Optimized synthesis, polymer conjugation, and proof-of-concept studies of the gd-IgA1 epitope for antibody-scavenging therapies in IgA nephropathy.

IgA nephropathy (IgAN) is the most common glomerular autoimmune disease and has severe long-term consequences for patients, with 40% of the patients eventually progressing to end-stage renal disease. Despite the severity, no causal treatment is currently available. While the pathogenesis of IgAN is complex, disease severity is linked to autoantibodies against the gd-IgA1 epitope, a stretch in the hinge region of IgA1 that lacks O-glycans and is found in the characteristic immune complexes deposited in the kidneys of IgAN patients. One elegant, causal approach would be to remove the anti-gd-IgA1 autoantibodies and consequently reduce the immune complex burden on the kidneys. The administration of synthetic polymers that present autoantigens in a multivalent manner have been established as promising therapeutic strategies in other autoimmune diseases and may be applied to IgAN. We here present an improved protocol for the synthesis of the gd-IgA1 epitope, its successful coupling to a poly-L-lysine polymer and proof-of-concept experiments that the polymer-bound synthetic glycopeptide is able to capture the IgAN autoantibodies, making this approach a promising way forward for developing a targeted treatment option for IgAN patients.

Decrease in Serum Anti-MAG Autoantibodies Is Associated With Therapy Response in Patients With Anti-MAG Neuropathy: Retrospective Study.

BACKGROUND AND OBJECTIVES: The objective of the retrospective analysis was to test the hypothesis that changes in serum anti-myelin-associated glycoprotein (MAG) autoantibodies are associated with clinical response to immunotherapy in patients with anti-MAG neuropathy. METHODS: As of January 29, 2020, we used anti-myelin-associated glycoprotein-related search strings in the Medline database to identify studies that provided information on anti-MAG immunoglobulin M (IgM) autoantibodies and clinical outcomes during immunotherapies. The relative change in anti-MAG IgM titers, paraprotein levels, or total IgM was determined before, during, or posttreatment, and the patients were assigned to "responder," "nonresponder,"' or "acute deteriorating" category depending on their clinical response to treatment. The studies were qualified as "supportive" or "not supportive" depending on the percentage of patients exhibiting an association between relative change of anti-MAG antibody titers or levels and change in clinical outcomes. RESULTS: Fifty studies with 410 patients with anti-MAG neuropathy were included in the analysis. Forty studies with 303 patients supported the hypothesis that a "responder" patient had a relative reduction of anti-MAG antibody titers or levels that is associated with clinical improvements and "nonresponder" patients exhibited no significant change in anti-MAG IgM antibodies. Six studies with 93 patients partly supported, and 4 studies with 26 patients did not support the hypothesis. DISCUSSION: The retrospective analysis confirmed the hypothesis that a relative reduction in serum anti-MAG IgM antibodies is associated with a clinical response to immunotherapies; a sustained reduction of at least 50% compared with pretreatment titers or levels could be a valuable indicator for therapeutic response.

CARD10 cleavage by MALT1 restricts lung carcinoma growth in vivo.

CARD-CC complexes involving BCL10 and MALT1 are major cellular signaling hubs. They govern NF-κB activation through their scaffolding properties as well as MALT1 paracaspase function, which cleaves substrates involved in NF-κB regulation. In human lymphocytes, gain-of-function defects in this pathway lead to lymphoproliferative disorders. CARD10, the prototypical CARD-CC protein in non-hematopoietic cells, is overexpressed in several cancers and has been associated with poor prognosis. However, regulation of CARD10 remains poorly understood. Here, we identified CARD10 as the first MALT1 substrate in non-hematopoietic cells and showed that CARD10 cleavage by MALT1 at R587 dampens its capacity to activate NF-κB. Preventing CARD10 cleavage in the lung tumor A549 cell line increased basal levels of IL-6 and extracellular matrix components in vitro, and led to increased tumor growth in a mouse xenograft model, suggesting that CARD10 cleavage by MALT1 might be a built-in mechanism controlling tumorigenicity.

Pharmacological Inhibition of MALT1 Protease Leads to a Progressive IPEX-Like Pathology.

Genetic disruption or short-term pharmacological inhibition of MALT1 protease is effective in several preclinical models of autoimmunity and B cell malignancies. Despite these protective effects, the severe reduction in regulatory T cells (Tregs) and the associated IPEX-like pathology occurring upon congenital disruption of the MALT1 protease in mice has raised concerns about the long-term safety of MALT1 inhibition. Here we describe the results of a series of toxicology studies in rat and dog species using MLT-943, a novel potent and selective MALT1 protease inhibitor. While MLT-943 effectively prevented T cell-dependent B cell immune responses and reduced joint inflammation in the collagen-induced arthritis rat pharmacology model, in both preclinical species, pharmacological inhibition of MALT1 was associated with a rapid and dose-dependent reduction in Tregs and resulted in the progressive appearance of immune abnormalities and clinical signs of an IPEX-like pathology. At the 13-week time point, rats displayed severe intestinal inflammation associated with mast cell activation, high serum IgE levels, systemic T cell activation and mononuclear cell infiltration in multiple tissues. Importantly, using thymectomized rats we demonstrated that MALT1 protease inhibition affects peripheral Treg frequency independently of effects on thymic Treg output and development. Our data confirm the therapeutic potential of MALT1 protease inhibitors but highlight the safety risks and challenges to consider before potential application of such inhibitors into the clinic.

Immune cell landscaping reveals a protective role for regulatory T cells during kidney injury and fibrosis.

Acute kidney injury (AKI) and chronic kidney diseases are associated with high mortality and morbidity. Although the underlying mechanisms determining the transition from acute to chronic injury are not completely understood, immune-mediated processes are critical in renal injury. We have performed a comparison of 2 mouse models leading to either kidney regeneration or fibrosis. Using global gene expression profiling we could identify immune-related pathways accounting for the majority of the observed transcriptional changes during fibrosis. Unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed major changes in tissue-resident macrophages and T cells. Following injury, there was a marked increase in tissue-resident IL-33R+ and IL-2Ra+ regulatory T cells (Tregs). Expansion of this population before injury protected the kidney from injury and fibrosis. Transcriptional profiling of Tregs showed a differential upregulation of regenerative and proangiogenic pathways during regeneration, whereas in the fibrotic environment they expressed markers of hyperactivation and fibrosis. Our data point to a hitherto underappreciated plasticity in Treg function within the same tissue, dictated by environmental cues. Overall, we provide a detailed cellular and molecular characterization of the immunological changes during kidney injury, regeneration, and fibrosis.

Requirement of Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 Protease Activity for Fcγ Receptor-Induced Arthritis, but Not Fcγ Receptor-Mediated Platelet Elimination, in Mice.

OBJECTIVE: Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases. METHODS: Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP). RESULTS: MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP. CONCLUSION: Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.

Malt1 Protease Deficiency in Mice Disrupts Immune Homeostasis at Environmental Barriers and Drives Systemic T Cell-Mediated Autoimmunity.

The paracaspase Malt1 is a key regulator of canonical NF-κB activation downstream of multiple receptors in both immune and nonimmune cells. Genetic disruption of Malt1 protease function in mice and MALT1 mutations in humans results in reduced regulatory T cells and a progressive multiorgan inflammatory pathology. In this study, we evaluated the altered immune homeostasis and autoimmune disease in Malt1 protease-deficient (Malt1PD) mice and the Ags driving disease manifestations. Our data indicate that B cell activation and IgG1/IgE production is triggered by microbial and dietary Ags preferentially in lymphoid organs draining mucosal barriers, likely as a result of dysregulated mucosal immune homeostasis. Conversely, the disease was driven by a polyclonal T cell population directed against self-antigens. Characterization of the Malt1PD T cell compartment revealed expansion of T effector memory cells and concomitant loss of a CD4+ T cell population that phenotypically resembles anergic T cells. Therefore, we propose that the compromised regulatory T cell compartment in Malt1PD animals prevents the efficient maintenance of anergy and supports the progressive expansion of pathogenic, IFN-γ-producing T cells. Overall, our data revealed a crucial role of the Malt1 protease for the maintenance of intestinal and systemic immune homeostasis, which might provide insights into the mechanisms underlying IPEX-related diseases associated with mutations in MALT1.

GEF-H1 Signaling upon Microtubule Destabilization Is Required for Dendritic Cell Activation and Specific Anti-tumor Responses.

Dendritic cell (DC) activation is a critical step for anti-tumor T cell responses. Certain chemotherapeutics can influence DC function. Here we demonstrate that chemotherapy capable of microtubule destabilization has direct effects on DC function; namely, it induces potent DC maturation and elicits anti-tumor immunity. Guanine nucleotide exchange factor-H1 (GEF-H1) is specifically released upon microtubule destabilization and is required for DC activation. In response to chemotherapy, GEF-H1 drives a distinct cell signaling program in DCs dominated by the c-Jun N-terminal kinase (JNK) pathway and AP-1/ATF transcriptional response for control of innate and adaptive immune responses. Microtubule destabilization, and subsequent GEF-H1 signaling, enhances cross-presentation of tumor antigens to CD8 T cells. In absence of GEF-H1, anti-tumor immunity is hampered. In cancer patients, high expression of the GEF-H1 immune gene signature is associated with prolonged survival. Our study identifies an alternate intracellular axis in DCs induced upon microtubule destabilization in which GEF-H1 promotes protective anti-tumor immunity.

Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade.

Targeted drug delivery with antibody-drug conjugates such as the HER2-directed ado-trastuzumab emtansine (T-DM1) has emerged as a powerful strategy for cancer therapy. We show that T-DM1 is particularly effective in eliciting antitumor immunity in patients with early breast cancer (WSG-ADAPT trial) and in a HER2-expressing orthotopic tumor model. In the latter, despite primary resistance to immunotherapy, combined treatment with T-DM1 and anti-CTLA-4/PD-1 (cytotoxic T lymphocyte-associated protein-4/programmed cell death protein-1) was curative because it triggered innate and adaptive immunity. Tumor rejection was accompanied by massive T cell infiltration, TH1 (T helper 1) cell polarization, and, notably, a substantial increase in regulatory T cells. Depletion of regulatory T cells resulted in inflammation and tissue damage, implying their essential role in protecting the host during therapy. This study provides insights into the mechanisms of T-DM1's therapeutic activity and a rationale for potential therapeutic combination strategies with immunotherapy.

Modulation of APC Function and Anti-Tumor Immunity by Anti-Cancer Drugs.

Professional antigen-presenting cells (APCs), such as dendritic cells (DCs), are central to the initiation and regulation of anti-cancer immunity. However, in the immunosuppressive environment within a tumor APCs may antagonize anti-tumor immunity by inducing regulatory T cells (Tregs) or anergy of effector T cells due to lack of efficient costimulation. Hence, in an optimal setting, anti-cancer drugs have the power to reduce tumor size and thereby may induce the release of tumor antigens and, at the same time, modulate APC function toward efficient priming of antigen-specific effector T cells. Selected cytotoxic agents may revert APC dysfunction either by directly maturing DCs or through induction of immunogenic tumor cell death. Furthermore, specific cytotoxic agents may support adaptive immunity by selectively depleting regulatory subsets, such as Tregs or myeloid-derived suppressor cells. Perspectively, this will allow developing effective combination strategies with novel immunotherapies to exert complementary pressure on tumors via direct toxicity as well as immune activation. We, here, review our current knowledge on the capacity of anti-cancer drugs to modulate APC functions to promote durable anti-cancer immune responses.

The microtubule-depolymerizing agent ansamitocin P3 programs dendritic cells toward enhanced anti-tumor immunity.

In addition to direct tumor cell cytotoxicity, chemotherapy can mediate tumor reduction through immune modulation of the tumor microenvironment to promote anti-tumor immunity. Mature dendritic cells (DCs) play key roles in priming robust immune responses in tumor-bearing hosts. Here, we screened a panel of 21 anticancer agents with defined molecular targets for their ability to induce direct maturation of DCs. We identified ansamitocin P3, a microtubule-depolymerizing agent, as a potent inducer of phenotypic and functional maturation of DCs. Exposure of both murine spleen-derived and human monocyte-derived DCs to ansamitocin P3 triggered up-regulation of maturation markers and production of pro-inflammatory cytokines, resulting in an enhanced T cell stimulatory capacity. Local administration of ansamitocin P3 induced maturation of skin Langerhans cells in vivo and promoted antigen uptake and extensive homing of tumor-resident DCs to tumor-draining lymph nodes. When used as an adjuvant in a specific vaccination approach, ansamitocin P3 dramatically increased activation of antigen-specific T cells. Finally, we demonstrate that ansamitocin P3, due to its immunomodulatory properties, acts in synergy with antibody-mediated blockade of the T cell inhibitory receptors PD-1 and CTLA-4. The combination treatment was most effective and induced durable growth inhibition of established tumors. Mechanistically, we observed a reduced regulatory T cell frequency and improved T cell effector function at the tumor site. Taken together, our study unravels an immune-based anti-tumor mechanism exploited by microtubule-depolymerizing agents, including ansamitocin P3, and paves the way for future clinical trials combining this class of agents with immunotherapy.

Microtubule-depolymerizing agents used in antibody-drug conjugates induce antitumor immunity by stimulation of dendritic cells.

Antibody-drug conjugates (ADC) are emerging as powerful treatment strategies with outstanding target-specificity and high therapeutic activity in patients with cancer. Brentuximab vedotin represents a first-in-class ADC directed against CD30(+) malignancies. We hypothesized that its sustained clinical responses could be related to the stimulation of an anticancer immune response. In this study, we demonstrate that the dolastatin family of microtubule inhibitors, from which the cytotoxic component of brentuximab vedotin is derived, comprises potent inducers of phenotypic and functional dendritic cell (DC) maturation. In addition to the direct cytotoxic effect on tumor cells, dolastatins efficiently promoted antigen uptake and migration of tumor-resident DCs to the tumor-draining lymph nodes. Exposure of murine and human DCs to dolastatins significantly increased their capacity to prime T cells. Underlining the requirement of an intact host immune system for the full therapeutic benefit of dolastatins, the antitumor effect was far less pronounced in immunocompromised mice. We observed substantial therapeutic synergies when combining dolastatins with tumor antigen-specific vaccination or blockade of the PD-1-PD-L1 and CTLA-4 coinhibitory pathways. Ultimately, treatment with ADCs using dolastatins induces DC homing and activates cellular antitumor immune responses in patients. Our data reveal a novel mechanism of action for dolastatins and provide a strong rationale for clinical treatment regimens combining dolastatin-based therapies, such as brentuximab vedotin, with immune-based therapies.

Cancer chemotherapy agents target intratumoral dendritic cells to potentiate antitumor immunity.

Cytotoxic drugs capable of killing cancer cells in conjunction with targeted conversion of tumor resident, tolerogenic dendritic cells (DCs) into efficient antigen presenting cells (APCs) are highly complementary therapeutic routes to boost antitumor immunity. Our data suggest that the microtubule-depolymerizing compounds Dolastatin 10 and Ansamitocin P3 may serve as prototypes for a class of agents that display this binary mode of action.