VISTA is an activating receptor in human monocytes.

As indicated by its name, V-domain Ig suppressor of T cell activation (VISTA) is thought to serve primarily as an inhibitory protein that limits immune responses. VISTA antibodies can dampen the effects of several concomitantly elicited activation signals, including TCR and TLR activation, but it is currently unclear if VISTA agonism could singly affect immune cell biology. In this study, we discovered two novel VISTA antibodies and characterized their effects on human peripheral blood mononuclear cells by scRNA/CITE-seq. Both antibodies appeared to agonize VISTA in an Fc-functional manner to elicit transcriptional and functional changes in monocytes consistent with activation. We also used pentameric VISTA to identify Syndecan-2 and several heparan sulfate proteoglycan synthesis genes as novel regulators of VISTA interactions with monocytic cells, adding further evidence of bidirectional signaling. Together, our study highlights several novel aspects of VISTA biology that have yet to be uncovered in myeloid cells and serves as a foundation for future research.

Phase I study of ABBV-428, a mesothelin-CD40 bispecific, in patients with advanced solid tumors.

BACKGROUND: CD40 agonist immunotherapy can potentially license antigen-presenting cells to promote antitumor T-cell activation and re-educate macrophages to destroy tumor stroma. Systemic administration of CD40 agonists has historically been associated with considerable toxicity, providing the rationale for development of tumor-targeted immunomodulators to improve clinical safety and efficacy. This phase I study assessed the safety, tolerability, preliminary antitumor activity, and preliminary biomarkers of ABBV-428, a first-in-class, mesothelin-targeted, bispecific antibody designed for tumor microenvironment-dependent CD40 activation with limited systemic toxicity. METHODS: ABBV-428 was administered intravenously every 2 weeks to patients with advanced solid tumors. An accelerated titration (starting at a 0.01 mg/kg dose) and a 3+3 dose escalation scheme were used, followed by recommended phase II dose cohort expansions in ovarian cancer and mesothelioma, tumor types associated with high mesothelin expression. RESULTS: Fifty-nine patients were treated at doses between 0.01 and 3.6 mg/kg. The maximum tolerated dose was not reached, and 3.6 mg/kg was selected as the recommended phase II dose. Seven patients (12%) reported infusion-related reactions. Treatment-related grade ≥3 treatment-emergent adverse events were pericardial effusion, colitis, infusion-related reaction, and pleural effusion (n=1 each, 2%), with no cytokine release syndrome reported. The pharmacokinetic profile demonstrated roughly dose-proportional increases in exposure from 0.4 to 3.6 mg/kg. Best response was stable disease in 9/25 patients (36%) treated at the recommended phase II dose. CD40 receptor occupancy >90% was observed on peripheral B-cells starting from 0.8 mg/kg; however, no consistent changes from baseline in intratumoral CD8+ T-cells, programmed death ligand-1 (PD-L1+) cells, or immune-related gene expression were detected post-ABBV-428 treatment (cycle 2, day 1). Mesothelin membrane staining showed greater correlation with progression-free survival in ovarian cancer and mesothelioma than in the broader dose escalation population. CONCLUSIONS: ABBV-428 monotherapy exhibited dose-proportional pharmacokinetics and an acceptable safety profile, particularly for toxicities characteristic of CD40 agonism, illustrating that utilization of a tumor-targeted, bispecific antibody can improve the safety of CD40 agonism as a therapeutic approach. ABBV-428 monotherapy had minimal clinical activity in dose escalation and in a small expansion cohort of patients with advanced mesothelioma or ovarian cancer. TRIAL REGISTRATION NUMBER: NCT02955251.

Epitope and Fc-Mediated Cross-linking, but Not High Affinity, Are Critical for Antitumor Activity of CD137 Agonist Antibody with Reduced Liver Toxicity.

CD137 (TNFRSF9, 4-1BB) agonist antibodies (mAb) have demonstrated potent antitumor activity with memory response while causing hepatotoxicity in mouse models. In clinical trials, the degrees of liver toxicity of anti-CD137 vary from grade 4 transaminitis (urelumab) to nonexistent (utomilumab). To exploit the antitumor potential of CD137 signaling, we identified a new class of CD137 agonist mAbs with strong antitumor potency without significant transaminitis in vivo compared with CD137 agonists previously reported. These mAbs are cross-reactive to mouse and cynomolgus monkey and showed cross-linking-dependent T-cell costimulation activity in vitro Antitumor efficacy was maintained in Fc gamma receptor (FcγR) III-deficient mice but diminished in FcγRIIB-deficient mice, suggesting the critical role for FcγRIIB to provide cross-linking in vivo Interestingly, a single dose of an affinity-reduced variant was sufficient to control tumor growth, but a higher affinity variant did not improve efficacy. These observations suggest that binding epitope and FcγR interaction, but not necessarily high affinity, are important for antitumor efficacy and reduced liver toxicity of CD137 mAb. Our study suggests the possibility of CD137 agonist therapy with improved safety profile in humans.

A Bispecific Molecule Targeting CD40 and Tumor Antigen Mesothelin Enhances Tumor-Specific Immunity.

Agonistic CD40 monoclonal antibodies (mAb) have demonstrated some clinical activity, but with dose-limiting toxicity. To reduce systemic toxicity, we developed a bispecific molecule that was maximally active in the presence of a tumor antigen and had limited activity in the absence of the tumor antigen. LB-1 is a bispecific molecule containing single-chain Fv domains targeting mouse CD40 and the tumor antigen mesothelin. LB-1 exhibited enhanced activity upon binding to cell-surface mesothelin but was less potent in the absence of mesothelin binding. In a mouse model implanted with syngeneic 4T1 tumors expressing cell-surface mesothelin, LB-1 demonstrated comparable antitumor activity as an agonistic CD40 mAb but did not cause elevation of serum cytokines and liver enzymes, as was observed in anti-CD40-treated mice. The results from our study of LB-1 were used to develop a human cross-reactive bispecific molecule (ABBV-428) that targeted human CD40 and mesothelin. ABBV-428 demonstrated enhanced activation of antigen-presenting cells and T cells upon binding to cell-surface mesothelin, and inhibition of cultured or implanted PC3 tumor cell growth after immune activation. Although expression of cell-surface mesothelin is necessary, the bispecific molecules induced immune-mediated antitumor activity against both mesothelin+ and mesothelin- tumor cells. ABBV-428 represents a class of bispecific molecules with conditional activity dependent on the binding of a tumor-specific antigen, and such activity could potentially maximize antitumor potency while limiting systemic toxicity in clinical studies.

LRRC15 Is a Novel Mesenchymal Protein and Stromal Target for Antibody-Drug Conjugates.

Progress in understanding tumor stromal biology has been constrained in part because cancer-associated fibroblasts (CAF) are a heterogeneous population with limited cell-type-specific protein markers. Using RNA expression profiling, we identified the membrane protein leucine-rich repeat containing 15 (LRRC15) as highly expressed in multiple solid tumor indications with limited normal tissue expression. LRRC15 was expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 expression was induced by TGFß on activated fibroblasts (αSMA+) and on mesenchymal stem cells. These collective findings suggested LRRC15 as a novel CAF and mesenchymal marker with utility as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate (ADC) directed against LRRC15, and it demonstrated robust preclinical efficacy against LRRC15 stromal-positive/cancer-negative, and LRRC15 cancer-positive models as a monotherapy, or in combination with standard-of-care therapies. ABBV-085's unique mechanism of action relied upon the cell-permeable properties of MMAE to preferentially kill cancer cells over LRRC15-positive CAF while also increasing immune infiltrate (e.g., F4/80+ macrophages) in the tumor microenvironment. In summary, these findings validate LRRC15 as a novel therapeutic target in multiple solid tumor indications and support the ongoing clinical development of the LRRC15-targeted ADC ABBV-085.Significance: These findings identify LRRC15 as a new marker of cancer-associated fibroblasts and cancers of mesenchymal origin and provide preclinical evidence for the efficacy of an antibody-drug conjugate targeting the tumor stroma. Cancer Res; 78(14); 4059-72. ©2018 AACR.

Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells.

Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

Elotuzumab enhances natural killer cell activation and myeloma cell killing through interleukin-2 and TNF-α pathways.

Elotuzumab is a humanized monoclonal antibody specific for signaling lymphocytic activation molecule-F7 (SLAMF7, also known as CS1, CD319, or CRACC) that enhances natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) of SLAMF7-expressing myeloma cells. This study explored the mechanisms underlying enhanced myeloma cell killing with elotuzumab as a single agent and in combination with lenalidomide, to support ongoing phase III trials in patients with relapsed/refractory or newly-diagnosed multiple myeloma (MM). An in vitro peripheral blood lymphocyte (PBL)/myeloma cell co-culture model was developed to evaluate the combination of elotuzumab and lenalidomide. Expression of activation markers and adhesion receptors was evaluated by flow cytometry, cytokine expression by Luminex and ELISPOT assays, and cytotoxicity by myeloma cell counts. Elotuzumab activated NK cells and promoted myeloma cell death in PBL/myeloma cell co-cultures. The combination of elotuzumab plus lenalidomide demonstrated superior anti-myeloma activity on established MM xenografts in vivo and in PBL/myeloma cell co-cultures in vitro than either agent alone. The combination enhanced myeloma cell killing by modulating NK cell function that coincided with the upregulation of adhesion and activation markers, including interleukin (IL)-2Rα expression, IL-2 production by CD3(+)CD56(+) lymphocytes, and tumor necrosis factor (TNF)-α production. In co-culture assays, TNF-α directly increased NK cell activation and myeloma cell death with elotuzumab or elotuzumab plus lenalidomide, and neutralizing TNF-α decreased NK cell activation and myeloma cell death with elotuzumab. These results demonstrate that elotuzumab activates NK cells and induces myeloma cell death via NK cell-mediated ADCC, which is further enhanced when combined with lenalidomide.

Nuclear Factor κB is Required for Tumor Growth Inhibition Mediated by Enavatuzumab (PDL192), a Humanized Monoclonal Antibody to TweakR.

TweakR is a TNF receptor family member, whose natural ligand is the multifunctional cytokine TWEAK. The growth inhibitory activity observed following TweakR stimulation in certain cancer cell lines and the overexpression of TweakR in many solid tumor types led to the development of enavatuzumab (PDL192), a humanized IgG1 monoclonal antibody to TweakR. The purpose of this study was to determine the mechanism of action of enavatuzumab's tumor growth inhibition and to provide insight into the biology behind TweakR as a cancer therapeutic target. A panel of 105 cancer lines was treated with enavatuzumab in vitro; and 29 cell lines of varying solid tumor backgrounds had >25% growth inhibition in response to the antibody. Treatment of sensitive cell lines with enavatuzumab resulted in the in vitro and in vivo (xenograft) activation of both classical (p50, p65) and non-classical (p52, RelB) NFκB pathways. Using NFκB DNA binding functional ELISAs and microarray analysis, we observed increased activation of NFκB subunits and NFκB-regulated genes in sensitive cells over that observed in resistant cell lines. Inhibiting NFκB subunits (p50, p65, RelB, p52) and upstream kinases (IKK1, IKK2) with siRNA and chemical inhibitors consistently blocked enavatuzumab's activity. Furthermore, enavatuzumab treatment resulted in NFκB-dependent reduction in cell division as seen by the activation of the cell cycle inhibitor p21 both in vitro and in vivo. The finding that NFκB drives the growth inhibitory activity of enavatuzumab suggests that targeting TweakR with enavatuzumab may represent a novel cancer treatment strategy.

Expression of TweakR in breast cancer and preclinical activity of enavatuzumab, a humanized anti-TweakR mAb.

BACKGROUND: The receptor for the cytokine TWEAK (TweakR) is a cell surface member of the tumor necrosis factor receptor superfamily with diverse biological roles. TNFRSF family members are appealing therapeutic targets in oncology due to their aberrant expression and function in tumor cells. The goal of the current study was to examine the potential of TweakR as a therapeutic target in breast cancer. METHODS: Expression of TweakR in primary breast cancer tissues and metastases was characterized using immunohistochemistry. To determine the functional relevance of TweakR, breast cancer cell lines were treated in vitro and in vivo with enavatuzumab, a humanized mAb against TweakR. RESULTS: Overexpression of TweakR was observed in infiltrating tumors compared to normal adjacent breast tissues, and strong staining of TweakR was observed in all subtypes of invasive ductal breast cancer. In addition, a positive correlation of TweakR and HER2 expression and co-localization were observed, irrespective of ER status. TweakR expression was also observed in bone metastasis samples from primary breast cancer but rarely in benign tumors. Enavatuzumab inhibited the in vitro growth of TweakR-expressing breast cancer cell lines, and this activity was augmented by cross-linking the mAb. In addition, enavatuzumab significantly inhibited the in vivo growth of multiple breast cancer xenograft models including a model of metastasis. CONCLUSIONS: TweakR is highly expressed in all subtypes of invasive ductal breast cancer, and enavatuzumab administration exhibited a dose-dependent inhibition of primary tumor growth and lung metastasis and enhanced the antitumor activity of several chemotherapy agents currently used to treat breast cancer. These data provide the rationale to evaluate enavatuzumab as a potential therapy for the treatment of breast cancer.

Antibodies to TWEAK receptor inhibit human tumor growth through dual mechanisms.

PURPOSE: Targeted therapeutics have significantly changed the outcome for patients diagnosed with cancer. Still, effective therapeutic intervention does not exist for many cancers and much remains to be done. The objective of this study was to identify novel genes that potentially regulate tumor growth, to target these gene products with monoclonal antibodies, and to examine the therapeutic potential of these antibodies. EXPERIMENTAL DESIGN: Using cDNA microarray analysis, we identified genes overexpressed in several solid malignancies. We generated a mouse monoclonal antibody, 19.2.1, and its humanized counterpart, PDL192, to one such target, TweakR (TWEAK receptor, Fn14, TNFRSF12A, CD266), and characterized the antitumor activities in vitro and in mouse xenograft models. RESULTS: Both 19.2.1 (mouse IgG2a) and PDL192 (human IgG1), like TWEAK, the natural ligand of TweakR, inhibited the growth of several TweakR-expressing cancer cell lines in anchorage-dependent and anchorage-independent assays in vitro. Both antibodies showed significant antitumor activity in multiple mouse xenograft models. PDL192 and 19.2.1 also induced antibody-dependent cellular cytotoxicity (ADCC) of cancer cell lines in vitro. A chimeric version of 19.2.1 containing the mouse IgG1 Fc region (19.2.1 x G1) exhibited significantly less ADCC than 19.2.1. However, 19.2. 1x G1 showed differential activity in vivo, with activity equivalent to 19.2.1 in one model, but significantly less efficacy than 19.2.1 in a second model. These results indicate that PDL192 and 19.2.1 mediate their antitumor effects by signaling through TweakR, resulting in reduced tumor cell proliferation, and by ADCC.

Dissociation of efficacy and cytokine release mediated by an Fc-modified anti-CD3 mAb in a chronic experimental autoimmune encephalomyelitis model.

Humanization and modification of the Fc region of anti-human CD3 mAbs have greatly expanded their potential use in chronic T cell mediated diseases. However, low levels of cytokine release and immunogenicity may still impact a chronic dosing strategy. We investigated the use of an Fc-modified murine chimeric anti-mouse CD3 (N297A) in the chronic MOG(35-55)-induced EAE mouse model of MS. Two daily doses of 10 microg at the onset of clinical symptoms led to both a reduction in T cell numbers in the blood and a significant, prolonged reduction in the symptoms. Histological examination of the spinal cords at the peak of efficacy confirmed a reduction of infiltrating T cells in the CNS. Analysis of the cerebral spinal fluid from EAE mice showed biologically active levels of N297A. Analysis of the cytokine/chemokine levels in cerebrospinal fluid showed a decrease in GM-CSF, IL-6 and IP-10. The combination of N297A dosing with cyclosporine A (CSA) pretreatment showed a significant decrease of TNFalpha, IL-6 and IP-10 without effect on clinical efficacy. However, pretreatment of CSA significantly reduced the immunogenic response observed following a second course of N297A treatment. Therefore, the side effects of an Fc-modified anti-CD3 mAb may be modulated without affecting efficacy.

Functional characterization of N297A, a murine surrogate for low-Fc binding anti-human CD3 antibodies.

Several low- or non-FcR binding anti-human CD3 monoclonal antibodies have been under investigation for the treatment of autoimmune diseases. To model the mechanism of action of these anti-human CD3 mAbs in the murine system, an Fc-modified anti-mouse CD3 antibody (N297A) was generated. N297A exhibited similar biological effects as Fc-modified anti-human CD3 antibodies including rapid, reversible reduction in peripheral leukocyte numbers, differential modulation of activated versus resting T cells, and reduced levels of induced cytokine release compared to the non-Fc-modified parent antibody. In an in vivo model of colitis induced by adoptive transfer of IL-10-deficient cells, administration of N297A significantly reduced body weight loss. As N297A shared many functional characteristics of non-FcR binding anti-human CD3 mAbs both in vitro and in vivo, it provides a means to model the mechanisms of action of Fc-modified anti-human CD3 antibodies in mouse.

A function blocking anti-mouse integrin alpha5beta1 antibody inhibits angiogenesis and impedes tumor growth in vivo.

BACKGROUND: Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin alpha5beta1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200), inhibits endothelial cell growth and movement in vitro, independent of the growth factor milieu, and inhibits tumor growth in vivo in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine alpha5beta1, precluding its use in standard mouse xenograft models. METHODS: We generated a panel of rat-anti-mouse alpha5beta1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for alpha5beta1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis in vitro. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models. RESULTS: A panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin alpha5beta1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC50 = 5.3 nM). In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40-60% (p < 0.05) and this inhibition correlates with a concomitant decrease in vessel density. CONCLUSION: The results herein demonstrate that 339.1, like volociximab, exhibits potent anti-alpha5beta1 activity and confirms that inhibition of integrin alpha5beta1 impedes angiogenesis and slows tumor growth in vivo.