Antigenic Modulation Research Articles

Antiandrogen modulation of prostate-specific membrane antigen (PSMA): Dynamics and synergy with PSMA-targeted therapy.

e16007 Background: Recent approvals of four new prostate cancer (PCa) drugs and a growing number of pipeline agents have created opportunities for designing rational drug combinations. Potent antiandrogens such as enzalutamide and abiraterone affect expression of a host of androgen-regulated molecules, including those that represent targets for therapy. One such target is PSMA, a well-characterized cell-surface antigen abundant on prostate cancer cells. PSMA ADC is a PSMA-targeted antibody-drug conjugate currently in phase II clinical testing, comprised of a fully human IgG1 mAb conjugated to vcMMAE (valine-citrulline monomethylauristatin E). Here we examined the kinetics and reversibility of PSMA induction by potent antiandrogens and their associated effects on the preclinical activity of PSMA ADC. Methods: Androgen-dependent and -independent PCa cell lines with varying basal levels of PSMA expression were cultured for up to one month in the presence of enzalutamide or abiraterone, followed by drug washout. Cells were tested for PSMA expression over time and for susceptibility to cytotoxicity by PSMA ADC. Potential drug synergy or antagonism was evaluated using the combination index method. Results: Enzalutamide (1 mM) increased PSMA expression by approximately 2.5-fold in LNCaP (androgen-dependent) and C4-2 (androgen-independent) cells, with maximal expression observed after approximately 4 weeks’ culture. PSMA expression returned to basal levels within days following removal of enzalutamide from the culture. Enzalutamide and PSMA ADC exhibited synergistic antitumor activity in LNCaP and C4-2 cells (P < 0.05). Similar results were observed for abiraterone in C4-2 cells. Less induction of PSMA expression by antiandrogens and modest effects on cytotoxicity were observed using 22Rv1 cells, an androgen-independent cell line with low basal expression of PSMA. Conclusions: Enzalutamide and abiraterone significantly and reversibly augmented PSMA expression and potentiated the activity of PSMA ADC in PCa cell lines in vitro. The findings support clinical exploration of regimens that combine potent antiandrogens and PSMA-targeted therapies.

B-cell subpopulations in humans and their differential susceptibility to depletion with anti-CD20 monoclonal antibodies

In humans, different B-cell subpopulations can be distinguished in peripheral blood and other tissues on the basis of differential expression of various surface markers. These different subsets correspond to different stages of maturation, activation and differentiation. B-cell depletion therapy based on rituximab, an anti-CD20 mAb, is widely used in the treatment of various malignant and autoimmune diseases. Rituximab induces a very significant depletion of B-cell subpopulations in the peripheral blood usually for a period of 6 to 9 months after one cycle of therapy. Cells detected circulating during depletion are mainly CD20 negative plasmablasts. Data on depletion of CD20-expressing B cells in solid tissues are limited but show that depletion is significant but not complete, with bone marrow and spleen being more easily depleted than lymph nodes. Factors influencing depletion are thought to include not only the total drug dose administered and distribution into various tissues, but also B-cell intrinsic and microenvironment factors influencing recruitment of effector mechanisms and antigen and effector modulation. Available studies show that the degree of depletion varies between individuals, even if treated with the same dose, but that it tends to be consistent in the same individual. This suggests that individual factors are important in determining the final extent of depletion.

PADI4 Haplotypes in Association with RA Mexican Patients, a New Prospect for Antigen Modulation

Peptidyl arginine deiminase IV (PAD 4) is the responsible enzyme for a posttranslational modification called citrullination, originating the antigenic determinant recognized by anti-cyclic citrullinated peptide antibodies (ACPA). Four SNPs (single nucleotide polymorphisms) have been described in PADI4 gene to form a susceptibility haplotype for rheumatoid arthritis (RA); nevertheless, results in association studies appear contradictory in different populations. The aim of the study was to analyze if the presence of three SNPs in PADI4 gene susceptibility haplotype (GTG) is associated with ACPA positivity in patients with RA. This was a cross-sectional study that included 86 RA patients and 98 healthy controls. Polymorphisms PADI4_89, PADI4_90, and PADI4_92 in the PADI4 gene were genotyped. The susceptibility haplotype (GTG) was more frequent in RA patients; interestingly, we found a new haplotype associated with RA with a higher frequency (GTC). There were no associations between polymorphisms and high scores in Spanish HAQ-DI and DAS-28, but we did find an association between RARBIS index and PADI4_89, PADI4_90 polymorphisms. We could not confirm an association between susceptibility haplotype presence and ACPA positivity. Further evidence about proteomic expression of this gene will determine its participation in antigenic generation and autoimmunity.

A quantitative flow cytometric assay for determining binding characteristics of chimeric, humanized and human antibodies in whole blood: Proof of principle with rituximab and ofatumumab

Clinical successes of antibody-based drugs has led to extensive (pre-) clinical development of human(ized) monoclonal antibodies in a great number of diseases. The high specificity of targeted therapy with antibodies makes it ideally suited for personalized medicine approaches in which treatments needs are tailored to individual patients. One aspect of patient stratification pertains to the accurate determination of target occupancy and target expression to determine individual pharmacodynamic properties as well as the therapeutic window. The availability of reliable tools to measure target occupancy and expression on diseased and normal cells is therefore essential. Here, we evaluate a novel human antibody detection assay (Human-IgG Calibrator assay), which allows the flow cytometric quantification of therapeutic antibodies bound to the surface of cells circulating in whole blood. This assay not only permits the determination of the number of specific antibody bound per cell (sABC), but, when combined with quantification of exogenously added mouse antibody, also provides information on binding kinetics and antigen modulation. Our data indicate that the calibrator assay has all properties required for a pharmacodynamic tool to quantify target occupancy of chimeric, humanized and human therapeutic antibodies during therapy, as well as to collect valuable information on both antibody and antigen kinetics.

High-yield and scalable cell-free assembly of virus-like particles by dilution

Virus-like particles (VLPs) have been developed as safe and efficacious vaccines, and are increasingly used as carriers for foreign peptide epitopes in modular architectures. An emerging technology for low-cost and rapid-response VLP vaccine manufacture is based on controlled cell-free assembly of capsomeres, which are expressed in Escherichia coli, into VLPs presenting pharmaceutically relevant antigenic modules. A key bioprocessing challenge in this technology is VLP self-assembly, which has until now been studied using qualitative laboratory methods and without sufficient quantitation. In this work, the yield and size distribution of VLPs assembled by dialysis or by ten-fold dilution were compared using quantitative metrics. Membrane-based steps used for dialysis and particle concentration were identified as the key inefficiencies in each method, resulting in 13–18% protein loss. Key inefficiencies were circumvented through process intensification that led to development of a two-fold dilution assembly method. The new process eliminated a unit operation, improved the final concentration of assembly products by a factor of five and reduced buffer consumption nine-fold. Using this process, modular capsomeres presenting a group A Streptococcus (GAS) antigenic module were assembled into high-quality VLPs with a final yield of 54%, which was 18–22 percentage points higher than obtained using conventional methods described in the literature. This study demonstrates the feasibility of manufacturing VLP vaccines in cell-free reactors at high yield, with high structural integrity, using a scalable and simple process. Ongoing development based on these results will be conducive to process-intensified VLP bioprocessing for low-cost vaccine delivery at global scale.

Tolerance induction by monoclonal CD3 antibodies is dependent on indoleamine 2,3 dioxygenase (IDO) (56.5)

Abstract Experimental data demonstrated that a short treatment with CD3 antibodies (CD3 Ab) promotes immune tolerance in primed hosts. Successful clinical transfer of this strategy has been achieved in autoimmune diabetes, and on this basis, CD3 Abs are currently under pharmaceutical development. The tolerogenic effect of CD3 Ab evolves in two consecutive phases: a first phase of “induction” during Ab treatment, involving transient T cell depletion as well as antigenic modulation and a second phase of “maintenance” that persists after CD3 Ab clearance and that correlates with the presence of active and transferable tolerance involving TGF-β dependent Tregs. We focused on the causal links relating the “induction” and “maintenance” phases of tolerance using spontaneously diabetic NOD mice and human CD3ϵ transgenic NOD mice, a novel preclinical model that we established. It is well known that abnormalities in dendritic cells (DCs) contribute to autoimmune diabetes in NOD mice, e.g. indoleamine 2,3 dioxygenase (IDO) induction in CD8+ DCs of NOD mice by IFN-γ is defective. Our results show that depletion of DCs or inhibition of IDO during CD3 Ab therapy abrogates the tolerogenic effect. TGF-β has been shown to induce IDO and we reported that remission from diabetes after CD3 Ab therapy is TGF-β-dependent. We propose that CD3 Ab mediated tolerance ensues from a local immune privilege in the pancreas driven by induction of TGF-β and tolerogenic DCs expressing IDO.

Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.

Modular engineering of a microbially-produced viral capsomere vaccine for influenza

Faster and cheaper vaccine manufacture based on modern technologies is increasingly needed to effectively mitigate the burden of disease caused by highly contagious and mutagenic pathogens, such as influenza viruses. This study describes an approach to synthetically engineer a new influenza vaccine system by antigenic modularization of a carrier viral capsomere, coupled with microbial processing of the sub-unit vaccine. This approach leads to a system optimized with respect to both biological and process criteria. Murine polyomavirus VP1 protein, which self assembles into a pentameric sub-unit capsomere of a virus-like particle (VLP), was engineered to inhibit VLP assembly and to allow modular insertion of influenza M2e antigen at multiple sites within the protein. The yield, solubility, and immunogenicity of the resulting modular capsomeres could be optimized by varying the module insertion site and the number of M2e modules per site. This study demonstrated, for the first time, an innovative strategy of inserting multiple antigenic modules, up to 45 M2e modules, in a single capsomere. Modularization of M2e antigen was shown to improve its immunogenicity by more than an order of magnitude over that attained by immunization with an equivalent mass of non-modularized M2e peptide. Vaccination of mice using modular capsomeres induced high antigen-specific antibody levels suggestive of protective efficacy. This modular vaccine design approach, inspired by synthetic biology approaches to new system development, is conducive to technologies rapidly adaptable to pathogenic variations.

Heterogeneity in clonal nature in the smoldering subtype of adult T-cell leukemia: continuity from carrier status to smoldering ATL

To better understand indeterminate HTLV-1 carriers and smoldering (SM) subtype of adult T-cell leukemia (ATL), HTLV-1 proviral integrated status, proviral load (PVL) and ATL-related biomarkers were examined in 57 smoldering cases, including unusual carriers with a percentage of ATL-like cells. We found that according to Southern blot hybridization analytic features, 28 patients with SM ATL could be divided into 3 groups consisting of 16 (57.4%) patients with a monoclonal band, 6 (21.4%) with oligoclonal bands and the remaining 6 with smears. Although no clinical differences were observed among the 3 SM subtypes, HTLV-1-infected CD4 T-cell counts increased in order of poly-, oligo- and monoclonal subtypes. This trend began in the carrier stage and also was observed in PVL, CD25 and CCR4, indicating that a clone consisting of leukemic phenotypic cells was continuously growing. Moreover, the antigen modulation rates of CD26 and CD7 and the increasing rate of CD25 and CCR4 cells were closely correlated to growing clonal size, indicating that these markers had the possibility to predict a monoclonal band. In particular, CD26 or the ratio of CD26/CD25 had a validity differential for leukemic nature and predictive detection of clonal band. Conclusively, the present study shows that smoldering ATL is heterogeneous in the leukemogenic process, and the behavior of CD26 plays a central role in the evolution from early occult to overt smoldering ATL.

“Mycoplasmal Antigen Modulation,” a Novel Surface Variation Suggested for a Lipoprotein Specifically Localized on Mycoplasma mobile

Mycoplasma mobile, a pathogen of freshwater fish, glides easily across surfaces, colonizes on the fish gill, and causes necrosis. The cell surface is differentiated into three parts: the head, neck, and body. Mobile variable surface proteins (Mvsps) localizing at each of these parts may be involved in surface variation including phase variation and antigenic variation, although no proof exists. In this study, we examined this possibility by focusing on MvspI, the largest Mvsp. Immunofluorescence microscopy showed that MvspI is expressed on the surfaces of all cells. When anti-MvspI antibody was added at concentrations over 0.8 nM, MvspI was observed to decrease over time. After 72 h of cultivation with the antibody, the fluorescence intensity and amount of MvspI decreased up to 13 and 39%, respectively, compared to those of cells grown without antibody. These changes were reversed by the removal of the antibody. Such effects were not observed when another antibody targeting other Mvsps was used, suggesting that the decrease is specific to the relationship between MvspI and the antibody. Cell growth was also inhibited by the antibody, but the decrease in MvspI could not be explained by the selective growth of MvspI-negative variants or by the inhibition of growth with other conditions. The decrease in MvspI caused by the antibody binding may suggest a novel type of surface variation, designated here as "mycoplasmal antigen modulation."

Both activating and inhibitory Fc gamma receptors mediate rituximab-induced trogocytosis of CD20 in mice

Antigenic modulation by trogocytosis during anti-CD20 mAb treatment with rituximab (RTX) leads to loss of CD20 and therefore can compromise therapy. During trogocytosis, effector cells, such as macrophages, remove CD20 from the surface of antibody-coated cells in an Fc receptor-dependent manner. Importantly, Fcγ receptors (FcγRs) are also crucial in the anti-tumor effects of RTX by inducing antibody dependent cell-mediated cytotoxicity (ADCC). Here we studied the role of FcγR during RTX-induced trogocytosis of CD20 in an intraperitoneal tumor model with EL4-CD20 cells. We found marked RTX-induced trogocytosis of CD20 in FcγRI- or FcγRIII-deficient mice, similar to wild type mice, demonstrating a redundancy for activating FcγR in trogocytosis. Interestingly, in FcRγ-chain-deficient mice, trogocytosis was still apparent, indicating that the inhibitory receptor FcγRIIB alone can also mediate trogocytosis. These data were confirmed by in vitro analysis with blocking antibodies. Decreasing the amount of RTX in vivo resulted in less trogocytosis of CD20, supporting clinical studies with lower RTX dose. Importantly, we show that cells which undergo in vivo trogocytosis can still be killed ex vivo by ADCC but not by complement-mediated cytotoxicity (CDC), underscoring the clinical relevance of trogocytosis.Taken together, our study provides more insights into the mechanism and consequences of RTX-induced trogocytosis of CD20.

Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods

Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.

Loss of CD20 and Bound CD20 Antibody from Opsonized B Cells Occurs More Rapidly Because of Trogocytosis Mediated by Fc Receptor-Expressing Effector Cells Than Direct Internalization by the B Cells

We previously reported that 1 h after infusion of CD20 mAb rituximab in patients with chronic lymphocytic leukemia (CLL), >80% of CD20 was removed from circulating B cells, and we replicated this finding, based on in vitro models. This reaction occurs via an endocytic process called shaving/trogocytosis, mediated by FcγR on acceptor cells including monocytes/macrophages, which remove and internalize rituximab-CD20 immune complexes from B cells. Beers et al. reported that CD20 mAb-induced antigenic modulation occurs as a result of internalization of B cell-bound mAb-CD20 complexes by the B cells themselves, with internalization of ∼40% observed after 2 h at 37°C. These findings raise fundamental questions regarding the relative importance of shaving versus internalization in promoting CD20 loss and have substantial implications for the design of mAb-based cancer therapies. Therefore, we performed direct comparisons, based on flow cytometry, to determine the relative rates and extent of shaving versus internalization. B cells, from cell lines, from patients with CLL, and from normal donors, were opsonized with CD20 mAbs rituximab or ofatumumab and incubated for varying times and then reacted with acceptor THP-1 monocytes to promote shaving. We find that shaving induces considerably greater loss of CD20 and bound mAb from opsonized B cells in much shorter time periods (75-90% in <45 min) than is observed for internalization. Both shaving/trogocytosis and internalization could contribute to CD20 loss when CLL patients receive rituximab therapy, but shaving should occur more rapidly and is most likely to be the key mechanism of CD20 loss.

Neutralizing Anti-gH Antibody of Varicella-Zoster Virus Modulates Distribution of gH and Induces Gene Regulation, Mimicking Latency

The anti-glycoprotein H (gH) monoclonal antibody (anti-gH-MAb) that neutralizes varicella-zoster virus (VZV) inhibited cell-to-cell infection, resulting in a single infected cell without apoptosis or necrosis, and the number of infectious cells in cultures treated with anti-gH-MAb declined to undetectable levels in 7 to 10 days. Anti-gH-MAb modulated the wide cytoplasmic distribution of gH colocalized with glycoprotein E (gE) to the cytoplasmic compartment with endoplasmic reticulum (ER) and Golgi markers near the nucleus, while gE retained its cytoplasmic distribution. Thus, the disintegrated distribution of gH and gE caused the loss of cellular infectivity. After 4 weeks of treatment with anti-gH-MAb, no infectious virus was recovered, even after cultivation without anti-gH-MAb for another 8 weeks or various other treatments. Cells were infected with Oka varicella vaccine expressing hepatitis B surface antigen (ROka) and treated with anti-gH-MAb for 4 weeks, and ROka was recovered from the quiescently infected cells by superinfection with the parent Oka vaccine. Among the genes 21, 29, 62, 63, and 66, transcripts of gene 63 were the most frequently detected, and products from the genes 63 and 62, but not gE, were detected mainly in the cytoplasm of quiescently infected cells, in contrast to their nuclear localization in lytically infected cells. The patterns of transcripts and products from the quiescently infected cells were similar to those of latent VZV in human ganglia. Thus, anti-gH-MAb treatment resulted in the antigenic modulation and dormancy of infectivity of VZV. Antigenic modulation by anti-gH-MAb illuminates a new aspect in pathogenesis in VZV infection and the gene regulation of VZV during latency in human ganglia.

The clinical and biological role of CD20 membrane antigen modulation under immunotherapy with anti-CD20 monoclonal antibody rituximab in lymphoprolipherative neoplastic disorders

Immunotherapy using an antibody (rituximab) targeting CD20 antigen in combination with chemotherapy has been recently associated with significantly improved response rate and survival in patients with various types of CD20-positive B-cell lymphoproliferative disorders. This treatment may induce the disappearance of CD20 surface expression on neoplastic B-cells. Several mechanisms have been proposed to explain CD20 loss after rituximab therapy, while the clinical significance (if any) of this phenomenon is still not clear. We have produced a brief overview of the biological aspects of CD20 modulation after rituximab treatment and its possible clinical implications.

Reverse Genetics Modification of Cytomegalovirus Antigenicity and Immunogenicity by CD8 T‐Cell Epitope Deletion and Insertion

The advent of cloning herpesviral genomes as bacterial artificial chromosomes (BACs) has made herpesviruses accessible to bacterial genetics and has thus revolutionised their mutagenesis. This opened all possibilities of reverse genetics to ask scientific questions by introducing precisely accurate mutations into the viral genome for testing their influence on the phenotype under study or to create phenotypes of interest. Here, we report on our experience with using BAC technology for a designed modulation of viral antigenicity and immunogenicity with focus on the CD8 T-cell response. One approach is replacing an intrinsic antigenic peptide in a viral carrier protein with a foreign antigenic sequence, a strategy that we have termed “orthotopic peptide swap”. Another approach is the functional deletion of an antigenic peptide by point mutation of its C-terminal MHC class-I anchor residue. We discuss the concepts and summarize recently published major scientific results obtained with immunological mutants of murine cytomegalovirus.

Weak Sensitivity of Myeloma Cells to CD38 or CD138-ADCC

AbstractAbstract 4072The development of novel treatments such as those based on human monoclonal antibodies (mAb) are currently being evaluated in preclinical and early clinical studies in cancers. There is convincing evidence supporting a key role for antibody-dependent cell-mediated cytotoxicity (ADCC) in the clinical response of several therapeutic monoclonal antibodies. In myeloma, CD38 and CD138, both highly expressed by all myeloma cells, represent the best targets for ADCC and humanized Abs are currently in development for both antigens. The aim of the present study was to assess the CD38 and CD138-ADCC sensitivity of a large panel of human myeloma cell lines (HMCLs) representative of the disease heterogeneity.To reproductively assess ADCC, we used mouse FcgRIII expressing human T lymphocytes as cytotoxic effector cells. We measured ADCC sensitivity of 20 HMCLs with two mouse IgG1 anti-CD38 (clones T16 and HIT2), with two mouse IgG1 anti-CD138 (clones BB4 and MI15) and with a mouse IgG2a anti-HLA class-I (clone W6.32) by a standard 51Cr release assays performed at an effector to target ratio of 10:1.Unexpectedly, despite high level of CD38, only 3 cells lines among 20 were sensitive to anti-CD38/ADCC. No correlation was observed between anti-CD38/ADCC sensibility and the CD38 expression level. No correlation was observed too between sensitivity and representative molecular heterogeneity of HMCLs. The absence anti-CD38/ADCC was not due to intrinsic resistance lysis through ADCC since these cell lines were lysed when using an anti-HLA class-I mAb (or anti-CD20 mAbs for one CD20+ HMCL). Furthermore, absence of ADCC was not due to antibody-induced antigenic modulation. Surprisingly too, none HMCL was lysed by anti-CD138 mAbs despite high expression of CD138. Moreover, primary myeloma cells purified from peripheral blood of a patient were also resistant to both anti-CD38 and anti-CD138 mAbs.These results show that ADCC activity mediated by a particular monoclonal antibody varies according to the target cell and independently of the antigen expression level, suggesting that the antigen “environment” might affect the interactions between antibody, Fc receptor and effector cells. Further studies will be required to determine the CD38 and CD138 “environment” factors on myeloma cell that can influence ADCC. Disclosures:No relevant conflicts of interest to declare.

Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection

AbstractRituximab, a monoclonal antibody that targets CD20 on B cells, is now central to the treatment of a variety of malignant and autoimmune disorders. Despite this success, a substantial proportion of B-cell lymphomas are unresponsive or develop resistance, hence more potent anti-CD20 monoclonal antibodies (mAbs) are continuously being sought. Here we demonstrate that type II (tositumomab-like) anti-CD20 mAbs are 5 times more potent than type I (rituximab-like) reagents in depleting human CD20 Tg B cells, despite both operating exclusively via activatory Fcγ receptor–expressing macrophages. Much of this disparity in performance is attributable to type I mAb-mediated internalization of CD20 by B cells, leading to reduced macrophage recruitment and the degradation of CD20/mAb complexes, shortening mAb half-life. Importantly, human B cells from healthy donors and most cases of chronic lymphatic leukemia and mantle cell lymphoma, showed rapid CD20 internalization that paralleled that seen in the Tg mouse B cells, whereas most follicular lymphoma and diffuse large B-cell lymphoma cells were far more resistant to CD20 loss. We postulate that differences in CD20 modulation may play a central role in determining the relative efficacy of rituximab in treating these diseases and strengthen the case for focusing on type II anti-CD20 mAb in the clinic.

In vitro characterization of an acetylcholine receptor–transferrin fusion protein for the treatment of myasthenia gravis

SHG2210, a fusion protein containing the N-terminus of human nicotinic acetylcholine receptor α (AchR-α; aa1-210) and human transferrin (TF), was characterized as a potential therapeutic for myasthenia gravis (MG) caused predominately by α subunit autoantibodies. SHG2210 was shown to be able to bind to α subunit autoantibodies and the TF receptor (TFR). SHG2210 and SHG2210–anti-AchR antibody complex are internalized through TFR-mediated endocytosis. The SHG2210 and SHG2210–anti-AchR antibody complex is present in Lamp1-positive lysosomal compartments after internalization; however, neither SHG2210 nor SHG2210–antibody complex is present in Rab11-positive recycling endosomes. SHG2210 bound to α subunit of AChR autoantibodies may be cleared by the lysosome, resulting in short cellular half-life relative to SHG2210. SHG2210 is shown to have a protective effect on antigenic modulation of the AChR induced by serum from select patients with MG, suggesting that a fusion protein approach may be an effective therapeutic for treating MG.

CXCR4 Mediated Chemotaxis Is Regulated by 5T4 Oncofetal Glycoprotein in Mouse Embryonic Cells

5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site.