ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells.

Despite recent advances in immunotherapies targeting single tumor-associated antigens, patients with multiple myeloma eventually relapse. ISB 2001 is a CD3+ T cell engager (TCE) co-targeting BCMA and CD38 designed to improve cytotoxicity against multiple myeloma. Targeting of two tumor-associated antigens by a single TCE resulted in superior cytotoxic potency across a variable range of BCMA and CD38 tumor expression profiles mimicking natural tumor heterogeneity, improved resistance to competing soluble factors and exhibited superior cytotoxic potency on patient-derived samples and in mouse models. Despite the broad expression of CD38 across human tissues, ISB 2001 demonstrated a reduced T cell activation profile in the absence of tumor cells when compared to TCEs targeting CD38 only. To determine an optimal first-in-human dose for the ongoing clinical trial ( NCT05862012 ), we developed an innovative quantitative systems pharmacology model leveraging preclinical data, using a minimum pharmacologically active dose approach, therefore reducing patient exposure to subefficacious doses of therapies.

Preclinical characterization of ISB 1342, a CD38 × CD3 T-cell engager for relapsed/refractory multiple myeloma.

Although treatment of multiple myeloma (MM) with daratumumab significantly extends the patient's lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/r MM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high-affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38, including those with a lower sensitivity to daratumumab. In a killing assay where multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity toward MM cells compared with daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in 2 therapeutic mouse models, unlike daratumumab. Finally, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/r MM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently being developed in a phase 1 clinical study.

Increased efflux of amyloid-ß peptides through the blood-brain barrier by muscarinic acetylcholine receptor inhibition reduces pathological phenotypes in mouse models of brain amyloidosis.

The formation and accumulation of toxic amyloid-ß peptides (Aß) in the brain may drive the pathogenesis of Alzheimer's disease. Accordingly, disease-modifying therapies for Alzheimer's disease and related disorders could result from treatments regulating Aß homeostasis. Examples are the inhibition of production, misfolding, and accumulation of Aß or the enhancement of its clearance. Here we show that oral treatment with ACI-91 (Pirenzepine) dose-dependently reduced brain Aß burden in AßPPPS1, hAßPPSL, and AßPP/PS1 transgenic mice. A possible mechanism of action of ACI-91 may occur through selective inhibition of muscarinic acetylcholine receptors (AChR) on endothelial cells of brain microvessels and enhanced Aß peptide clearance across the blood-brain barrier. One month treatment with ACI-91 increased the clearance of intrathecally-injected Aß in plaque-bearing mice. ACI-91 also accelerated the clearance of brain-injected Aß in blood and peripheral tissues by favoring its urinal excretion. A single oral dose of ACI-91 reduced the half-life of interstitial Aß peptide in pre-plaque mhAßPP/PS1d mice. By extending our studies to an in vitro model, we showed that muscarinic AChR inhibition by ACI-91 and Darifenacin augmented the capacity of differentiated endothelial monolayers for active transport of Aß peptide. Finally, ACI-91 was found to consistently affect, in vitro and in vivo, the expression of endothelial cell genes involved in Aß transport across the Blood Brain Brain (BBB). Thus increased Aß clearance through the BBB may contribute to reduced Aß burden and associated phenotypes. Inhibition of muscarinic AChR restricted to the periphery may present a therapeutic advantage as it avoids adverse central cholinergic effects.

Efficacy and safety of a liposome-based vaccine against protein Tau, assessed in tau.P301L mice that model tauopathy.

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimer's disease. The unmet need of effective therapy for Alzheimer's disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimer's disease.

TLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T cell-independent isotype switch in mice.

Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell-independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-ß (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease.

Discovery and structure activity relationship of small molecule inhibitors of toxic ß-amyloid-42 fibril formation.

Increasing evidence implicates Aß peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aß aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aß42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the ß-sheet conformation of Aß42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aß42. The efficacy of these compounds on inhibiting Aß fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aß42 leading to decreased cell toxicity.

An effector-reduced anti-ß-amyloid (Aß) antibody with unique aß binding properties promotes neuroprotection and glial engulfment of Aß.

Passive immunization against ß-amyloid (Aß) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aß monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aß, protected against Aß1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aß oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aß, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aß antibody that takes advantage of a unique Aß binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases.

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule ß-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing ß-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized ß-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.

APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells.

The persistence of serum IgG antibodies elicited in human infants is much shorter than when such responses are elicited later in life. The reasons for this rapid waning of antigen-specific antibodies elicited in infancy are yet unknown. We have recently shown that adoptively transferred tetanus toxoid (TT)-specific plasmablasts (PBs) efficiently reach the bone marrow (BM) of infant mice. However, TT-specific PBs fail to persist in the early-life BM, suggesting that they fail to receive the molecular signals that support their survival/differentiation. Using a proliferation-inducing ligand (APRIL)- and B-cell activating factor (BAFF) B-lymphocyte stimulator (BLyS)-deficient mice, we demonstrate here that APRIL is a critical factor for the establishment of the adult BM reservoir of anti-TT IgG-secreting cells. Through in vitro analyses of PB/plasma cell (PC) survival/differentiation, we show that APRIL induces the expression of Bcl-X(L) by a preferential binding to heparan sulfate proteoglycans at the surface of CD138(+) cells. Last, we identify BM-resident macrophages as the main cells that provide survival signals to PBs and show that this function is slowly acquired in early life, in parallel to a progressive acquisition of APRIL expression. Altogether, this identifies APRIL as a critical signal for PB survival that is poorly expressed in the early-life BM compartment.

Reduced ability of neonatal and early-life bone marrow stromal cells to support plasmablast survival.

In human infants (<1 year), circulating IgG Abs elicited in response to most T-dependent Ags rapidly decline and return to baseline within a few months after immunization for yet-unknown reasons. In mice immunized between 1 and 4 wk of age, a limited establishment of the bone marrow (BM) pool of long-lived plasma cells is observed. In this study, we show that tetanus toxoid (TT)-specific plasmablasts generated in the spleen are efficiently attracted in vitro and in vivo toward early-life BM stromal cells, which express adult levels of CXCL12. Similarly, adoptively transferred TT plasmablasts efficiently reach the BM compartment of 2-wk-old and adult mice. In contrast, TT plasmablasts fail to persist in the early-life BM compartment, as indicated by the persistence of a significantly lower number of TT plasmablasts in the early-life compartment than in the adult BM compartment 48 h after transfer. This limited persistence is associated with an increased rate of in vivo apoptosis of TT-specific plasmablasts that have reached the early-life BM and with a significantly lower survival rate of TT-specific plasmablasts cocultured on early-life BM stromal cells compared with adult BM stromal cells. Thus, early-life BM stromal cells fail to provide the molecular signals that support plasmablast survival and differentiation into surviving plasma cells.

Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white x C57BL/6)F(1)-bcl-2 transgenic mice.

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) x C57BL/6)F(1) mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW x C57BL/6)F(1)-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW x C57BL/6)F(1)-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.

CpG-motifs enhance initial and sustained primary tetanus-specific antibody secreting cell responses in spleen and bone marrow, but are more effective in adult than in neonatal mice.

Unmethylated CpG oligonucleotides (CpG-ODN) increase adult and neonatal primary antibody responses to T-dependent antigens, at yet unidentified stages of antigen-specific B cell differentiation. In adult mice, a single dose of CpG-ODN adjuvanted tetanus toxoid (TT) vaccine markedly enhanced and prolonged splenic TT-specific antibody-secreting-cell (ASC) responses and significantly increased the size of the bone marrow (BM) ASC pool. Surprisingly, this was not associated with changes of germinal center (GC) numbers, size, apoptosis or function. In 1-week-old mice, CpG-ODN also enhanced TT-specific splenic ASC responses, but failed to correct limitations of the GC reaction and of the development of the BM ASC pool.

Unresponsiveness to lymphoid-mediated signals at the neonatal follicular dendritic cell precursor level contributes to delayed germinal center induction and limitations of neonatal antibody responses to T-dependent antigens.

The factors limiting neonatal and infant IgG Ab responses to T-dependent Ags are only partly known. In this study, we assess how these B cell responses are influenced by the postnatal development of the spleen and lymph node microarchitecture. When BALB/c mice were immunized with alum-adsorbed tetanus toxoid at various stages of their immune development, a major functional maturation step for induction of serum IgG, Ab-secreting cells, and germinal center (GC) responses was identified between the second and the third week of life. This correlated with the development of the follicular dendritic cell (FDC) network, as mature FDC clusters only appeared at 2 wk of age. Adoptive transfer of neonatal splenocytes into adult SCID mice rapidly induced B cell follicles and FDC precursor differentiation into mature FDC, indicating effective recruitment and signaling capacity of neonatal B cells. In contrast, adoptive transfer of adult splenocytes into neonatal SCID mice induced primary B cell follicles without any differentiation of mature FDC and failed to correct limitations of tetanus toxoid-induced GC. Thus, unresponsiveness to lymphoid-mediated signals at the level of neonatal FDC precursors delays FDC maturation and GC induction, thus limiting primary Ab-secreting cell responses to T-dependent Ags in early postnatal life.