InVivoMAb human IgG1 isotype control

Tissue regenerative responses involve complex interactions between resident structural and immune cells. Recent reports indicate that accumulation of senescent cells during injury repair contributes to pathological tissue fibrosis. Using tissue-based spatial transcriptomics and proteomics, we identified upregulation of the immune checkpoint protein, cytotoxic T lymphocyte-associated protein 4 (CTLA4), on CD8+ T cells adjacent to regions of active fibrogenesis in human idiopathic pulmonary fibrosis and in a repetitive bleomycin lung injury murine model of persistent fibrosis. In humanized CTLA4-knockin mice, treatment with ipilimumab, an FDA-approved drug that targets CTLA4, resulted in accelerated lung epithelial regeneration and diminished fibrosis from repetitive bleomycin injury. Ipilimumab treatment resulted in the expansion of Cd3e+ T cells, diminished accumulation of senescent cells, and robust expansion of type 2 alveolar epithelial cells, facultative progenitor cells of the alveolar epithelium. Ex vivo activation of isolated CTLA4-expressing CD8+ cells from mice with established fibrosis resulted in enhanced cytolysis of senescent cells, suggesting that impaired immune-mediated clearance of these cells contributes to persistence of lung fibrosis in this murine model. Our studies support the concept that endogenous immune surveillance of senescent cells may be essential in promoting tissue regenerative responses that facilitate the resolution of fibrosis.

The PD-L1/PD-1 pathway is crucial for immune regulation and has become a target in cancer immunotherapy. However, in order to improve patient selection for immune checkpoint blockade (ICB) therapies, better selection criteria are needed. This study explores how the N-glycosylation of PD-L1 affects its interaction with PD-1 and ICB efficacy, focusing on its four N-linked glycosylation sites: N35, N192, N200, and N219.

Tafasitamab is a novel humanized anti-CD19 monoclonal antibody, designed for the treatment of B-cell malignancies. Our study aims to enhance the direct, non-immune-mediated, activity of tafasitamab (TAFA) with the combination of metronomic chemotherapy (mCHEMO), including vinorelbine (mVNR) and etoposide (mETO), in preclinical models of diffuse large B-cell lymphoma (DLBCL). In vitro, the 144 h exposure of thrice-weekly mVNR, daily mETO, and single-dose TAFA significantly inhibited the viability of human CD19+ DLBCL cell lines (i.e., Toledo, OCI-LY3, and SU-DHL10) in a concentration-dependent manner. In all cell lines, the concomitant treatment with TAFA and mVNR or mETO showed a marked synergism, except for TAFA + mETO on SU-DHL10 cells. The TAFA + mCHEMO treatments promoted apoptosis, and the TAFA + mVNR combination significantly inhibited, already after 24 h, the phosphorylation of GSK3α/β, mTOR, p70S6K, RPS6, and TSC2 proteins in DLBCL cells. TAFA significantly increased the VNR and ETO intracellular concentrations in all DLBCL cells after 24 h, except for ETO levels in SU-DHL10. The TAFA + mCHEMO treatment strongly reduced the ABCB1, ABCG2, and c-MYC gene expression in SU-DHL10 cells. In vivo, the TAFA + mVNR combination was well tolerated, significantly reduced the volumes of subcutaneous DLBCL masses, and increased the overall survival of mice affected by systemic DLBCL. We report additional mechanisms to enhance the direct activity of TAFA with mCHEMO synergistically in DLBCL cells in vitro and in vivo, suggesting the use of this combination schedule into future clinical trials.

Regulatory T cells (Treg) play an important role in regulating immune homeostasis in health and disease. Traditionally their suppressive function has been assayed by mixing purified cell populations, which does not provide an accurate picture of a physiologically relevant response. To overcome this limitation, we here develop 'single cell suppression profiling of human Tregs' (scSPOT). scSPOT uses a 52-marker CyTOF panel, a cell division detection algorithm, and a whole PBMC system to assess the effect of Tregs on all other cell types simultaneously. In this head-to-head comparison, we find Tregs having the clearest suppressive effects on effector memory CD8 T cells through partial division arrest, cell cycle inhibition, and effector molecule downregulation. Additionally, scSPOT identifies a Treg phenotypic split previously observed in viral infection and propose modes of action by the FDA-approved drugs Ipilimumab and Tazemetostat. scSPOT is thus scalable, robust, widely applicable, and may be used to better understand Treg immunobiology and screen for therapeutic compounds.

The activated B-cell-like subtype of diffuse large B-cell lymphoma (ABC-DLBCL) displays a worse outcome than the germinal center B-cell-like subtype (GCB-DLBCL). Currently, targeting the tumor microenvironment (TME) is the most promising approach to cure DLBCL with profound molecular heterogeneity; however, the factors affecting the tumor-promoting TME of ABC-DLBCL remain elusive. Here, cytokine interleukin-16 (IL-16) is expressed in tumor cells of ABC-DLBCL and secreted by the cleavage of active caspase-3. The serum IL-16 levels are not only a sensitive marker of treatment response, but also positively correlated with unfavorable prognosis in DLBCL patients. While IL-16 shows few direct promotional effects on tumor cell growth in vitro, its bioactive form significantly promotes tumor progression in vivo. Mechanically, IL-16 increases the infiltration of macrophages by the chemotaxis of CD4+ monocytes in the TME, enhancing angiogenesis and the expression of cytokine IL-6 and IL-10, as well as decreasing T-cell infiltration to accelerate tumor progression. This study demonstrates that IL-16 exerts a novel role in co-ordinating the bidirectional interactions between tumor progression and the TME. IMM0306, a fusion protein of CD20 mAb with the CD47 binding domain of SIRPα, reverses the tumor-promoting effects of IL-16, providing new insights into treatment strategy in ABC-DLBCL.

The cell membrane transport capacity and surface targets of multiple myeloma (MM) cells heavily influence chemotherapy and immunotherapy. Here, it is found that caveolin-1 (CAV1), a primary component of membrane lipid rafts and caveolae, is highly expressed in MM cells and is associated with MM progression and drug resistance. CAV1 knockdown decreases MM cell adhesion to stromal cells and attenuates cell adhesion-mediated drug resistance to bortezomib. CAV1 inhibition in MM cells enhances natural killer cell-mediated cytotoxicity through increasing CXCL10, SLAMF7, and CD112. CAV1 suppression reduces mitochondrial membrane potential, increases reactive oxygen species, and inhibits autophagosome-lysosome fusion, resulting in the disruption of redox homeostasis. Additionally, CAV1 knockdown enhances glutamine addiction by increasing ASCT2 and LAT1 and dysregulates glutathione metabolism. As a result of CAV1 inhibition, MM cells are more sensitive to starvation, glutamine depletion, and glutamine transporter inhibition, and grow more slowly in vivo in a mouse model treated with bortezomib. The observation that CAV1 inhibition modulated by 6-mercaptopurine, daidzin, and statins enhances the efficacy of bortezomib in vitro and in vivo highlights the translational significance of these FDA-approved drugs in improving MM outcomes. These data demonstrate that CAV1 serves as a potent therapeutic target for enhancing chemotherapy and immunotherapy for MM.

The SARS-CoV-2 pandemic alerted the potential for significant harm due to future cross-species transmission of various animal coronaviruses to human. There is a significant need of antibody-based drugs to treat patients infected with previously unseen coronaviruses. In this study, we generated CV804, an antibody that binds to the S2 domain of SARS-CoV-2 spike protein, which is highly conserved across the coronavirus family and less susceptible to mutations. CV804 demonstrated broad cross-reactivities not only disease-associated human beta coronaviruses including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1 and with existing mutant strains of SARS-CoV-2 and but also with 20 representative animal-origin coronaviruses. CV804 exhibits strong antibody-dependent cellular cytotoxicity (ADCC) to SARS-CoV-2 spike protein expressed on cells in vitro, while completely lacks virus-neutralization activity. In animal models, CV804 suppressed disease progression caused by SARS-CoV-2 infection. Structural studies using HDX-MS combined with reactivity analysis with point mutants of recombinant spike proteins revealed that CV804 binds to a unique conformational epitope within the S2 domain of the spike proteins that is highly conserved among various coronaviruses. Overall, obtained data suggest that the non-neutralizing CV804 antibody recognizes the conformational structure of the spike protein displayed on the surface of infected cells and weakens the viral virulence by supporting the host immune cells' attack through ADCC activity in vivo. The CV804 epitope information revealed in this study is useful for designing pan-corona antibody therapeutics and universal coronavirus vaccines for preparing potential future pandemics.

T-cell immunoglobulin and mucin domain containing molecule-4 (TIM-4) is a scavenger receptor best known for its role in recognizing dying cells. TIM-4 orchestrates phagocytosis allowing for cellular clearance of apoptotic cells, termed efferocytosis. It was previously shown that TIM-4 directly interacts with AMPKα1, activating the autophagy pathway, leading to degradation of ingested tumors, and effectively reducing antigen presentation.

Mounting evidence indicates that antibodies can contribute towards control of tuberculosis (TB). However, the underlying mechanisms of humoral immune protection and whether antibodies can be exploited in therapeutic strategies to combat TB are relatively understudied. Here we engineered the receptor-binding Fc (fragment crystallizable) region of an antibody recognizing the Mycobacterium tuberculosis (Mtb) capsule, to define antibody Fc-mediated mechanism(s) of Mtb restriction. We generated 52 Fc variants that either promote or inhibit specific antibody effector functions, rationally building antibodies with enhanced capacity to promote Mtb restriction in a human whole-blood model of infection. While there is likely no singular Fc profile that universally drives control of Mtb, here we found that several Fc-engineered antibodies drove Mtb restriction in a neutrophil-dependent manner. Single-cell RNA sequencing analysis showed that a restrictive Fc-engineered antibody promoted neutrophil survival and expression of cell-intrinsic antimicrobial programs. These data show the potential of Fc-engineered antibodies as therapeutics able to harness the protective functions of neutrophils to promote control of TB.

This study sought to elucidate the mechanisms underlying the impact of the interferon signaling pathway on Ferroptosis in tumor cells and its correlation with CD8 + T cell exhaustion. Using mouse models and single-cell sequencing, the researchers studied the interaction between CD8 + T cells and the interferon signaling pathway. Differential gene analysis revealed key genes involved in CD8 + T cell exhaustion, and their downstream factors were explored using bioinformatics tools. The expression levels of interferon-related genes associated with Ferroptosis were analyzed using data from the TCGA database, and their relevance to tumor tissue Ferroptosis and patients' prognosis was determined. In vitro experiments were conducted to measure the levels of IFN-γ, MDA, and LPO, as well as tumor cell viability and apoptosis. In vivo validation using a mouse tumor model confirmed the results obtained from the in vitro experiments, highlighting the potential of silencing HSPA6 or DNAJB1 in enhancing the efficacy of PD-1 therapy and inhibiting tumor growth and migration.

Labeled antibodies are excellent imaging agents in oncology to non-invasively visualize cancer-related antigens expression levels. However, tumor tracer uptake (TTU) of specific antibodies in-vivo may be inferior to non-specific IgG in some cases.

Vitamin B12 is critical for hematopoiesis and myelination. Deficiency can cause neurologic deficits including loss of coordination and cognitive decline. However, diagnosis relies on measurement of vitamin B12 in the blood, which may not accurately reflect the concentration in the brain. Using programmable phage display, we identified an autoantibody targeting the transcobalamin receptor (CD320) in a patient with progressive tremor, ataxia, and scanning speech. Anti-CD320 impaired cellular uptake of cobalamin (B12) in vitro by depleting its target from the cell surface. Despite a normal serum concentration, B12 was nearly undetectable in her cerebrospinal fluid (CSF). Immunosuppressive treatment and high-dose systemic B12 supplementation were associated with increased B12 in the CSF and clinical improvement. Optofluidic screening enabled isolation of a patient-derived monoclonal antibody that impaired B12 transport across an in vitro model of the blood-brain barrier (BBB). Autoantibodies targeting the same epitope of CD320 were identified in seven other patients with neurologic deficits of unknown etiology, 6% of healthy controls, and 21.4% of a cohort of patients with neuropsychiatric lupus. In 132 paired serum and CSF samples, detection of anti-CD320 in the blood predicted B12 deficiency in the brain. However, these individuals did not display any hematologic signs of B12 deficiency despite systemic CD320 impairment. Using a genome-wide CRISPR screen, we found that the low-density lipoprotein receptor serves as an alternative B12 uptake pathway in hematopoietic cells. These findings dissect the tissue specificity of B12 transport and elucidate an autoimmune neurologic condition that may be amenable to immunomodulatory treatment and nutritional supplementation.

Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes. Antibodies against individual RGD-binding integrins are desirable for investigating their specific functions, and were selected here from a synthetic yeast-displayed Fab library. We discovered 11 antibodies that exhibit high specificity and affinity toward their target integrins, i.e. αVβ3, αVβ5, αVβ6, αVβ8, and α5β1. Of these, six are function-blocking antibodies and contain a ligand-mimetic R(G/L/T)D motif in their CDR3 sequences. We report antibody-binding specificity, kinetics, and binding affinity for purified integrin ectodomains, as well as intact integrins on the cell surface. We further used these antibodies to reveal binding preferences of the αV subunit for its 5 β-subunit partners: β6 = β8 > β3 > β1 = β5.

The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide. We used limited proteolysis-mass spectrometry (LiP-MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP-MS detects well-characterized PPIs, including antibody-target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson's disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies. We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states.

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody-dependent cell-mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single-particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor-binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan-protective mAbs against SARS-CoV-2.

Tax-interacting protein 1 (TIP1) is a cancer-specific radiation-inducible cell surface antigen that plays a role in cancer progression and resistance to therapy. This study aimed to develop a novel anti-TIP1 human antibody for noninvasive PET imaging in patients with cancer.

Preclinical murine data indicate that fragment crystallizable (Fc)-dependent depletion of intratumoral regulatory T cells (Treg) is a major mechanism of action of anti-CTLA-4. However, the two main antibodies administered to patients (ipilimumab and tremelimumab) do not recapitulate these effects. Here, we investigate the underlying mechanisms responsible for the limited Treg depletion observed with these therapies. Using an immunocompetent murine model humanized for CTLA-4 and Fcγ receptors (FcγR), we show that ipilimumab and tremelimumab exhibit limited Treg depletion in tumors. Immune profiling of the tumor microenvironment (TME) in both humanized mice and humans revealed high expression of the inhibitory Fc receptor, FcγRIIB, which limits antibody-dependent cellular cytotoxicity/phagocytosis. Blocking FcγRIIB in humanized mice rescued the Treg-depleting capacity and antitumor activity of ipilimumab. Furthermore, Fc engineering of antibodies targeting Treg-associated targets (CTLA-4 or CCR8) to minimize FcγRIIB binding significantly enhanced Treg depletion, resulting in increased antitumor activity across various tumor models. Our results define the inhibitory FcγRIIB as an immune checkpoint limiting antibody-mediated Treg depletion in the TME, and demonstrate Fc engineering as an effective strategy to overcome this limitation and improve the efficacy of Treg-targeting antibodies.

Immunotherapy is a promising approach for treating metastatic breast cancer (MBC), offering new possibilities for therapy. While checkpoint inhibitors have shown great progress in the treatment of metastatic breast cancer, their effectiveness in patients with bone metastases has been disappointing. This lack of efficacy seems to be specific to the bone environment, which exhibits immunosuppressive features. In this study, we elucidate the multiple roles of the sialic acid-binding Ig-like lectin (Siglec)-15/sialic acid glyco-immune checkpoint axis in the bone metastatic niche and explore potential therapeutic strategies targeting this glyco-immune checkpoint. Our research reveals that elevated levels of Siglec-15 in the bone metastatic niche can promote tumor-induced osteoclastogenesis as well as suppress antigen-specific T cell responses. Next, we demonstrate that antibody blockade of the Siglec-15/sialic acid glyco-immune checkpoint axis can act as a potential treatment for breast cancer bone metastasis. By targeting this pathway, we not only aim to treat bone metastasis but also inhibit the spread of metastatic cancer cells from bone lesions to other organs.