The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, ...depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.
B7-H3 is a novel protein structurally related to the B7 family of ligands by the presence of a single set of immunoglobulin-V-like and immunoglobulin-C-like (VC) domains. By multiplex PCR, the ...dominantly expressed form of human B7-H3 was found to be a splice variant containing tandemly duplicated VC domains (VCVC). In contrast, mouse B7-H3 cDNA contained only one single VC form due to an exon structure corresponding to V–(pseudoexon C)–(pseudoexon V)–C. Comparisons of human, monkey, mouse, and hamster genomic
B7-H3 reveal that primates, but not rodents, exhibited a higher degree of intramolecular sequence similarity between VC duplications than between molecules. Both VC and VCVC forms of human B7-H3 inhibited CD4
+ T cell proliferation and downregulated cytokine production upon TCR activation. These results suggest independent, but convergent, paths of B7-H3 active domain duplication followed by divergent histories of exon degeneration in rodents and exon maintenance by humans.
BackgroundLeukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4) is an immunoinhibitory protein expressed on the surface of myeloid cells that has been increasingly recognized as a therapeutic ...target of interest in immuno-oncology (IO). Upon binding its ligands, MHC I molecules (e.g. HLA-G/HLA-A), LILRB2 inhibits myeloid cell activation and promotes an M2-like (anti-inflammatory) state. LILRB2 was the first target prioritized from a macrophage discovery effort leading to the development of JTX-8064, a humanized monoclonal antibody that specifically binds to and antagonizes LILRB2. JTX-8064 has been shown to induce an M1-like (pro-inflammatory; anti-tumor) functional state in macrophages. Rodents do not express LILRB proteins limiting their usefulness as a model for preclinical study of JTX-8064. To overcome this limitation, we conducted an ex vivo human tumor histoculture study to assess the pharmacodynamic effects of LILRB2 antagonism. Protein and/or gene expression analysis of matched tumor samples enabled the discovery of predictive biomarkers associated with the induction of specific pharmacodynamic signatures in ex vivo-cultured human tumors in response to JTX-8064. Finally, tumor types were identified that had a high prevalence of these predictive biomarkers suggesting they may be priority indications for JTX-8064 therapy.MethodsMore than 100 fresh treatment-naïve human tumor samples obtained post-surgery from kidney, lung, and head and neck cancer were treated with JTX-8064 or isotype control antibody for 24 hrs in the histoculture system. RNA was isolated from tumors prior to any treatment as well as from JTX-8064 and isotype control treated samples. Gene expression was analyzed using the NanoString nCounter® and qPCR assays. Additional IHC analyses were performed on baseline untreated tumor samples.ResultsJTX-8064 was shown to induce pharmacodynamic responses to treatment significantly above isotype control indicative of macrophage polarization, IFNg-signaling, and T cell inflammation. To identify predictive biomarkers of pharmacodynamic response to JTX-8064, matched untreated samples were characterized by gene expression analysis and by IHC (CD8, CD163, and HLA-G proteins). Numerous LILRB2 pathway-related molecules (e.g. HLA-A, HLA-B, CD163, LILRB2) and gene signatures were found to be statistically significantly higher in the untreated kidney, head and neck, and lung cancer samples of matched pharmacodynamic responders compared to non-responders. Further bioinformatics analysis revealed additional cancer subtypes where these biomarkers are enriched.ConclusionsThese data will inform indication selection and combination strategies for JTX-8064 to maximize potential therapeutic benefit for patients with solid tumor malignancies.
Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of ...many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in V
L
CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Fas
lpr
mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC0
-∞
) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2-3-fold or 4-7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Fas
lpr
mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.
We describe a simple, rapid technique for simultaneously isolating large numbers of cDNAs encoding secreted proteins. The technique makes use of a facile genetic selection performed in a strain of
...Saccharomyces cerevisiae deleted for its endogenous invertase gene. A cDNA cloning vector which carries a modified invertase gene lacking its leader sequence is used in conjunction with this strain. Heterologous secreted genes fused appropriately upstream of this defective invertase provide the necessary signals to restore secretion, allowing the yeast to grow on sugars such as sucrose or raffinose. This microbial growth selection facilitates scanning cDNA libraries containing millions of clones, enabling the wholesale identification of novel secreted proteins without the need for specific bioassays. The technique is similar to one previously described (
Klein et al. (1996)Proc. Natl. Acad. Sci. USA 93, 7108–7113). We describe results using a cDNA library derived from activated human peripheral blood mononuclear cells (PBMC). Genes identified from this library encoded signal sequences of proteins of diverse structure, function, and cellular location such as cytokines, type 1 and type 2 transmembrane proteins, and proteins found in intracellular organelles. In addition, a number of novel secreted proteins were identified, including a chemokine and a novel G-protein-coupled receptor. Since signal sequences possess features conserved throughout evolution, the procedure can be used to isolate genes encoding secreted proteins from both eukaryotes and prokaryotes.
Anti-IL-21R antibodies are potential therapeutics for the treatment of autoimmune diseases. This study evaluated correlations between the pharmacodynamic (PD) activity, pharmacokinetics, and ...anti-product antibody responses of human anti-IL-21R antibodies Ab-01 and Ab-02 following IV administration to cynomolgus monkeys.
The PD assay was based on the ability of recombinant human IL-21 (rhuIL-21) to induce expression of the IL-2RA gene in cynomolgus monkey whole blood samples ex vivo. Monkeys screened for responsiveness to rhuIL-21 stimulation using the PD assay, were given a single 10 mg/kg IV dosage of Ab-01, Ab-02, or a control antibody (3/group), and blood samples were evaluated for PD activity (inhibition of IL-2RA expression) for up to 148 days. Anti-IL-21R antibody concentrations and anti-product antibody responses were measured in serum using immunoassays and flow cytometry.
Following IV administration of Ab-01 and Ab-02 to cynomolgus monkeys, PD activity was observed as early as 5 minutes (first time point sampled). This PD activity had good correlation with the serum concentrations and anti-product antibody responses throughout the study. The mean terminal half-life (t1/2) was approximately 10.6 and 2.3 days for Ab-01 and Ab-02, respectively. PD activity was lost at approximately 5-13 weeks for Ab-01 and at approximately 2 weeks for Ab-02, when serum concentrations were relatively low. The estimated minimum concentrations needed to maintain PD activity were approximately 4-6 nM for Ab-01 and approximately 2.5 nM for Ab-02, and were consistent with the respective KD values for binding to human IL-21R. For Ab-01, there was noticeable inter-animal variability in t1/2 values (approximately 6-14 days) and the resulting PD profiles, which correlated with the onset of anti-product antibody formation. While all three Ab-01-dosed animals were positive for anti-Ab-01 antibodies, only one monkey (with the shortest t1/2 and the earliest loss of PD activity) had evidence of neutralizing anti-Ab-01 antibodies. All three Ab-02-dosed monkeys developed neutralizing anti-Ab-02 antibodies.
For anti-IL-21R antibodies Ab-01 and Ab-02, there was good correlation between PD activity and PK profiles following IV administration to cynomolgus monkeys. Compared with Ab-01, Ab-02 was eliminated markedly faster from the circulation, which correlated with a shorter duration of PD activity.
Tumor-associated macrophages (TAM) play an important role in maintaining the immunosuppressive state of the tumor microenvironment (TME). High levels of CD163+ TAMs specifically are associated with ...poor prognosis in many solid tumor types. Targeting TAMs may represent a key approach in development of the next generation of cancer immune therapeutics. Members of the leukocyte immunoglobulin-like receptor B (LILRB) family, including LILRB2 (ILT4), are known to transmit inhibitory signals in macrophages and other myeloid cells. Leveraging bulk and single cell RNA-sequencing datasets, as well as extensive immunophenotyping of human tumors, we found that LILRB2 is highly expressed on CD163+ CD11b+ cells in the TME and that LILRB2 expression correlates with CD163 expression across many tumor types. To target LILRB2, we have developed JTX-8064, a highly potent and selective antagonistic mAb. JTX-8064 blocks LILRB2 binding to its cognate ligands, including classical and nonclassical MHC molecules. In vitro, JTX-8064 drives the polarization of human macrophages and dendritic cells toward an immunostimulatory phenotype. As a result, human macrophages treated with a LILRB2 blocker are reprogrammed to increase the activation of autologous T cells in co-culture systems. Furthermore, JTX-8064 significantly potentiates the activity of anti-PD-1 in allogeneic mixed lymphocyte reaction. In a human tumor explant culture, pharmacodynamic activity of JTX-8064 was observed in monotherapy and in combination with anti-PD-1. Collectively, our work provides strong translational and preclinical rationale to target LILRB2 in cancer.
We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. ...Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concentrations. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogues are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approximately 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.
BackgroundImmune checkpoint therapy has achieved durable clinical responses, but only in a subset of cancer patients. Immunosuppressive myeloid cells, a heterogenous group of innate immune cells, ...have emerged as key contributors to resistance to T cell based immune checkpoint therapy. Leukocyte immunoglobulin-like receptor B4 (LILRB4), also known as immunoglobulin-like transcript 3 (ILT3), is an inhibitory receptor selectively expressed on myeloid cells, enriched in the tumor microenvironment and contributes to myeloid-driven immunosuppression. Recently, fibronectin has been identified as a functional ligand for LILRB4, and the LILRB4-fibronectin interaction was proposed as a stromal checkpoint suppressing myeloid cell anti-tumor activity. Targeting LILRB4 could represent a strategy to reprogram immunosuppressive myeloid cells and promote anti-tumor response. We developed JTX-1484, a highly selective, high-affinity humanized monoclonal antibody that binds to and antagonizes LILRB4 and blocks its interaction with fibronectin.MethodsA diverse panel of high affinity monoclonal antibodies that bind specifically to LILRB4 was generated. JTX-1484 activity alone or in combination with an anti-PD1 antibody was evaluated in vitro in different functional assays. Human primary myeloid-derived suppressor cells (MDSCs) were used in a T cell suppression assay and treated with JTX-1484. Primary monocyte-derived tolerogenic dendritic cells (tDCs) were utilized in mixed lymphocyte reactions with T cells and treated with JTX-1484 in combination with anti-PD1. JTX-1484’s ability to block fibronectin inhibitory activity on tDCs and THP-1 cells was also tested. Finally, the pharmacodynamic effect of anti-LILRB4 treatment in human tumor samples was evaluated by assessing gene expression changes in an ex vivo histoculture system.ResultsJTX-1484 reprogramed tDCs to a stimulatory phenotype as evidenced by increased pro-inflammatory cytokine production and increased ability to induce T cell activation in combination with anti-PD1. LILRB4 antagonism by JTX-1484 also reversed fibronectin-mediated inhibition of tDC activation and reduced MDSC-mediated T cell immunosuppression. Moreover, LILRB4 blockade in ex vivo human tumor samples induced pharmacodynamic responses consistent with increased immune activation and reduced myeloid immunosuppression.ConclusionsResults from our preclinical studies demonstrate that JTX-1484 is a highly specific and potent antagonist of LILRB4 that leads to myeloid cell reprogramming and more efficient T cell activation that could result in enhanced anti-tumor responses. JTX-1484 immunostimulatory properties towards myeloid cells could be complementary to immune checkpoint blockade therapy. Our data therefore support clinical development of JTX-1484. Indication selection will be guided by multiple factors including predictive biomarkers such as target and ligand abundance, as well as complementarity and combination potential with other therapies.Ethics ApprovalHuman blood and tumor samples were acquired from commercial providers and from the CHTN and NDRI networks respectively. Specimens were collected under each provider's human subject research institutional review board approved protocols and were fully anonymized or otherwise permanently de-identified to recipient investigators.
BackgroundLeukocyte immunoglobulin-like receptors (LILRBs) are cell surface immunoinhibitory proteins that have been recently recognized as therapeutic targets of interest in immuno-oncology. LILRB1, ...LILRB2 and LILRB4 among them are broadly expressed on myeloid cells; however, their individual expression patterns have been difficult to define due to high sequence homology. To devise rational treatment options, it is important to develop a better understanding of LILRB1, LILRB2 and LILRB4 specific expression patterns as well as functional overlap and differences, as suggested by their diverse ligand binding partners. Here we characterize expression of these receptors on human immune cells in tumor and blood samples across different cancer indications. In addition, we performed pharmacodynamic analysis and unbiased clustering after individual LILRBs (1,2 or 4) were blocked in primary human tumor ex vivo systems to understand the roles of these receptors in complex cellular environments.MethodsCell-specific anchoring across multiple single cell RNAseq data sets was performed to characterize temporal and spatial LILRB gene expression on individual cells. Proteins were detected on human tumor specimens using ligand, cell type and LILRB-specific IHC antibodies. LILRB protein levels on immune cells from dissociated human tumors and peripheral blood mononuclear cells (PBMCs) were quantified using flow cytometry. Fresh treatment-naïve human tumor samples were treated with anti-LILRB1, anti-LILRB2, anti-LILRB4 or isotype control antibodies and gene expression analysis was performed on treated samples.ResultsLILRBs (1,2,4) are expressed on myeloid cells but distinct expression patterns also emerge. LILRB1 is expressed by T, B and NK cells; LILRB4 is highly expressed on plasmacytoid dendritic cells (pDCs) and a subset of conventional DCs (cDC2); LILRB2 is prominently expressed on immunosuppressive macrophages as well as monocytes, and within that population, intermediate monocytes display higher levels of LILRB2 than classical monocytes. In complex ex vivo systems, treatment with blocking anti-LILRB molecules induced both overlapping and distinct patterns of gene expression changes.ConclusionsLILRB1, LILRB2 and LILRB4 each display unique cell-specific expression patterns and blocking their ligand binding activity in ex vivo systems suggests that they have non-redundant functions. Thus, context-dependent inhibition of one or more of these molecules can contribute to the optimal activation of anti-tumor immunity.Ethics ApprovalHuman blood and tumor samples were acquired from commercial providers and from the CHTN and NDRI networks respectively. Specimens were collected under each provider's human subject research institutional review board approved protocols and were fully anonymized or otherwise permanently de-identified to recipient investigators.
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