Novel signatures associated with systemic lupus erythematosus clinical response to IFN-α/-ω inhibition.

OBJECTIVES: We aimed to identify transcriptional gene signatures predictive of clinical response, for pharmacodynamic evaluation, and to provide mechanistic insight into JNJ-55920839, a human IgG1κ neutralizing mAb targeting IFN-α/IFN-ω, in participants with systemic lupus erythematosus (SLE). METHODS: Blood samples were obtained from SLE participants at baseline and up to Day 130, who received six 10 mg/kg IV doses of JNJ-55920839/placebo every 2 weeks. Participants with mild-to-moderate SLE who achieved clinical responses using SLE Disease Activity Index 2000 Responder Index 4-point change were considered responders. Transcriptional signatures from longitudinally collected blood were generated by RNA-Seq; signatures were generated by microarray from baseline blood samples exposed in vitro to JNJ-55920839 versus untreated. RESULTS: Two gene signatures (IFN-I Signaling and Immunoglobulin Immune Response) exhibited pharmacodynamic changes among JNJ-55920839 responders. The Immunoglobulin signature, but not the IFN-I signature, was elevated at baseline in JNJ-55920839 responders. A gene cluster associated with neutrophil-mediated immunity was reduced at baseline in JNJ-55920839 responders, substantiated by lower neutrophil counts in responders. An IFN-I signature was suppressed by JNJ-55920839 in vitro treatment versus untreated blood to a greater extent in responders before in vivo dosing. CONCLUSIONS: These signatures may enable enrichment for treatment responders when using IFN-I-suppressing treatments in SLE.

Added benefit of raxibacumab to antibiotic treatment of inhalational anthrax.

Although antibiotics treat bacteremia in inhalational anthrax, pathogenesis is mainly driven by bacterial exotoxins. Raxibacumab, an IgG1 monoclonal antibody, binds the protective antigen (PA) of Bacillus anthracis, thus blocking toxin effects and leading to improved survival in the rabbit and monkey models of inhalational anthrax. To assess raxibacumab's added benefit over levofloxacin (LVX) alone, rabbits surviving to 84 h after a challenge with 200 times the median (50%) lethal dose of B. anthracis spores were randomized to receive 3 daily intragastric LVX doses of 50 mg/kg of body weight, with the first LVX dose administered just prior to administration of a single intravenous dose of placebo or 40 mg/kg raxibacumab. The percentages of animals alive at 28 days following the last LVX dose were compared between the 2 treatment groups using a two-sided likelihood-ratio chi-square test. The 82% survival rate for the LVX-raxibacumab combination was higher than the 65% survival rate for LVX alone (P=0.0874). There were nearly 2-fold fewer deaths for the combination (7 deaths; n=39) than for LVX alone (13 deaths; n=37), and the survival time was prolonged for the combination (P=0.1016). Toxin-neutralizing-activity titers were similar for both treatment groups, suggesting that survivors in both groups were able to mount a toxin-neutralizing immune response. Microscopic findings considered consistent with anthrax were present in animals that died or became moribund on study in both treatment groups, and there were no anthrax-related findings in animals that survived. Overall, raxibacumab provided a meaningful benefit over antibiotic alone when administered late in the disease course.

Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs.

Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.

Development of systemic lupus erythematosus in NZM 2328 mice in the absence of any single BAFF receptor.

OBJECTIVE: To determine the necessity for any individual BAFF receptor in the development of systemic lupus erythematosus (SLE). METHODS: Bcma-, Taci-, and Br3-null mutations were introgressed into NZM 2328 mice. NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice were evaluated for lymphocyte phenotype and BAFF receptor expression by flow cytometry; for B cell responsiveness to BAFF by in vitro culture; for serum levels of BAFF and total IgG and IgG anti-double-stranded DNA (anti-dsDNA) by enzyme-linked immunosorbent assay; for renal immunopathology by immunofluorescence and histopathology; and for clinical disease. RESULTS: BCMA, TACI, and B lymphocyte stimulator receptor 3 (BR3) were not surface-expressed in NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice, respectively. Transitional and follicular B cells from NZM.Br3-/- mice were much less responsive to BAFF than were the corresponding cells from wild-type, NZM.Bcma-/-, or NZM.Taci-/- mice. In comparison with wild-type mice, NZM.Bcma-/- and NZM.Taci-/- mice harbored an increased number of spleen B cells, T cells, and plasma cells, whereas serum levels of total IgG and IgG anti-dsDNA were similar to those in wild-type mice. Despite their paucity of B cells, NZM.Br3-/- mice had an increased number of T cells, and the numbers of plasma cells and levels of IgG anti-dsDNA were similar to those in wild-type mice. Serum levels of BAFF were increased in NZM.Taci-/- and NZM.Br3-/- mice but were decreased in NZM.Bcma-/- mice. Despite their phenotypic differences, NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice had renal immunopathology and clinical disease that were at least as severe as that in wild-type mice. CONCLUSION: Any single BAFF receptor, including BR3, is dispensable for the development of SLE in NZM mice. Development of disease in NZM.Br3-/- mice demonstrates that BAFF-BCMA and/or BAFF-TACI interactions contribute to SLE, and that a profound, life-long reduction in the numbers of B cells does not guarantee protection against SLE.

Evaluating the safety, tolerability, and immunogenicity of a 24-valent pneumococcal conjugate vaccine (VAX-24) in healthy adults aged 18 to 64 years: a phase 1/2, double-masked, dose-finding, active-controlled, randomised clinical trial.

BACKGROUND: Despite substantial reductions in pneumococcal disease with the availability of pneumococcal conjugate vaccines, a significant burden of pneumococcal disease remains due to the diversity of serotypes combined with serotype replacement. We developed a new vaccine candidate, VAX-24 (24-valent pneumococcal conjugate vaccine), using cell-free protein synthesis to produce a variant of cross-reactive material 197 (eCRM) as the carrier protein, increasing serotype coverage while minimising carrier suppression. The aim of this clinical trial was to assess the safety, tolerability, and immunogenicity of three different doses of VAX-24 compared to pneumococcal 20-valent conjugate vaccine (PCV20). METHODS: This was a phase 1/2, randomised, double-masked study of VAX-24 versus PCV20 conducted in the USA. Key inclusion criteria included being a male or female aged 18 to 64 years in good health; key exclusion criteria included previous history of pneumococcal disease, receipt of a licensed or investigational pneumococcal vaccine, or immunosuppressive therapy. Participants were randomly allocated in a 1:1:1:1 ratio by permuted block to receive one dose of VAX-24 (1·1 µg of each antigen, 2·2 µg of each antigen, or 2·2 µg of 17 antigens mixed with 4·4 µg of seven antigens), or PCV20. The safety population included all participants with safety data. The immunogenicity population was as per-treatment in phase 2. Primary outcome measures included solicited and unsolicited adverse events. Secondary outcomes included serotype-specific opsonophagocytic activity (OPA) geometric mean titres (GMT), and IgG geometric mean concentrations (GMC) were measured 1 month postvaccination. Traditional non-inferiority criteria included OPA geometric mean ratio (GMR), with a lower bound of the two sided 95% CI of greater than 0·5 for shared serotypes. This completed trial is registered at ClinicalTrials.gov, NCT05266456. FINDINGS: Safety profiles were comparable among the treatment groups, with 170 of 209 participants (81%, 95% CI 75·2-86·2) to 178 of 207 participants (86%, 80·5-90·4) reporting at least one solicited adverse event among the three VAX-24 groups. 24 of 207 participants (12%, 7·6-16·8) to 32 of 209 of participants (15%, 10·7-20·9) experiened an unsolicited treatment emergent adverse event within 1 month postvaccination. VAX-24 2·2 µg met traditional OPA GMR non-inferiority criteria for all 20 shared serotypes; 16 serotypes elicited GMR point estimates greater than 1·0, and four reached the lower bound of the two-sided 95% CI greater than 1·0. INTERPRETATION: VAX-24 had a safety profile similar to PCV20 at all doses, with the 2·2 µg dose showing increased serotype coverage with decreased carrier suppression. FUNDING: Vaxcyte.

Dispensability of APRIL to the development of systemic lupus erythematosus in NZM 2328 mice.

OBJECTIVE: To determine the role of APRIL in the development of systemic lupus erythematosus (SLE) in mice. METHODS: Wild-type (WT) NZM 2328, NZM. April(-/-) , NZM.Baff(-/-) , and NZM.Baff(-/-) .April(-/-) mice were evaluated for lymphocyte phenotype by flow cytometry, for serum total IgG and IgG autoantibody levels by enzyme-linked immunosorbent assay, for glomerular deposition of IgG and C3 by immunofluorescence, for renal changes by histopathology, and for clinical disease by laboratory assessment (severe proteinuria). RESULTS: In comparison to WT mice, NZM.April(-/-) mice harbored increased spleen B cells, T cells, and plasma cells (PCs), increased serum levels of IgG antichromatin antibodies, and decreased numbers of bone marrow (BM) PCs. Glomerular deposition of IgG and C3 was similar in NZM.April(-/-) mice and WT mice, renal changes on histopathology tended to be more severe in NZM.April(-/-) mice than in WT mice, and development of clinical disease was identical in NZM.April(-/-) mice and WT mice. BM (but not spleen) PCs and serum IgG antichromatin and anti-double-stranded DNA antibody levels were lower in NZM.Baff(-/-) .April(-/-) mice than in NZM.Baff(-/-) mice, whereas renal immunopathology in each cohort was equally mild. CONCLUSION: APRIL is dispensable for the development of full-blown SLE in NZM mice. Moreover, the elimination of both APRIL and BAFF had no discernible effect on the development of renal immunopathology or clinical disease beyond that of elimination of BAFF alone. The reduction in BM PCs in hosts doubly deficient in APRIL and BAFF beyond that in hosts deficient only in BAFF raises concern that combined antagonism of APRIL and BAFF may lead to greater immunosuppression without a concomitant increase in therapeutic efficacy.

Belimumab reduces autoantibodies, normalizes low complement levels, and reduces select B cell populations in patients with systemic lupus erythematosus.

OBJECTIVE: To assess the effects of the B lymphocyte stimulator (BLyS)-specific inhibitor belimumab on immunologic biomarkers, including B cell and T cell populations, and maintenance of antibody titers to prior vaccines in autoantibody-positive systemic lupus erythematosus (SLE) patients. METHODS: Pooled data from 2 phase III trials, the Study of Belimumab in Subjects with SLE 52-week (BLISS-52) and 76-week (BLISS-76) trials, comparing belimumab 1 mg/kg or 10 mg/kg versus placebo (plus standard SLE therapy for each group) were analyzed for changes in autoantibody, immunoglobulin, and complement levels. BLISS-76 patients were also analyzed for changes in B cell and T cell populations and effects on prior vaccine-induced antibody levels. RESULTS: Belimumab-treated patients experienced significant sustained reductions in IgG and autoantibodies and improvement in C3/C4 levels, resulting in greater positive-to-negative conversion rates for IgG anti-double-stranded DNA (anti-dsDNA), anti-Sm, anticardiolipin, and anti-ribosomal P autoantibodies and normalization of hypergammaglobulinemia and low C3/C4 levels. Belimumab-treated patients experienced significant decreases in the numbers of naive and activated B cells, as well as plasma cells, whereas memory B cells and T cell populations did not decrease. Belimumab did not substantially affect preexisting antipneumococcal or anti-tetanus toxoid antibody levels. Post hoc analysis showed greater reductions in SLE disease activity and the risk of severe flares in patients treated with belimumab 10 mg/kg (P≤0.01) who were anti-dsDNA positive and had low C3/C4 levels at baseline. Normalization of the C3 or anti-dsDNA level by 8 weeks, irrespective of therapy, was predictive of a reduced risk of severe flare over 52 weeks. CONCLUSION: Belimumab appears to promote normalization of serologic activity and reduce BLyS-dependent B cell subsets in serologically and clinically active SLE. Greater serologic activity may predict a better treatment response to belimumab.

Cellular competition independent of BAFF/B lymphocyte stimulator results in low frequency of an autoreactive clonotype in mature polyclonal B cell compartments.

The peripheral B cell prosurvival cytokine BAFF/B lymphocyte stimulator (BLyS) has been proposed to participate in the regulation of immunological tolerance. Selective elimination or reconstitution of B cells expressing transgene-encoded, autoreactive BCRs upon systemic BLyS depletion or supplementation, respectively, was observed in two separate studies. Such findings led to a model positing a higher dependency of autoreactive B cells on BLyS. We tested this model by exploiting two targeted IgH transgenic mice (H chain knock-in [HKI]) that produce large numbers of follicular (FO) B cells that are either weakly or strongly autoreactive with nuclear autoantigens. Even though HKI B cells do not exhibit classical features of anergy, we found that mature, naive, autoreactive HKI B cells are outcompeted for representation in the periphery by a polyclonal B cell population. However, this is not due to a higher dependency of HKI B cells on BLyS for survival. Additionally, excess BLyS does not rescue HKI B cells from selective elimination. These findings suggest that some autoreactive FO B cells can fully develop while in competition with non-autoreactive cells for BLyS, but remain at a competitive disadvantage for other trophic factors that regulate peripheral stability. As such, our data indicate the existence of peripheral tolerance mechanisms that regulate the frequency of autoreactive FO B cells independent of the BLyS pathway.

Protective antigen antibody augments hemodynamic support in anthrax lethal toxin shock in canines.

BACKGROUND: Anthrax-associated shock is closely linked to lethal toxin (LT) release and is highly lethal despite conventional hemodynamic support. We investigated whether protective antigen-directed monoclonal antibody (PA-mAb) treatment further augments titrated hemodynamic support. METHODS AND RESULTS: Forty sedated, mechanically ventilated, instrumented canines challenged with anthrax LT were assigned to no treatment (controls), hemodynamic support alone (protocol-titrated fluids and norepinephrine), PA-mAb alone (administered at start of LT infusion [0 hours] or 9 or 12 hours later), or both, and observed for 96 hours. Although all 8 controls died, 2 of 8 animals receiving hemodynamic support alone survived (median survival times 65 vs 85 hours, respectively; P = .03). PA-mAb alone at 0 hour improved survival (5 of 5 animals survived), but efficacy decreased progressively with delayed treatment (9 hours, 2 of 3 survived; 12 hours, 0 of 4 survived) (P = .004 comparing survival across treatment times). However, combined treatment increased survival irrespective of PA-mAb administration time (0 hours, 4 of 5 animals; 9 hours, 3 of 3 animals; and 12 hours, 4 of 5 animals survived) (P = .95 comparing treatment times). Compared to hemodynamic support alone, when combined over PA-mAb treatment times (0, 9, and 12 hours), combination therapy produced higher survival (P = .008), central venous pressures, and left ventricular ejection fractions, and lower heart rates, norepinephrine requirements and fluid retention (P ≤ .03). CONCLUSIONS: PA-mAb may augment conventional hemodynamic support during anthrax LT-associated shock.

Raxibacumab for the treatment of inhalational anthrax.

BACKGROUND: Inhalational anthrax caused by Bacillus anthracis is associated with high mortality primarily due to toxin-mediated injury. Raxibacumab is a human IgG1lambda monoclonal antibody directed against protective antigen, a component of the anthrax toxin. METHODS: We evaluated the efficacy of raxibacumab as a prophylactic agent and after disease onset in a total of four randomized, placebo-controlled studies conducted in rabbits and monkeys. Animals were exposed to an aerosolized target exposure of B. anthracis spores that was approximately 100 times (in the prophylactic studies) and 200 times (in the therapeutic-intervention studies) the median lethal dose. In the therapeutic-intervention studies, animals were monitored for the onset of symptoms. Animals with detectable protective antigen in serum, a significant increase in temperature, or both received a single intravenous bolus of placebo or raxibacumab at a dose of either 20 mg per kilogram of body weight or 40 mg per kilogram. The primary end point was survival at day 14 (in rabbits) or at day 28 (in monkeys). Safety studies were conducted with intravenous raxibacumab (40 mg per kilogram) in 333 healthy human volunteers. RESULTS: In both rabbits and monkeys, the time to detection of protective antigen correlated with the time to bacteremia (r=0.9, P<0.001). In the therapeutic-intervention studies, the survival rate was significantly higher among rabbits that received raxibacumab at a dose of 40 mg per kilogram (44% [8 of 18]) than among rabbits that received placebo (0% [0 of 18]; P=0.003). Raxibacumab treatment also significantly increased survival in monkeys (64% [9 of 14], vs. 0% [0 of 12] with placebo; P<0.001). In human subjects, intravenous raxibacumab at a dose of 40 mg per kilogram had a half-life of 20 to 22 days and provided a maximum concentration of the drug in excess of levels that are protective in animals. Concentrations of raxibacumab provide a surrogate end point that should be predictive of clinical benefit. CONCLUSIONS: A single dose of raxibacumab improved survival in rabbits and monkeys with symptomatic inhalational anthrax. (ClinicalTrials.gov number, NCT00639678.)

B-cell homeostasis requires complementary CD22 and BLyS/BR3 survival signals.

Peripheral B-cell numbers are tightly regulated by homeostatic mechanisms that influence the transitional and mature B-cell compartments and dictate the size and clonotypic diversity of the B-cell repertoire. B-lymphocyte stimulator (BLyS, a trademark of Human Genome Sciences, Inc.) plays a key role in regulating peripheral B-cell homeostasis. CD22 also promotes peripheral B-cell survival through ligand-dependent mechanisms. The B-cell subsets affected by the absence of BLyS and CD22 signals overlap, suggesting that BLyS- and CD22-mediated survival are intertwined. To examine this, the effects of BLyS insufficiency following neutralizing BLyS mAb treatment in mice also treated with CD22 ligand-blocking mAb were examined. Combined targeting of the BLyS and CD22 survival pathways led to significantly greater clearance of recirculating bone marrow, blood, marginal zone and follicular B cells than either treatment alone. Likewise, BLyS blockade further reduced bone marrow, blood and spleen B-cell numbers in CD22(-/-) mice. Notably, BLyS receptor expression and downstream signaling were normal in CD22(-/-) B cells, suggesting that CD22 does not directly alter BLyS responsiveness. CD22 survival signals were likewise intact in the absence of BLyS, as CD22 mAb treatment depleted blood B cells from mice with impaired BLyS receptor 3 (BR3) signaling. Finally, enforced BclxL expression, which rescues BR3 impairment, did not affect B-cell depletion following CD22 mAb treatment. Thus, the current studies support a model whereby CD22 and BLyS promote the survival of overlapping B-cell subsets but contribute to their maintenance through independent and complementary signaling pathways.

BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact.

We have used an inhibiting antibody to determine whether preimmune versus antigen-experienced B cells differ in their requisites for BLyS, a cytokine that controls differentiation and survival. Whereas in vivo BLyS inhibition profoundly reduced naïve B cell numbers and primary immune responses, it had a markedly smaller effect on memory B cells and long-lived plasma cells, as well as secondary immune responses. There was heterogeneity within the memory pools, because IgM-bearing memory cells were sensitive to BLyS depletion whereas IgG-bearing memory cells were not, although both were more resistant than naïve cells. There was also heterogeneity within B1 pools, as splenic but not peritoneal B1 cells were diminished by anti-BLyS treatment, yet the number of natural antibody-secreting cells remained constant. Together, these findings show that memory B cells and natural antibody-secreting cells are BLyS-independent and suggest that these pools can be separately manipulated.

BLyS neutralization results in selective anti-HLA alloantibody depletion without successful desensitization.

Pre-existing anti-HLA allo-antibodies (allo-Abs) are a major barrier to successful kidney transplantation, resulting in an elevated risk for antibody-mediated rejection (AMR) and eventual graft loss. The cytokine B lymphocyte stimulator (BLyS) promotes B cell maturation and plasma cell survival; consequently, anti-BLyS therapy represents a potential therapeutic opportunity in diminishing pre-existing allo-Abs. Here we report that in our 1-year pilot trial, BLyS neutralization failed to reduce total anti-HLA allo-Ab levels in highly sensitized candidates awaiting kidney transplant in a clinically meaningful way. Additionally, we performed a post hoc analysis using sera from trial candidates which revealed selective depletion of anti-HLA class I and class II Abs in response to belimumab treatment, restricted to certain allele specificities and IgG subclasses. Altogether, we observed that BLyS blockade only results in selective depletion of anti-HLA Abs recognizing a few discrete HLA allele specificities.

Non-clinical immunological comparison of a Next-Generation 24-valent pneumococcal conjugate vaccine (VAX-24) using site-specific carrier protein conjugation to the current standard of care (PCV13 and PPV23).

Despite widespread utilization of pneumococcal conjugate vaccines (PCVs) and the resultant disease reduction, the development of PCVs containing additional serotypes remains a public health priority due to serotype replacement and the resultant shift to non-vaccine containing serotypes. However, incorporating additional serotypes to existing PCVs using conventional technologies has proven problematic. Immune responses to individual serotypes have consistently decreased as more polysaccharide-conjugates are added due to carrier suppression. Using our proprietary cell-free protein synthesis (CFPS) platform, we have successfully produced eCRM® based on the CRM197 sequence for use as an enhanced carrier protein to develop a 24-valent PCV. The eCRM carrier protein contains multiple non-native amino acids (nnAAs) located outside of the primary T-cell epitope regions, thereby enabling site-specific covalent conjugation of the pneumococcal polysaccharides to the nnAAs to consistently expose the critical T-cell epitopes. eCRM also serves to reduce structural heterogeneity associated with classic reductive-amination conjugation while promoting formation of the conjugate matrix structures, the hallmark of PCVs. This process serves to increase the overall polysaccharide:protein ratio, enabling the inclusion of more serotypes while minimizing carrier-mediated immunological interference. The aim of this non-clinical study was to construct a 24-valent PCV and evaluate its immunogenicity. Using the XPressCF® CFPS platform, the eCRM carrier protein was separately conjugated through nnAAs to each of the 24 pneumococcal polysaccharides through click chemistry and mixed with aluminum phosphate to produce VAX-24, Vaxcyte's proprietary PCV preclinical candidate. VAX-24, Prevnar13® and Pneumovax®23 were administered to New Zealand White rabbits to compare the resulting opsonophagocytic activity (OPA) and anti-capsular IgG antibodies. VAX-24 showed conjugate-like immune responses to all 24 serotypes based on comparable OPA and IgG responses to Prevnar13 and higher responses than Pneumovax 23. This study demonstrates the utility of site-specific conjugation technology in a preclinical setting and the potential for a PCV with improved serotype coverage.

In vivo BLyS/BAFF neutralization ameliorates islet-directed autoimmunity in nonobese diabetic mice.

B lymphocytes are required for the pathogenesis of autoimmune diabetes in NOD mice. Previous studies established that a lymphopenic transitional (TR) B cell compartment reduces the competitive constraint on the entry of newly emerging TR B cells into the splenic follicle (FO), thereby disrupting a peripheral negative selection checkpoint in NOD mice. Thus, development of clinically feasible immunotherapeutic approaches for restoration of appropriate negative selection is essential for the prevention of anti-islet autoimmunity. In this study we hypothesized that in vivo neutralization of the B lymphocyte stimulator (BLyS/BAFF) may enhance the stringency of TR-->FO selection by increasing TR B cell competition for follicular entry in NOD mice. This study demonstrated that in vivo BLyS neutralization therapy leads to the depletion of follicular and marginal zone B lymphocytes. Long-term in vivo BLyS neutralization caused an increased TR:FO B cell ratio in the periphery indicating a relative resistance to follicular entry. Moreover, in vivo BLyS neutralization: 1) restored negative selection at the TR-->FO checkpoint, 2) abrogated serum insulin autoantibodies, 3) reduced the severity of islet inflammation, 4) significantly reduced the incidence of spontaneous diabetes, 5) arrested the terminal stages of islet cell destruction, and 6) disrupted CD4 T cell activation in NOD mice. Overall, this study demonstrates the efficacy of B lymphocyte-directed therapy via in vivo BLyS neutralization for the prevention of autoimmune diabetes.

Circulating TNF-like protein 1A (TL1A) is elevated early in rheumatoid arthritis and depends on TNF.

BACKGROUND: The tumor necrosis factor (TNF) superfamily cytokine TNF-like protein 1A (TL1A) and its receptor DR3 are essential for diverse animal models of autoimmune disease and may be pathogenic in rheumatoid arthritis (RA). However, the relationship of TL1A to disease duration, activity, and response to anti-TNF and other therapies in RA is not clear. METHODS: We measured soluble TL1A in synovial fluid (SF), serum, or plasma from RA first-degree relatives (FDRs) and in early RA and established disease. We measured the effects of anti-TNF and methotrexate (MTX) therapy on circulating TL1A from multiple independent RA treatment trials. We also determined the ability of a blocking anti-TL1A antibody to inhibit clinical disease and articular bone destruction in the murine collagen-induced arthritis (CIA) model of human RA. RESULTS: Soluble TL1A was specifically elevated in the blood and SF of patients with RA compared to patients with other diseases and was elevated early in disease and in at-risk anti-cyclic citrullinated peptide (CCP) (+) first-degree relatives (FDRs). Therapeutic TNF inhibition reduced serum TL1A in both responders and non-responders, whereas TL1A declined following MTX treatment only in responders. In murine CIA, TL1A blockade was clinically efficacious and reduced bone erosions. CONCLUSIONS: TL1A is specifically elevated in RA from early in the disease course and in at-risk FDRs. The decline in TL1A after TNF blockade suggests that TL1A levels may be a useful biomarker for TNF activity in RA. These results support the further investigation of the relationship between TL1A and TNF and TL1A blockade as a potential therapeutic strategy in RA.

Treating Tissue Factor-Positive Cancers with Antibody-Drug Conjugates That Do Not Affect Blood Clotting.

The primary function of tissue factor (TF) resides in the vasculature as a cofactor of blood clotting; however, multiple solid tumors aberrantly express this transmembrane receptor on the cell surface. Here, we developed anti-TF antibody-drug conjugates (ADC) that did not interfere with the coagulation cascade and benchmarked them against previously developed anti-TF ADCs. After screening an affinity-matured antibody panel of diverse paratopes and affinities, we identified one primary paratope family that did not inhibit conversion of Factor X (FX) to activated Factor X (FXa) and did not affect conversion of prothrombin to thrombin. The rest of the antibody panel and previously developed anti-TF antibodies were found to perturb coagulation to varying degrees. To compare the anticancer activity of coagulation-inert and -inhibitory antibodies as ADCs, a selection of antibodies was conjugated to the prototypic cytotoxic agent monomethyl auristatin E (MMAE) through a protease-cleavable linker. The coagulation-inert and -inhibitory anti-TF ADCs both killed cancer cells effectively. Importantly, the coagulation-inert ADCs were as efficacious as tisotumab vedotin, a clinical stage ADC that affected blood clotting, including in patient-derived xenografts from three solid tumor indications with a need for new therapeutic treatments-squamous cell carcinoma of the head and neck (SCCHN), ovarian, and gastric adenocarcinoma. Furthermore, a subset of the anti-TF antibodies could also be considered for the treatment of other diseases associated with upregulation of membranous TF expression, such as macular degeneration. Mol Cancer Ther; 17(11); 2412-26. ©2018 AACR.

Prognostic significance of serum B-lymphocyte stimulator in Hodgkin's lymphoma.

B-lymphocyte stimulator (BLyS) plays a critical role in the survival of B-lymphocytes. In 50 patients with Hodgkin's lymphoma BLyS levels were higher in newly diagnosed patients (median 2.0 ng/mL, range <0.3-56.0) and relapsed patients (8.7 ng/mL, range 1.5-71.5) than in 93 healthy donors (<0.3 ng/mL, range <0.3-0.5). High serum BLyS levels (> or =2.0 ng/mL) in newly diagnosed patients were associated with resistance to therapy (p=0.01) and shorter progression-free survival (log-rank p=0.029, 2-year rate 64% vs 100%). Serum BLyS levels may have prognostic significance in Hodgkin's lymphoma.

TIP, a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model.

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.

Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation.

We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21-mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (TFH cells) provide a local source of BLyS. Whereas T cell-derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on TFH-derived BLyS for their persistence.