Search

SARS-CoV-2-neutralizing antibodies are promising therapeutics for COVID-19. However, little is known about the mechanisms of action of these antibodies or their effective dosing windows. We report the discovery and development of SC31, a potent SARS-CoV-2 neutralizing IgG1 antibody, originally isolated from a convalescent patient at day 27 after the onset of symptoms. Neutralization occurs via a binding epitope that maps within the ACE2 interface of the SARS-CoV-2 Spike protein, conserved across all common circulating SARS-CoV-2 mutants. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, SC31 demonstrated potent survival benefit by dramatically reducing viral load concomitant with attenuated pro-inflammatory responses linked to severe systemic disease, such as IL-6. Comparison with a Fc-null LALA variant of SC31 demonstrated that optimal therapeutic efficacy of SC31 requires intact Fc-mediated effector functions that can further induce an IFNγ-driven anti-viral immune response. Dose-dependent efficacy for SC31 was observed down to 5mg/kg when dosed before the activation of lung inflammatory responses. Importantly, despite FcγR binding, no evidence of antibody dependent enhancement was observed with the Fc-competent SC31 even at sub-therapeutic doses. Therapeutic efficacy was confirmed in SARS-CoV-2-infected hamsters, where SC31 again significantly reduced viral load, decreased lung lesions and inhibited progression to severe disease manifestations. This study underlines the potential for significant COVID-19 patient benefit for the SC31 antibody that justifies rapid advancement to the clinic, as well as highlighting the importance of appropriate mechanistic and functional studies during development.One Sentence SummaryAnti-SARS-CoV-2 IgG1 antibody SC31 controls infectionin vivoby blocking SP:ACE2 binding and triggering a Fc-mediated anti-viral response.

BackgroundV-domain Ig suppressor of T cell Activation (VISTA), an immune checkpoint regulator predominantly expressed on myeloid cells, represents a promising therapeutic target due to its role in suppressing pro-inflammatory, anti-tumor responses within the tumor microenvironment (TME). Based on VISTA’s broad expression across immune cell subtypes, HMBD-002 has been designed as a non-depleting, IgG4 monoclonal antibody with high affinity and specificity to VISTA across species (human, cynomolgus monkey, and rodent) that has the ability to block a predicted counter-structure binding site. In preclinical studies, HMBD-002 significantly inhibited tumor growth, both as a monotherapy and in combination with pembrolizumab, while decreasing infiltration of suppressive myeloid cells within the TME and increasing T cell activity. While rapid serum clearance and immune toxicities (e.g. cytokine release syndrome) have been reported for IgG1 antibodies, these were not observed preclinically with HMBD-002. In addition to VISTA expression on pro-inflammatory immune cells, examination of VISTA expression across cancer types has revealed that several malignancies, particularly human samples of triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC), express high levels of VISTA, thereby providing a rationale for exploring these indications in clinical studies.MethodsThis Phase 1, first in human study is being conducted in two parts and will evaluate multiple doses and schedules of intravenously (IV) administered HMBD-002, with or without pembrolizumab, in patients with advanced solid tumors. Part 1 (dose escalation) seeks to identify the maximum tolerated dose (MTD), or the maximum tested dose, of HMBD-002 as a monotherapy, and separately, in combination with pembrolizumab to define the recommend doses for subsequent disease directed studies (i.e., recommended phase 2 dose [RP2D]). Part 2 (dose expansion) will assess the anti-cancer activity of HMBD-002 as a monotherapy at the RP2D in previously treated patients with TNBC, and NSCLC, and in combination with pembrolizumab in patients with TNBC, NSCLC, and other VISTA-expressing malignancies. The size of the disease-directed cohorts will be determined based on an interim futility analysis conducted upon enrollment of 15 patients. Safety, efficacy, pharmacokinetic, and pharmacodynamic endpoints will be monitored and reported. Correlative studies will assess pre- and post-treatment markers of immune activity in the periphery and the tumor microenvironment.AcknowledgementsThis work was funded in part by the Cancer Prevention and Research Institute of Texas (CPRIT).Ethics ApprovalThe study was approved by each participating Institution’s Institutional Review Board.

Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate.

In recent years, HER3 has increasingly been implicated in the progression of a variety of tumor types and in acquired resistance to EGFR and HER2 therapies. Whereas EGFR and HER2 primarily signal through the MAPK pathway, HER3, as a heterodimer with EGFR or HER2, potently activates the PI3K pathway. Despite its critical role, previous attempts to target HER3 with neutralizing antibodies have shown disappointing efficacy in the clinic, most likely due to suboptimal and indirect mechanisms of action that fail to completely block heterodimerization; for example, tumors can escape inhibition of ligand binding by upregulating ligand-independent mechanisms of HER3 activation. We therefore developed 10D1F, a picomolar affinity, highly specific anti-HER3 neutralizing antibody that binds the HER3 heterodimerization interface, a region that was hitherto challenging to raise antibodies against. We demonstrate that 10D1F potently inhibits both EGFR:HER3 and HER2:HER3 heterodimerization to durably suppress activation of the PI3K pathway in a broad panel of tumor models. Even as a monotherapy, 10D1F shows superior inhibition of tumor growth in the same cell lines both in vitro and in mouse xenograft experiments, when compared with other classes of anti-HER3 antibodies. This includes models demonstrating ligand-independent activation of heterodimerization as well as constitutively activating mutations in the MAPK pathway. Possessing favorable pharmacokinetic and toxicologic profiles, 10D1F uniquely represents a new class of anti-HER3 neutralizing antibodies with a novel mechanism of action that offers significant potential for broad clinical benefit. 10D1F is a novel anti-HER3 antibody that uniquely binds the receptor dimerization interface to block ligand-dependent and independent heterodimerization with EGFR/HER2 and thus more potently inhibits tumor growth than existing anti-HER3 antibodies.

e14569 Background: Inhibition of immune checkpoints has been an effective strategy against several cancers, but efficacy gaps remain. VISTA is an immunoregulatory protein expressed on both hematopoietic and tumor cells and has been shown to induce an immunosuppressive environment by repressing T-cell activation and cytokine production. VISTA expression has been shown to be upregulated following treatment with immune checkpoint inhibitors. Hummingbird Bioscience is developing HMBD-002, an IgG4 antibody against VISTA with high binding specificity across species (human, cyno and rodent) and low pM affinity to the region of VISTA required for engagement with interaction partners, including VSIG3. As HMBD-002 is an IgG4 isotype, it uniquely blocks the inhibitory function of VISTA and releases immune suppression without depleting VISTA+ cells. Here we establish the preclinical efficacy and safety of HMBD-002 along with the rationale for development of HMBD-002 as a monotherapy and in combination with PD1 inhibitors in triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). Methods: The effect of HMBD-002 in an in vitro model of human immune activation was evaluated using an allogenic Mixed Lymphocyte Reaction (MLR) assay. Tumor growth inhibition studies were conducted in syngeneic murine cell-derived xenograft (CDX) models. The PK and tolerability of HMBD-002 was evaluated in rats and cynomolgus monkeys with a 21 or 28-day observation period. The expression profile of VISTA was examined in different cancers by IHC staining of FFPE TMAs of various solid cancers. Results: HMBD-002 induced a significant dose dependent increase in the levels of inflammatory cytokines, IFN-γ and TNF- α and suppressed the expansion of CD14+ monocytes. Treatment with HMBD-002 significantly inhibited tumor growth and improved survival in CDX models. HMBD-002 is well tolerated in rats and cynomolgus monkeys with acceptable serum half-life, no HMBD-002 related morbidity/mortality or clinical signs, and no HMBD-002 related changes were observed in body weight, food consumption, clinical chemistry, or hematology parameters. HMBD-002 did not induce cytokine release in human PBMCs or whole blood. TNBC and NSCLC patients were found to harbor the highest VISTA expression across cancer types tested, providing a rationale for exploring these indications in upcoming clinical studies. Conclusions: These studies provide the rationale for development of HMBD-002 as a monotherapy and in combination with pembrolizumab to treat patients with solid tumors expressing VISTA in the tumor microenvironment. A Phase 1 study will be initiated in 2021.

Immune checkpoint inhibitors (ICIs) have improved overall survival for cancer patients, however, optimal duration of ICI therapy has yet to be defined. Given ICIs were first used to treat patients with metastatic melanoma, a condition that at the time was incurable, little attention was initially paid to how much therapy would be needed for a durable response. As the early immunotherapy trials have matured past 10 years, a significant per cent of patients have demonstrated durable responses; it is now time to determine whether patients have been overtreated, and if durable remissions can still be achieved with less therapy, limiting the physical and financial toxicity associated with years of treatment. Well-designed trials are needed to identify optimal duration of therapy, and to define biomarkers to predict who would benefit from shorter courses of immunotherapy. Here, we outline key questions related to health, financial and societal toxicities of over treating with ICI and present four unique clinical trials aimed at exposing criteria for early cessation of ICI. Taken together, there is a serious liability to overtreating patients with ICI and future work is warranted to determine when it is safe to stop ICI.

Myeloid-derived suppressor cells (MDSCs) are a group of immature myeloid cells with potent immune suppressive activity. Accumulation of pathologically activated MDSCs within the tumor microenvironment has emerged as an important mechanism of primary and acquired resistance to immunotherapy. MDSCs have high surface expression levels of V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA), a co-inhibitory negative checkpoint regulator (NCR). Immune suppression by VISTA+ MDSCs has been implicated in the failure of, and resistance to, anti-PD1/PDL1 therapy and represents a unique axis for NCR targeting in the non-responder population. Elucidating the dynamics of VISTA+ MDSCs within tumors is imperative for predicting response and patient stratification. HMBD-002 is a novel anti-VISTA neutralizing monoclonal antibody, developed using Hummingbird Bioscience's proprietary Rational Antibody Discovery platform, against a specific functional epitope on VISTA predicted to be involved in ligand binding. HMBD002 binds human, cyno and murine VISTA protein with high affinity (kD To investigate predictive immune signatures for HMBD002 antibody efficacy, as a monotherapy and in combination with anti- PD-L1 therapy, we established multiple mouse models of tumor immune subtypes using syngeneic cell lines. Tumor biopsies were analyzed before and during treatment, to determine the evolution of immune response and its effect on tumor burden within responders and non-responders. The combination of HMBD-002 and anti-PD-L1 was more effective than either antibody alone, especially in tumors that showed abundant MDSC infiltration. Immune phenotyping revealed that this was associated with a significant increase in activated CD8+ T effector cells and concomitant increase in IFN-gamma. Profiling syngeneic models for deeper insights into the interplay between the tumor, immune system and therapy, will facilitate the clinical development of immune checkpoint and combination therapies. Hummingbird Bioscience anticipates commencing First-in-Human trials of HMBD002 in 2019. Citation Format: Jerome D. Boyd-Kirkup, Dipti Thakkar, Konrad Paszkiewicz, Piers J. Ingram. Integrative immune profiling of syngeneic tumor models provides predictive immune signatures for treatment response with HMBD002, a novel anti-VISTA neutralizing antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1729.

Background: Progressive cellular and extracellular matrix (ECM) changes related to age and disease severity have been demonstrated in rotator cuff tendon tears. Larger rotator cuff tears demonstrate structural abnormalities that potentially adversely influence healing potential. This study aimed to gain greater insight into the relationship of pathologic changes to tear size by analyzing gene expression profiles from normal rotator cuff tendons, small rotator cuff tears, and large rotator cuff tears. Methods: We analyzed gene expression profiles of 28 human rotator cuff tendons using microarrays representing the entire genome; 11 large and 5 small torn rotator cuff tendon specimens were obtained intraoperatively from tear edges, which we compared with 12 age-matched normal controls. We performed real-time polymerase chain reaction and immunohistochemistry for validation. Results: Torn rotator cuff tendons demonstrated upregulation of a number of key genes, such as matrix metalloproteinase 3, 10, 12, 13, 15, 21, and 25; a disintegrin and metalloproteinase (ADAM) 12, 15, and 22; and aggrecan. Amyloid was downregulated in all tears. Small tears displayed upregulation of bone morphogenetic protein 5. Chemokines and cytokines that may play a role in chemotaxis were altered; interleukins 3, 10, 13, and 15 were upregulated in tears, whereas interleukins 1, 8, 11, 18, and 27 were downregulated. Conclusions: The gene expression profiles of normal controls and small and large rotator cuff tear groups differ significantly. Extracellular matrix remodeling genes were found to contribute to rotator cuff tear pathogenesis. Rotator cuff tears displayed upregulation of a number of matrix metalloproteinase (3, 10, 12, 13, 15, 21, and 25), a disintegrin and metalloproteinase (ADAM 12, 15, and 22) genes, and downregulation of some interleukins (1, 8, and 27), which play important roles in chemotaxis. These gene products may potentially have a role as biomarkers of failure of healing or therapeutic targets to improve tendon healing.

V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA) also known as PD1 homolog (PD1H) is a co-inhibitory immune checkpoint receptor. VISTA is predominantly expressed on hematopoietic cells, particularly myeloid derived suppressor cells and antigen presenting cells, and at lower levels on CD4+ and CD8+ T cells and Foxp3 Tregs. Multiple studies have demonstrated VISTA can strongly suppress human T-cell activation, and the presence of high VISTA expressing cells in the tumor microenvironment has been postulated as playing a critical role in tumorigenesis and resistance to immunotherapy. VISTA neutralizing antibodies have been previously observed in pre-clinical models to increase the abundance of tumor infiltrating effector T cells as well as their effector functions, resulting in enhanced control of tumor growth, even in the absence of detectable expression of VISTA on the tumor cells, a potential advantage over PD1 or PDL1 blockade. There is an urgent need to develop the most clinically effective anti-VISTA antibodies however the limited characterization of the VISTA pathway and the uncertainty around the cognate VISTA ligand has limited the extent to which in vitro studies can support the selection of optimal antibodies for development. Hummingbird Bioscience’s proprietary Rational Antibody Development platform to the design of neutralizing antibodies against specific epitopes on VISTA. Extensive in silico analyses of the VISTA structure and comparative structural modeling against other B7 protein family members has enabled the prediction of key binding sites where antibody binding will inhibit function, while preserving necessary safety and developability profiles. Subsequently, a directed evolution strategy was used to isolate monoclonal antibodies specifically targeting these key predicted functional domains on the native folded VISTA protein. Hummingbird’s anti-VISTA antibodies bind human and murine VISTA protein with high affinity (kD 60% inhibition of tumor growth (after 15 days) with no observable toxicity. Further in vitro and in vivo validation experiments in other cancer models are ongoing, both as monotherapy and in combination with other immune therapies. The first in-human trial of HMBD-002 is planned for 2018. Citation Format: Piers J. Ingram, Dipti Thakkar, Jerome D. Boyd-Kirkup. HMBD002, a novel neutralizing antibody targeting a specific epitope on the co-inhibitory immune checkpoint receptor VISTA, displays potent anti-tumor effects in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 587. doi:10.1158/1538-7445.AM2017-587

Gastric cancer is the second highest cause of cancer mortality in Asia. Over-expression of ErbB3, a cell surface RTK, occurs in ~60% of mGC patients where it is significantly associated with poorer survival. Ligand binding to ErbB3 triggers heterodimerization and downstream signaling through Akt. Neutralizing antibodies that inhibit ligand binding show poor efficacy in clinical trials, likely due to high levels of ligand independent activation in many tumor types. Directly inhibiting heterodimerization could be more effective, however, current methods of antibody isolation offer limited control over the site of antibody binding and, therefore, mechanism of action, requiring extensive screening of antibodies and posing significant challenges to identifying suitable candidates for therapeutic development. Hummingbird Bioscience has used its proprietary Rational Antibody Development Platform for the design and development of novel therapeutic antibodies against ErbB3. Computational sequence and structural analyses predicted highly specific surface epitopes, with strong antigenicity and good safety profiles, where antibody binding would inhibit dimerization. Subsequently, Hummingbird’s directed evolution mouse immunization strategy efficiently isolated monoclonal antibodies that bound with low nM (Kd < 5nM) affinity to these epitopes on native folded ErbB3. Furthermore, these mAbs showed ELISA cross-reactivity to ErbB3 in model organisms (mouse, rat, monkey) but no binding to other EGFR family proteins. Antibody HMBD001 was found to strongly inhibit the phosphorylation of ErbB3 and decrease downstream signaling through the Akt pathway (90% decrease in p-ErbB3 and 60% decrease in p-AKT observed at 24 hrs) resulting in the inhibition of tumor cell proliferation in models of ErbB3+ gastric cancer (over 90% inhibition of NCI-N87 proliferation after 5 days) and breast cancer (up to 85% inhibition of BT474 proliferation after 5 days). In mouse NCI-N87 gastric cancer CDX models, weekly doses of HMBD001 achieved greater than 90% inhibition of tumor growth (at 25 days) with no observable adverse toxicity. The first in-human trial of HMBD001 is planned for 2018. Citation Format: Jerome D. Boyd-Kirkup, Dipti Thakkar, Piers J. Ingram. HMBD001, a novel anti-ErbB3 antibody with a unique mechanism of action, effectively inhibits tumor growth in pre-clinical models of ErbB3+ solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 24. doi:10.1158/1538-7445.AM2017-24

There is increasing evidence for a progressive extracellular matrix change in rotator cuff disease progression. Directly surrounding the cell is the pericellular matrix, where assembly of matrix aggregates typically occurs making it critical in the response of tendon cells to pathological conditions. Studies in animal models have identified type VI collagen, fibrillin-1 and elastin to be located in the pericellular matrix of tendon and contribute in maintaining the structural and biomechanical integrity of tendon. However, there have been no reports on the localization of these proteins in human tendon biopsies. This study aimed to characterize the distribution of these ECM components in human rotator cuffs and gain greater insight into the relationship of pathology to tear size by analyzing the distribution and expression profiles of these ECM components. Confocal microscopy confirmed the localization of these structural molecules in the pericellular matrix of the human rotator cuff. Tendon degeneration led to an increased visibility of these components with a significant disorganization in the distribution of type VI collagen. At the genetic level, an increase in tear size was linked to an increased transcription of type VI collagen and fibrillin-1 with no significant alteration in the elastin levels. This is the first study to confirm the localization of type VI collagen, elastin and fibrillin-1 in the pericellular region of human supraspinatus tendon and assesses the effect of tendon degeneration on these structures, thus providing a useful insight into the composition of human rotator cuff tears which can be instrumental in predicting disease prognosis.

A comprehensive proteomic study utilizing 2D-DIGE and MALDI-TOF was used to assess the effect of inhibiting two different regulatory mechanisms of telomerase in glioma. RNAi was used to target hTERT and hsp90α. Inhibition of telomerase activity resulted in downregulation of various cytoskeletal proteins with correlative evidence of the involvement of telomerase in regulating the expression of vimentin. Inhibition of telomerase via sihTERT resulted in the downregulation of vimentin expression in glioma cell lines in a grade-specific manner. This study identified novel downstream role of telomerase in regulating the expression of vimentin, thereby affecting tumor progression and metastasis.

Hsp90alpha's vital role in cell cycle progression and apoptosis together with its presence in gliomas and absence in normal tissue, make it a credible target for cancer therapy. Three sets of dsRNA oligos designed to align different regions of the hsp90alpha sequence were used to downregulate hsp90alpha. SiRNA 1, 2, and 3 resulted in significant levels of silencing of hsp90alpha after 48 hr treatment (p.0001). Concurrent treatment of the glioma cell line U87-MG with siRNA 1 and temozolomide (TMZ) resulted in a 13-fold reduction in the dose of TMZ required to achieve a similar effect if TMZ was used alone.

Telomerase expression strongly correlates with the grade of malignancy in glioma with inhibition illustrating a definite increase in chemosensitivity. This study was designed to investigate the effects of a green tea derivative, epigallocatechin-3-gallate (EGCG); together with either cisplatin or tamoxifen in glioma, and to investigate whether these effects are mediated through telomerase suppression. EGCG showed a significant cytotoxic effect on 1321N1 cells after 24 h and on U87-MG cells after 72 h (P < 0.001) without significantly affecting the normal astrocytes. Treatment with EGCG inhibited telomerase expression significantly (P < 0.01) and enhanced the effect of cisplatin and tamoxifen in both 1321N1 (P < 0.01) and U87-MG (P < 0.001) cells. EGCG, as a natural product has enormous potential to be an anti-cancer agent capable of enhancing tumour cell sensitivity to therapy.