate that BAFF plasma levels increased during acute malarial disease and reflected disease severity. The expression profiles for BAFF receptors on B cells agreed with rapid activation and differentiation of a proportion of B cells to plasma cells. However, BAFF receptor (BAFF-R) expression was reduced on all peripheral blood B cells during acute infection, but those children with the highest level of BAFF-R expression on B cells maintained schizont-specific immunoglobin G (IgG) over a period of 4 months, indicating that dysregulation of BAFF-R expression on B cells may contribute to short-lived antibody responses to malarial antigens in children. In summary, this study suggests a potential role for BAFF during malaria disease, both as a marker for disease severity and in shaping the differentiation pattern of antigen-specific B cells

Antibodies Against Human BLyS and APRIL Attenuate EAE Development in Marmoset Monkeys

B lymphocyte stimulator (BLyS, also indicated as BAFF (B-cell activating factor) and CD257), and A Proliferation Inducing Ligand (APRIL, CD256) are two members of the TNF superfamily with a central role in B cell survival. Antibodies against these factors have potential therapeutic relevance in autoimmune inflammatory disorders with a proven pathogenic contribution of B cells, such as multiple sclerosis (MS). In the current study we performed a multi-parameter efficacy comparison of monoclonal antibodies against human anti-BLyS and anti-APRIL in a common marmoset (Callithrix jacchus) model of experimental autoimmune encephalomyelitis (EAE). A MS-like disease was induced by immunization with recombinant human myelin/oligodendrocyte glycoprotein (rhMOG) in complete Freund's adjuvant. The results show that the anti-BLyS and anti-APRIL antibody cause significant depletion of circulating CD20+ B cells, but a small subset of CD20 + CD40highB cells was not depleted. Induction of CD20+ B cell depletion from lymph nodes was only observed in the anti-BLyS treated monkeys. Both antibodies had a significant inhibitory effect on disease development, but all monkeys developed clinically evident EAE. Anti-BLyS treated monkeys were sacrificed with the same clinical signs as saline-treated monkeys, but nevertheless displayed significantly reduced spinal cord demyelination. This effect was not observed in the anti-APRIL treated monkeys. The two antibodies had a different effect on T cell subset activation and the profiles of ex vivo released cytokines. In conclusion, treatment with anti-BLyS and anti-APRIL delays the development of neurological disease in a relevant preclinical model of MS. The two mAbs achieve this effect via different mechanisms

Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro–B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP–keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4+ T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell–activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations

Redirecting therapeutic T cells against myelin-specific T lymphocytes using a humanized myelin basic protein-HLA-DR2-zeta chimeric receptor.

Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes implicated in MS. We generated two heterodimeric receptors that genetically link the human MBP84-102 epitope to HLA-DR2 and either incorporate or lack a TCRzeta signaling domain. The Ag-MHC domain serves as a bait, binding the TCR of MBP-specific target cells. The zeta signaling region stimulates the therapeutic cell after cognate T cell engagement. Both receptors were well expressed on primary T cells or T hybridomas using a tricistronic (alpha, beta, green fluorescent protein) retroviral expression system. MBP-DR2-zeta-, but not MBP-DR2, modified CTL were specifically stimulated by cognate MBP-specific T cells, proliferating, producing cytokine, and killing the MBP-specific target cells. The receptor-modified therapeutic cells were active in vivo as well, eliminating Ag-specific T cells in a humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP84-102/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to counteract pathologic, self-specific T lymphocytes, and specifically validate humanized MBP-DR2-zeta chimeric receptors as a potential therapeutic in MS