Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models.

Inhibitors of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have dramatically changed the treatment landscape for patients with cancer. Clinical activity of anti-PD-(L)1 antibodies has resulted in increased median overall survival and durable responses in patients across selected tumor types. To date, 6 PD-1 and PD-L1, here collectively referred to as PD-(L)1, pathway inhibitors are approved by the US Food and Drug Administration for clinical use.

TSR-033, a Novel Therapeutic Antibody Targeting LAG-3, Enhances T-Cell Function and the Activity of PD-1 Blockade and .

Progressive upregulation of checkpoints on tumor-infiltrating lymphocytes promotes an immunosuppressive tumor microenvironment, severely compromising tumor immunity. Lymphocyte activation gene-3 (LAG-3) is a coinhibitory receptor associated with impaired T-cell function and is frequently coexpressed with programmed cell death protein-1 (PD-1) in the context of human cancers. Targeting LAG-3 in conjunction with PD-1 thus represents a strategy to amplify and broaden the therapeutic impact of PD-1 blockade alone.

Mammalian cell display and somatic hypermutation in vitro for human antibody discovery.

Human therapeutic antibody discovery has utilized a variety of systems, from in vivo immunization of human immunoglobulin-expressing mice, to in vitro display of antibody libraries. Of the in vitro antibody display technologies, mammalian cell display provides a number of advantages with the ability to co-select immunoglobulin molecules for high expression level in mammalian cells, native folding, and biophysical properties appropriate for drug development. Mammalian cell display has been achieved using either transient or stable expression systems, using a number of alternate transmembrane domains to present antibody on the cell surface.

Mammalian cell display for the discovery and optimization of antibody therapeutics.

Recent advances are described for the isolation and affinity maturation of antibodies that couple in vitro somatic hypermutation (SHM) with mammalian cell display, replicating key aspects of the adaptive immune system. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID). AID-directed SHM in vitro in non-B cells, combined with mammalian display of a library of human antibodies, initially naïve to SHM, can be used to isolate and affinity mature antibodies via iterative cycles of fluorescence-activated cell sorting (FACS) under increasingly stringent sort conditions.

EC144 is a potent inhibitor of the heat shock protein 90.

Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.

Transient depletion of B cells in young mice results in activation of regulatory T cells that inhibit development of autoimmune disease in adults.

B-cell depletion therapy can be effective for treating B-cell lymphomas as well as many human and murine autoimmune diseases. B-cell-deficient mice are normally resistant to spontaneous autoimmune thyroiditis (SAT), but they develop SAT if regulatory T cells are transiently depleted during the first 3-6 weeks after birth. This was also a critical time when B-cell depletion effectively inhibited development of SAT in adult mice.

B cell depletion enhances T regulatory cell activity essential in the suppression of arthritis.

The efficacy of B cell-depletion therapy in rheumatoid arthritis has driven interest in understanding the mechanism. Because the decrease in autoantibodies in rheumatoid arthritis does not necessarily correlate with clinical outcome, other mechanisms may be operative. We previously reported that in proteoglycan-induced arthritis (PGIA), B cell-depletion inhibits autoreactive T cell responses.

Loss of intra-islet CD20 expression may complicate efficacy of B-cell-directed type 1 diabetes therapies.

Objective: Consistent with studies in NOD mice, early clinical trials addressing whether depletion of B cells by the Rituximab CD20-specific antibody provides an effective means for type 1 diabetes reversal have produced promising results. However, to improve therapeutic efficacy, additional B-cell-depleting agents, as well as attempts seeking diabetes prevention, are being considered.

Research Design And Methods: Autoantibodies, including those against insulin (IAAs), are used to identify at-risk subjects for inclusion in diabetes prevention trials.

An acquired defect in IgG-dependent phagocytosis explains the impairment in antibody-mediated cellular depletion in Lupus.

B cells play important roles in autoimmune diseases ranging from multiple sclerosis to rheumatoid arthritis. B cells have also long been considered central players in systemic lupus erythematosus. However, anti-CD20-mediated B cell depletion was not effective in two clinical lupus studies, whereas anti-B lymphocyte stimulator, which inhibits B cell survival, was effective.

B cell depletion therapy exacerbates murine primary biliary cirrhosis.

Unlabelled: Primary biliary cirrhosis (PBC) is considered a model autoimmune disease due to the clinical homogeneity of patients and the classic hallmark of antimitochondrial antibodies (AMAs). Indeed, the presence of AMAs represents the most highly directed and specific autoantibody in autoimmune diseases. However, the contribution of B cells to the pathogenesis of PBC is unclear.

EC144, a synthetic inhibitor of heat shock protein 90, blocks innate and adaptive immune responses in models of inflammation and autoimmunity.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity.

G alpha q-containing G proteins regulate B cell selection and survival and are required to prevent B cell-dependent autoimmunity.

Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the G(alphaq) subunit of trimeric G proteins (Gnaq(-/-) mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq(-/-) B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive.

Prolonged effects of short-term anti-CD20 B cell depletion therapy in murine systemic lupus erythematosus.

Objective: Although B cells are implicated in the pathogenesis of systemic lupus erythematosus, the role of B cell depletion (BCD) as a treatment is controversial, given the variable benefit in human disease. This study was undertaken to test the effects of BCD therapy in a murine lupus model to better understand the mechanisms, heterogeneity, and effects on disease outcomes.

Methods: (NZB x NZW)F(1) female mice with varying degrees of disease severity were treated with an anti-mouse CD20 (anti-mCD20) antibody (IgG2a), BR3-Fc fusion protein (for BAFF blockade), or control anti-human CD20 monoclonal antibody (approximately 10 mg/kg each).

B-cell depletion with anti-CD20 ameliorates autoimmune cholangitis but exacerbates colitis in transforming growth factor-beta receptor II dominant negative mice.

Unlabelled: The treatment of primary biliary cirrhosis (PBC) with conventional immunosuppressive drugs has been relatively disappointing and there have been few efforts in defining a role for the newer biological agents useful in rheumatoid arthritis and other systemic autoimmune diseases. In this study we took advantage of transforming growth factor-beta (TGF-beta) receptor II dominant negative (dnTGF-betaRII) mice, a mouse model of autoimmune cholangitis, to address the therapeutic efficacy of B-cell depletion using anti-CD20. Mice were treated at either 4-6 weeks of age or beginning at 20-22 weeks of age with intraperitoneal injections of anti-CD20 every 2 weeks.

Dendritic cells and B cells maximize mucosal Th1 memory response to herpes simplex virus.

Although the importance of cytotoxic T lymphocytes and neutralizing antibodies for antiviral defense is well known, the antiviral mechanism of Th1 remains unclear. We show that Th1 cells mediate noncytolytic antiviral protection independent of direct lysis through local secretion of IFN-gamma after herpes simplex virus (HSV) 2 infection. IFN-gamma acted on stromal cells, but not on hematopoietic cells, to prevent further viral replication and spread throughout the vaginal mucosa.

Anti-CD20 antibody suppresses anti-HLA antibody formation in a HLA-A2 transgenic mouse model of sensitization.

Background: B cell depletion by anti-CD20 antibody is used in desensitization protocols and for treatment of antibody-mediated rejection (AMR). However, little is known about the efficacy and the mechanism(s) of action.

Methods: A mouse model of HLA sensitization was used to study the effectiveness of anti-CD20 treatment on B cell depletion and anti-HLA antibody suppression.

Systemic administration of an Fcgamma-Fc(epsilon)-fusion protein in house dust mite sensitive nonhuman primates.

Crosslinking Fc(epsilon)RI and FcgammaRIIB receptors inhibits mast cell and basophil activation, decreasing mediator release. In this study, a fusion protein incorporating human Fcgamma and Fc(epsilon) domains, hGE2, was shown to inhibit degranulation of human mast cells and basophils, and to exhibit efficacy in a nonhuman primate model of allergic asthma. hGE2 increased the provocative concentration of dust mite aeroallergen that induced an early phase asthmatic response.

B cell depletion inhibits spontaneous autoimmune thyroiditis in NOD.H-2h4 mice.

B cells are important for the development of most autoimmune diseases. B cell depletion immunotherapy has emerged as an effective treatment for several human autoimmune diseases, although it is unclear whether B cells are necessary for disease induction, autoantibody production, or disease progression. To address the role of B cells in a murine model of spontaneous autoimmune thyroiditis (SAT), B cells were depleted from adult NOD.

B-cell depletion using an anti-CD20 antibody augments antitumor immune responses and immunotherapy in nonhematopoetic murine tumor models.

The role played by B cells in cancer biology is complex and somewhat controversial. Previous studies using genetically engineered mice suggest that B cells may be immunosuppressive and inhibit tumor rejection. However, the effects of B-cell depletion employing an antibody in mice bearing solid tumors has not been tested owing to difficulties in making an effective antimouse CD20 antibody (similar to rituximab).

Suppression of proteoglycan-induced arthritis by anti-CD20 B Cell depletion therapy is mediated by reduction in autoantibodies and CD4+ T cell reactivity.

B cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) since the discovery of RA as an autoimmune disease. There is renewed interest in B cells in RA based on the clinical efficacy of B cell depletion therapy in RA patients. Although, reduced titers of rheumatoid factor and anti-cyclic citrullinated peptide Abs are recorded, the mechanisms that convey clinical improvement are incompletely understood.

A mouse Fcgamma-Fcepsilon protein that inhibits mast cells through activation of FcgammaRIIB, SH2 domain-containing inositol phosphatase 1, and SH2 domain-containing protein tyrosine phosphatases.

Background: A human Fcgamma-Fcepsilon fusion protein (GE2) designed to inhibit FcepsilonRI signaling by coaggregating FcepsilonRI with the inhibitory receptor FcgammaRIIB has been shown to inhibit mast cell activation and block cutaneous anaphylaxis. A critical issue remained as to whether the mechanism of GE2 inhibition is competition for IgE binding or inhibitory signaling through FcgammaRIIB.

Objective: Our aim was to define the in vitro and in vivo mechanism of action of a mouse homolog of GE2 (mGE) and to assess the potential of human GE2 (hGE2) for therapeutic administration.

Depletion of B cells in murine lupus: efficacy and resistance.

In mice, genetic deletion of B cells strongly suppresses systemic autoimmunity, providing a rationale for depleting B cells to treat autoimmunity. In fact, B cell depletion with rituximab is approved for rheumatoid arthritis patients, and clinical trials are underway for systemic lupus erythematosus. Yet, basic questions concerning mechanism, pathologic effect, and extent of B cell depletion cannot be easily studied in humans.

An intermediate pH unfolding transition abrogates the ability of IgE to interact with its high affinity receptor FcepsilonRIalpha.

The interaction between IgE-Fc (Fcepsilon) and its high affinity receptor FcepsilonRI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fcepsilon-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood.

Alloimmunity in primate heart recipients with CD154 blockade: evidence for alternative costimulation mechanisms.

Background: CD154 mediates key facets of humoral and cellular immunity to alloantigens, and is tolerogenic to influenza antigens in primates. Barriers to CD154-based tolerance induction for primate cardiac allografts have not previously been defined.

Methods: Heterotopic cardiac allograft outcomes in cynomolgus monkeys treated with a CD154 inhibitor, IDEC-131 (n=27), were compared to no treatment (n=4) or cyclosporine A (n=6).

Humoral immunity to vimentin is associated with cardiac allograft injury in nonhuman primates.

Immunity to autologous protein has not previously been described following nonhuman primate cardiac transplant. Native hearts and cardiac allografts from cynomolgus monkeys were assessed by immunohistology for vimentin, a highly conserved intermediate filament protein. IgM and IgG to vimentin were measured in serial sera from untreated (n = 4) or cyclosporine (CsA)-treated (n = 8, 2 with ATG) cardiac allograft recipients, and in groups treated with anti-CD154 antibody with (n = 6) or without ATG (n = 28).