Tumor Growth In Preclinical Studies Research Articles

PTHrP intracrine actions divergently influence breast cancer growth through p27 and LIFR

The role of parathyroid hormone (PTH)-related protein (PTHrP) in breast cancer remains controversial, with reports of PTHrP inhibiting or promoting primary tumor growth in preclinical studies. Here, we provide insight into these conflicting findings by assessing the role of specific biological domains of PTHrP in tumor progression through stable expression of PTHrP (-36-139aa) or truncated forms with deletion of the nuclear localization sequence (NLS) alone or in combination with the C-terminus. Although the full-length PTHrP molecule (-36-139aa) did not alter tumorigenesis, PTHrP lacking the NLS alone accelerated primary tumor growth by downregulating p27, while PTHrP lacking the NLS and C-terminus repressed tumor growth through p27 induction driven by the tumor suppressor leukemia inhibitory factor receptor (LIFR). Induction of p27 by PTHrP lacking the NLS and C-terminus persisted in bone disseminated cells, but did not prevent metastatic outgrowth, in contrast to the primary tumor site. These data suggest that the PTHrP NLS functions as a tumor suppressor, while the PTHrP C-terminus may act as an oncogenic switch to promote tumor progression through differential regulation of p27 signaling.

Characterization of methionine dependence in melanoma cells.

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of S-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

A phase 1 first-in-human clinical trial of HMBD-002, an IgG4 monoclonal antibody targeting VISTA, in advanced solid tumors.

TPS2664 Background: V-domain Ig Suppressor of T-cell Activation (VISTA), is an immune checkpoint regulator found on tumor, myeloid, and other immune cells. Its presence has been shown to enhance tumor growth, create an immunosuppressive microenvironment, and may potentially contribute to developing resistance to anti-CTLA-4 and anti-PD-1/PD-L1 therapies. Therefore, VISTA represents a promising therapeutic target. HMBD-002, a non-depleting, high-affinity IgG4 monoclonal antibody against VISTA, has demonstrated significant inhibition of tumor growth in preclinical studies, both as a monotherapy and in combination with pembrolizumab. HMBD-002 acts by increasing T-cell activity and reprogramming the suppressive tumor microenvironment to a proinflammatory antitumor phenotype. Cancer types including triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC), which exhibit high expression levels of VISTA in the TME, provide a rational basis for exploring these indications in clinical studies. Methods: This phase 1/2, open-label, multi-center trial is being conducted in two parts to evaluate the safety, pharmacokinetics, and antitumor activity of HMBD-002, an IgG4 monoclonal antibody targeting VISTA, as a monotherapy and in combination with pembrolizumab. The dose-escalation stage (Part 1) will follow a standard 3 + 3 study design with weekly dosing and adaptive dose-escalation increments to determine the recommended phase 2 dose (RP2D) for subsequent disease-directed studies. Monotherapy escalation cohorts 1-4 have been completed without dose-limiting toxicities (DLTs). The dose-expansion stage (Part 2) will evaluate the antitumor activity of HMBD-002 alone and combined with pembrolizumab in patients with TNBC and NSCLC, as well as a wide range of VISTA-expressing malignancies. Exploratory analyses will be conducted to assess treatment-induced immunological changes both systemically and within the tumor microenvironment. Longitudinal blood samples will be evaluated using single-cell mass cytometry (CYTOF) and multiplex cytokine analysis. Pre and on-treatment tumor tissue samples will be assessed for VISTA & PD-L1 expression using IHC. Additionally, tumor tissues will be evaluated using multiplex immunofluorescence, and gene-expression profiling. Biomarker correlations with clinical outcomes may lead to the identification of predictive markers of response to HMBD-002. Clinical trial information: NCT05082610 .

Monitoring autochthonous lung tumors induced by somatic CRISPR gene editing in mice using a secreted luciferase

BackgroundIn vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies.MethodsTo facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes.ResultsWe demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination.ConclusionsOur study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.

Abstract 5602: VTX-0811, a first-in-class PSGL-1 blocking monoclonal antibody, repolarizes tumor associated macrophages and induces inflammation in the tumor microenvironment, leading to suppression of tumor growth in pre-clinical studies

Abstract Suppressive myeloid cells in the tumor microenvironment (TME) are associated with poor survival of cancer patients and resistance to T cell checkpoint inhibitors. These myeloid cells shield cancer cells from the sentinel immune response and create a niche for tumor growth. Repolarizing immuno-suppressive myeloid populations in the TME attracted considerable interest from scientific community and several companies, and has produced early positive clinical data. One of the targets that potently sustains macrophage suppressive phenotypes and is highly expressed on tumor associated macrophages is PSGL-1. Blocking a previously unknown epitope on PSGL-1 triggers macrophage repolarization to a pro inflammatory state. We have determined that our therapeutic candidate VTX-0811, anti-PSGL-1 monoclonal antibody (mAb), repolarizes M2-like macrophages to a more M1-like state both phenotypically and functionally as assessed by primary in vitro macrophage assays. Transcriptomics profiling of M2c macrophages showed that VTX-0811 upregulated TNF-α/NF-κB and chemokine-mediated signaling, while downregulating oxidative phosphorylation, fatty acid metabolism, and Myc signaling pathways, consistent with a broad M2-to-M1 shift. Furthermore, repolarized macrophages enhanced inflammatory responses in complex primary multi-cellular assays.VTX-0811 also showed efficacy in a humanized mouse PDX model of melanoma. VTX-0811 suppressed tumor growth to a significantly greater degree compared with an anti-PD-1 mAb. At the cellular and molecular levels, the treatment led to an inflammatory microenvironment, including a reduced MDSC population, and increased systemic pro-inflammatory mediators. Compared with anti-PD-1 monotherapy, VTX-0811 alone and in combination with anti-PD-1 increased the fraction of CD8+ T cells among the infiltrating T cells. Significant combination effects of VTX-0811 plus anti-PD-1 were seen within the tumor tissue, spleen, and peripheral blood.Additionally, pre-clinical efficacy of VTX-0811 was demonstrated using ex vivo cultures of fresh patient-derived tumors that preserve cellular and molecular composition of the TME. VTX-0811 increased secretion of inflammatory cytokines and chemokines known to be involved in immune activation of the TME and fresh leukocyte recruitment, as well as linked to clinical response to T cell checkpoint inhibitors.VTX-0811 is a humanized high affinity IgG4/κ mAb that demonstrated safety in NHP primates up to 200mg/kg dosed weekly for 5 total doses. VTX-0811 does not induce activation of unstimulated PBMC or unstimulated blood-derived neutrophil or T cell cultures. These data provide biological and mechanistic support for advancing this program into the clinic with a first in human trial planned for early 2022. Citation Format: Igor Feldman, Tatiana Novobrantseva, Ani Nguyen, Jessica Ritter, Mohammad Zafari, Denise Manfra, Susan Low, Steve Sazinsky, Michael Brehm, Boris Klebanov. VTX-0811, a first-in-class PSGL-1 blocking monoclonal antibody, repolarizes tumor associated macrophages and induces inflammation in the tumor microenvironment, leading to suppression of tumor growth in pre-clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5602.

Selected Articles from This Issue

Colony stimulating factor 1 receptor (CSF1R)-dependent cells in the microenvironment (such as macrophages and osteoclasts) contribute to angiogenesis, tumor growth, metastasis, and bone degradation. In this First Disclosure, Smith and colleagues present the characterization of vimseltinib, a selective oral inhibitor of CSF1R in tenosynovial giant cell tumor (TGCT). Vimseltinib depleted macrophages and CSF1R-dependent cells and inhibited tumor growth in preclinical studies. Clinically, the authors present three patients whose CSF1R biomarkers and tumor burden were reduced. Their data supports the continued clinical development of vimseltinib for TGCT.The triple-negative subtyp. of breast cancer (TNBC) carries the highest rate of parenchymal brain metastasis (BrM) and leptomeningeal metastasis (LM). In this manuscript, Deng and colleagues disclose the nitrogen mustard-based compound QBS10072S. QBS10072S delayed tumor growth and reduced leptomeningeal dissemination in a preclinical brain metastasis model. Interestingly, the compound was able to target micro-metastases, which remains a difficult aspect of detecting and treating LM. As the compound was well-tolerated, the continued development of QBS10072S for TNBC LM patients is warranted.Brain metastases occur in approximately 30% of NSCLC patients, leading to significantly worse prognosis. Stereotactic radiotherapy and/or whole brain radiotherapy is currently standard of care for these patients. To increase the effectiveness of radiation therapy in NSCLC brain metastases, Baschnagel and colleagues administered the ATP-competitive ATR inhibitor M6620. M6620 radiosensitized patient-derived xenografts in mice by inhibiting pChk1 and reducing DNA double-strand break (DSB) repair. Ultimately, this resulted in longer overall survival in the preclinical model. Their results support the ongoing phase I clinical study of M6620 in combination with whole brain irradiation (NCT02589522).Adenosine acts as an immunosuppressor by suppressing T cells and dendritic cells and stimulating regulatory T cells. Adenosine is generated in the tumor microenvironment by CD73 from AMP released by cell turnover. In this study, Wurm and colleagues generate a humanized antagonistic CD73 antibody, mAb19. Combining mAb19 with PD-1 inhibition increased T cell activation in vitro. They also demonstrate the combination of mAb19 with the adenosine receptor agonist ADORA3 and doxorubicin, which is known to increase adenosine levels. mAb19 therefore inhibits immunosuppression from the adenosine pathway and is suited for continued development as a cotherapy with immune-targeted or chemotherapy.

Abstract 1629: IO-202, a first-in-class LILRB4 antagonist antibody, activates dendritic cells and inhibits solid tumor growth in preclinical studies

Abstract LILRB4, also known as ILT3, is an immune inhibitory transmembrane protein found on monocytes and antigen presenting cells, including dendritic cells (DCs). LILRB4 upregulation in DCs induces a tolerogenic phenotype that facilitates the generation of antigen-specific T regulatory cells. LILRB4 is also expressed on acute myeloid leukemia (AML) with monocytic differentiation, in which it promotes T cell suppression and tumor infiltration. IO-202 is a first-in-class LILRB4 antagonist antibody that is being evaluated in a Phase I trial (NCT04372433) for the treatment of AML and chronic myelomonocytic leukemia (CMML). RNA-seq data from TCGA database indicates that LILRB4 expression is upregulated in many solid tumor types. Therefore, the therapeutic potential of IO-202 in solid tumors was investigated in this study. Using computational biology approaches, we found that high LILRB4 expression in solid tumors from TCGA database is associated with macrophage infiltration in the tumor microenvironment. Flow cytometric analysis of cancer patients' blood and tumor samples confirmed LILRB4 expression on tumor-associated macrophages (TAMs) and on monocytic myeloid-derived suppressor cells (M-MDSCs) and DCs in tumor and periphery. These data raised the hypothesis that LILRB4 functions as a myeloid checkpoint and contributes to tumor immune evasion, not only in AML, but also in many solid tumor types. LILRB4 is not present in rodents. As such, the functional activity and anti-tumor efficacy of IO-202 were evaluated using human primary immune cells and immunocompetent LILRB4 transgenic mice, respectively. In healthy donor monocyte-derived DCs, IO-202 treatment promoted DC maturation, activation and an antigen presenting phenotype, as well as enhanced their ability to activate allogeneic T cells. In the radiation therapy (RT)-resistant Lewis lung carcinoma (LLC) syngeneic model, IO-202 in combination with RT resulted in tumor growth inhibition, which was associated with changes in the relative abundance of tumor-infiltrating immune cells, evidenced by a reduction in M-MDSCs and an increase in T cells, along with increased T cell activation. In conclusion, our data suggest that LILRB4 functions as a myeloid checkpoint in multiple cancer types and that IO-202, the first-in-class LILRB4 antagonist antibody, enhances dendritic cell function and T cell activation in vitro and anti-tumor immunity in a solid tumor model in vivo. These data demonstrate the therapeutic potential of IO-202 as a myeloid checkpoint inhibitor for solid tumors. Citation Format: Maria José Costa, Tao Huang, Jing-Tyan Ma, Caroline Bonnans, Azita Tabrizi, Xiaoling C. Liao, Paul Woodard, Benjamin P. Chen, Ching-Cheng Hsu, Mi Deng, Cheng Cheng Zhang, An Song. IO-202, a first-in-class LILRB4 antagonist antibody, activates dendritic cells and inhibits solid tumor growth in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1629.

Phase Ib study of decitabine in combination with gemcitabine in treatment of advanced soft tissue and bone sarcomas.

11550 Background: Sarcomas are a heterogeneous group of tumors and are associated with high rates of metastases leading to poor prognosis. Numerous epigenetic changes including hypermethylation have been identified in several sarcoma subtypes. Restoration of normal methylation patterns using DNA hypomethylating agent when combined with chemotherapy has shown to slow tumor growth in preclinical studies. Low continuous dosing of hypomethylating agent has epigenetic modulating effect with less toxicity. We conducted a Phase 1b study evaluating safety & tolerability and identifying the recommended phase 2 dose (RP2D) of subcutaneous (SQ) decitabine (DEC) given with fixed dose infusion gemcitabine (GEM) in patients with advanced soft tissue sarcomas (STS) and bone sarcomas. Methods: Eligible patients at least age 18 yrs with metastatic histologically confirmed STS or bone sarcoma after progression on one line or if refused adriamycin for STS were included. Prior GEM use was permitted. A modified 3+3 dose escalation design was used exploring two dose cohorts of DEC, 0.1 and 0.2 mg/kg SQ administered on a twice weekly schedule for three weeks and GEM given as 900 mg/m2, IV over 90 min on days 1, 8 & 15 of a 28-day cycle. Treatment was continued until disease progression or unacceptable toxicity. Dose limiting toxicity (DLT) was defined as any drug related non-hematological grade 3 or 4 toxicity per CTCAE v4.0. Disease assessment was performed every 8 weeks using RECIST v1.1. Results: 31 patients (25 STS & 6 bone sarcomas) were enrolled of which 7 were non-evaluable. There were 12 evaluable patients in each dosing cohort. Of the total 744 adverse events (AE) 17.2% were grade 3/4 and most were neutropenia without neutropenic fever. 45.7% AEs were possibly (44.6%) or probably (1.1%) attributed to DEC use. The toxicities were not significantly different between DEC doses. No DLTs were observed. One patient died due to progressive disease. Conclusions: Combination of fixed dose infusion GEM with low dose subcutaneous DEC is moderately toxic. Most toxicities were hematological. While there were few responses, the RP2D of DEC selected was 0.1 mg/kg as it showed prolonged disease stabilization. Clinical trial information: NCT02959164 . [Table: see text]

The effect of lysosomal-associated membrane protein-1-targeting of Epstein–Barr virus nuclear antigen 1 (EBNA1) on immune response and tumor growth in preclinical studies.

74 Background: Epstein-Barr virus (EBV) is an oncogenic γ-herpes virus that infects > 90% of the global population. EBV is the primary cause of infectious mononucleosis and is associated with several epithelial and lymphoid malignancies, including nasopharyngeal carcinoma, gastric carcinoma, non-Hodgkin’s and Hodgkin’s lymphoma. Of these, nasopharyngeal carcinoma is particularly prevalent in East and Southeast Asia and is highly metastatic. Current treatment strategies for nasopharyngeal carcinoma are limited to radiation and chemotherapy, demonstrating a need for new, targeted therapies. Most nasopharyngeal carcinomas express EBNA1, a DNA-binding protein involved in maintenance of the episomal virus genome that is required for EBV latency and associated transformation. Targeting EBNA1 allows for an immunotherapeutic approach, by exploiting the potential of the immune system to recognize tumor cells through their expression of this viral antigen. Methods: We designed a DNA vaccine encoding EBNA1 using the UNITE (UNiversal Intracellular Targeted Expression) platform. The UNITE platform is based in part on lysosomal targeting technology which results in enhanced antigen presentation and a balanced T cell response. Results: We report that the EBNA1-UNITE vaccine induced IFNγ-producing effector memory (EM) CD4+ T and CD8+ T cells with complete rejection of EBNA1-expressing tumors observed in 50% of mice. Mice rejecting tumors were protected from rechallenge with CT26-EBNA1, demonstrating that antigen-specific memory was induced in these animals. Tumor microenvironment (TME) analysis showed vaccine-induced mobilization of effector cells, including tumor infiltration of IL-12-producing type 1 dendritic cells, iNOS-producing M1 macrophages, activated EM CD4+T, and CD8+TNFα+T cells. Furthermore, increased PD-1+CD8+T cells in the TME suggests that a combination strategy with PD-1/PD-L1 blockade may be beneficial in a therapeutic setting. Conclusions: This pre-clinical data suggests that the EBNA1-UNITE vaccine has the potential to be used as an immunotherapeutic agent against EBV-associated cancers.

Abstract 5787: Ipatasertib impairs tumor growth in androgen sensitive and castrate resistant prostate cancer: Evidence in preclinical models

Abstract Introduction: Therapeutic targeting the PI3K/AKT signaling pathway is an attractive strategy to combat solid tumors that exhibit hyper-AKT activity. Ipatasertib, a potent pan-AKT inhibitor, has exhibited significant inhibitory effects on tumor growth in preclinical studies, including models of Androgen Receptor (AR)-independent prostate cancer (PCa) that lack PTEN, a negative regulator of the PI3K/AKT pathway. It is currently being investigated in several ongoing clinical trials, including evaluating its efficacy in combination with abiraterone in castrate resistant PCa (CRPC) (NCT01485861). There is cross-talk between androgen signaling and PI3K/AKT pathway activation, however the optimal setting for AKT inhibition in PCa is not clear. Here we seek to determine the benefit of ipatasertib for androgen sensitive (AS) PCa alone or in combination with androgen deprivation (AD), as well as the benefit of ipatasertib alone or in combination with the antiandrogen enzalutamide in CRPC. Methods: In vitro studies utilized human androgen sensitive PCa cell lines LAPC, LAPC4shPTEN, and LNCAP, as well as two LNCaP-derived, castrate resistant cell lines. Cell viability was determined using CellTitre Glo assay and apoptosis by flow cytometry. Inhibitory effects of AKT signaling pathway was determined by western blot assays against AKT phosphorylation sites and downstream targets as well as detection for hormone receptor expression. In vivo studies utilized LuCaP 136 and LuCaP 136-derived castrate resistant (LuCaP 136CR) patient derived xenograft (PDX) models. Cells were injected into athymic mice and grouped into 4 arms. Intact mice bearing AS tumors were treated with vehicle, ipatasertib (100mg/Kg), AD, or ipatasertib + AD. Castrated mice bearing castrate-resistant tumors were treated with vehicle, ipatasertib, enzalutamide (10mg/Kg), or ipatasertib + enzalutamide. Tumor growth was determined twice/week via blinded caliper measurements. Results: Ipatasertib effectively inhibited AKT signaling, as indicated by significant decrease in phosphorylation of the downstream targets P70S6K and 4E-BP1. Our in vitro data indicate significant anti-proliferation effects of ipatasertib across all PCa models, both androgen sensitive and castrate resistant. Interestingly, this was irrespective of PTEN status. In addition, we observed a decrease in glucocorticoid receptor (GR) expression in enzalutamide resistant lines, although AR expression was not significantly altered. Preclinical animal studies showed significant response to tumor growth inhibition with ipatasertib monotherapy, which was enhanced with combination treatment. Conclusion: Ipatasertib is beneficial in models of androgen sensitive and castrate resistant PCa and can be effective in the treatment of PCa alone or in combination with current standard therapies targeting the androgen receptor. Citation Format: Remi M. Adelaiye-Ogala, Yen T. Nguyen, Jose A. Rodriguez-Nieves, Aian Neil Alilin, Supreet Agarwal, David Vanderweele. Ipatasertib impairs tumor growth in androgen sensitive and castrate resistant prostate cancer: Evidence in preclinical models [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 5787.

Combining expression of GPC3 in tumors and CD16 on NK cells from peripheral blood to identify patients responding to codrituzumab.

BackgroundCodrituzumab, a monoclonal antibody targeting an oncofetal protein glypican-3 (GPC3) expressed on cell surface of hepatocellular carcinoma (HCC) induces antibody-dependent cellular cytotoxicity (ADCC) and inhibits tumor growth in preclinical studies. Based on this mechanism, tumor GPC3 expression and CD16 expression on NK cells, which are the effector cells of ADCC, were investigated to correlate with codrituzumab's clinical efficacy in patients with advanced HCC.ResultsJoint analyses of the two biomarkers revealed that both high levels of GPC3 and CD16 were required for patients to benefit from codrituzumab; lack of either one of them would lead to a loss of the therapeutic effect.ConclusionsThese results suggest the combination of tumor GPC3 expression and CD16 expression on NK cells from peripheral blood at baseline as a composite biomarker to select HCC patients for codrituzumab.ImpactThe conclusion warrants a future study in an HCC population with both high GPC3 expression and high levels of CD16 at baseline to establish codrituzumab's therapeutic benefit in HCC.MethodsData from a phase II clinical trial of codrituzumab were used for the analyses. GPC3 expression in baseline tumor biopsies was determined by immunohistochemistry (IHC) analysis, and baseline CD16 expression on NK cells were quantified by peripheral blood lymphocyte immunophenotyping. According to high or low expression of GPC3 and CD16, different patient subgroups were formed; for each subgroup, overall survival of patients having high codrituzumab exposure was compared to that of patients receiving placebo.

Abstract 3944: Combining tumor glypican-3 expression and CD16 Expression on NK cells from peripheral blood to identify patients responding to codrituzumab/GC33/RO5137382

Abstract Background & Aims: Codrituzumab (GC33 or RO5137382), a monoclonal antibody targeting an oncofetal protein glypican-3 (GPC3) expressed on the cell surface of hepatocellular carcinoma (HCC), induces antibody-dependent cellular cytotoxicity (ADCC) and inhibits tumor growth in preclinical studies. Based on this mechanism of codrituzumab, two biomarkers GPC3 and CD16 of the NK cells, which are the effector cells of ADCC, were investigated to correlate with clinical efficacy of codrituzumab in patients with advanced HCC. Methods: Data from a phase II clinical trial of codrituzumab were used for this analysis. GPC3 expression was determined by immunohistochemistry (IHC) analysis of tumor biopsies, and CD16 expression on NK cells were quantified by a flow cytometry analysis of peripheral blood mononuclear cells. Overall survival of patients with high exposure of codrituzumab or placebo (n = 113) was used to compare different patient subgroups stratified by high or low expression of GPC3 and CD16. Results: In individual-biomarker analyses, longer survival after codrituzumab treatment correlates with either high expression level of GPC3 in tumors (n = 97, HR = 0.39, 95%CI = 0.21 - 0.70, p = 0.00081) or a relatively high level of CD16 expressed on NK cells (n = 80, HR = 0.44, 95%CI = 0.23 - 0.83, p = 0.0056) at baseline. Furthermore, joint analyses of the two biomarkers reveal that both high levels of GPC3 and CD16 are required for patients to benefit from codrituzumab treatment (n = 67, HR = 0.29, 95%CI = 0.13 - 0.62, p = 0.00074), and lack of either one of them leads to a loss of codrituzumab therapeutic effect. Conclusions: The retrospective analysis supports the mechanism of ADCC, in which the combination of high GPC3 expression in tumors and high CD16 expression in NK cells from peripheral blood is associated with prolonged overall survival given treatment of codrituzumab. This result supports the usage of both GPC3 and CD16 as potential biomarkers to select HCC patients for codrituzumab treatment. Citation Format: Gong Chen, Ya-Chi Chen, Bernhard Reis, Anton Belousov, Lori Jukofsky, Christine Rossin, Axel Muehlig, Chao Xu, Laurent Essioux, Toshihiko Ohtomo, Laura Di Laurenzio, Oscar Puig, Ray Lee. Combining tumor glypican-3 expression and CD16 Expression on NK cells from peripheral blood to identify patients responding to codrituzumab/GC33/RO5137382. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3944.

Abstract A67: Phase 1 study of VX15/2503, an immunomodulatory antibody to Semaphorin 4D that reverses tumor growth in preclinical studies

Abstract Semaphorin 4D (SEMA4D, CD100) normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, SEMA4D promotes tumor progression and metastasis. SEMA4D and its receptor plexin B1 are broadly expressed in murine and human tumors; expression correlates with invasive disease in several human tumors. SEMA4D is highly expressed on T cells and is also released as a biologically active soluble molecule by activated inflammatory cells. Preclinical studies demonstrate a novel immunomodulatory function of SEMA4D, whereby SEMA4D influences the recruitment and distribution of leukocytes in the tumor microenvironment. Antibody blockade of SEMA4D with the murine progenitor of VX15/2503 regulates the balance and activity of inflammatory and tolerance-inducing cells and cytokines to effectively delay tumor growth and promote durable tumor rejection in syngeneic colon and breast cancer models. Combination of anti-SEMA4D antibody with immune checkpoint inhibitor anti-CTLA-4 enhances anti-tumor immune activity and synergistically increases tumor rejection. Combinations with anti-PD-1 or cyclophosphamide, an immunomodulatory chemotherapy reported to differentially affect regulatory T cells, also increases efficacy and frequency of tumor rejection. Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote immune infiltration and distribution in the tumor microenvironment and inhibit tumor progression. The humanized anti-SEMA4D antibody, VX15/2503, has successfully completed a Phase I multiple ascending dose trial in adult patients with advanced refractory solid tumors. Patients were administered weekly IV doses of VX15/2503 until progression. Dose levels were 0.3 to 20 mg/kg. Tumors were assessed by RECIST 1.1 after each 8 dose cycle. The study has concluded (n=42 Pts); sex 40%M/60%F. Mean age (yrs) 64.8; ECOG 0/1/2 are 28.6%/69%/2.4%. No MTD was found. One DLT (grade 3 GGT elevation; 15 mg/kg) was reported in a pancreatic cancer patient with disease progression. The most frequent treatment-related AE's (n=42 pts) included grade 1/2 nausea (14.3%) and fatigue (11.9%) and 15 unrelated SAE's were reported in 12 patients. Thirteen of 42 pts at all dose levels exhibited stable disease for at least 8 weeks. Patients with the longest duration of treatment, 48-55 weeks, included colorectal (9 mg/kg); breast (15 mg/kg); and papillary thyroid (20 mg/kg)—this patient had a partial response by RECIST and stable disease for at least 6 months following cessation of treatment at 48 weeks. VX15/2503 serum concentrations of ≥ 0.3 µg/mL produced complete saturation of membrane SEMA4D on circulating T cells. HAHA responses (titer > 100) with possible effects on PK were observed in 4 of 41 patients (10%); in only 1 patient (2%) was an effect of HAHA on PD observed. VX15/2503 was well tolerated at dose levels up to 20 mg/kg, with 459 doses administered to 42 patients. A phase 1b/2a trial of combination therapy with anti-CTLA-4 is planned. Citation Format: Elizabeth E. Evans, Terrence L. Fisher, Cynthia Edington, Holm Bussler, Sebold Torno, Alan S. Jonason, Jr., Janaki Veeraraghavan, Christine Reilly, Michael A. Doherty, Jennifer Seils, Laurie A. Winter, Tracy Pandina, Crystal Mallow, Renee Kirk, Alan Howell, Sue Giralico, Maria Scrivens, Katya Klimatcheva, William J. Bowers, Mark Paris, Drew Warren Rasco, Ramesh K. Ramanathan, Amita Patnaik, Glen J. Weiss, Danielle Mutz, Lisa Blaydorn, Anthony W. Tolcher, Valerie Iddison, Ernest S. Smith, John E. Leonard, Maurice Zauderer. Phase 1 study of VX15/2503, an immunomodulatory antibody to Semaphorin 4D that reverses tumor growth in preclinical studies. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A67.

Novel analogs of antitumor agent calixarene 0118: Synthesis, cytotoxicity, click labeling with 2-[(18)F]fluoroethylazide, and in vivo evaluation.

Calixarene 0118 is a potent anti-angiogenic agent that effectively inhibited tumor growth in preclinical studies, and is currently being evaluated in a phase I clinical trial. We have designed two close mimetics of calixarene 0118 containing a terminal alkynyl-functional group, and developed an optimized semi-automated procedure for radiolabeling with 2-[(18)F]fluoroethylazide using click chemistry. Following semi-preparative HPLC purification and formulation, the lower-rim modified analog [(18)F]6 and the equatorially labeled [(18)F]13 were obtained in >97% radiochemical purity and overall decay-corrected isolated radiochemical yields of 18.7±2.7% (n=4) and 10.2±5.0% (n=4), respectively, in a total synthesis time of about 2h. Preliminary invivo studies in nude mice bearing human tumor xenografts revealed highest accumulation of both tracers in the liver, followed by spleen, kidney, lung and bone, with no substantial uptake in the tumor. Still, these first-in-class radiotracers are a valuable tool for pharmacokinetic profiling and improvement of calixarene-based anti-angiogenic therapeutics in the future, as similar radiolabeling strategies may be applied to other compounds in the calixarene series. The cold reference compounds of the radiotracers were characterized in terms of cytotoxicity and anti-proliferative effects on HUVEC cells and on MA148 human ovarian carcinoma cells, along with the respective precursors, a small series of 0118 analogs modified with short-chain linear alkyl substituents, and a PEG3-spaced calixarene dimer. While all of the new analogs proved at least equipotent to parent 0118, some of them inhibited HUVEC and MA148 cell growth almost 4- and 10-fold more effectively, rendering these analogs promising candidates for further evaluation in anti-angiogenic cancer therapy.

Abstract 4734: Genes cooperatively downregulated by combined mTOR/histone deactylase (HDAC) inhibition are overexpressed in myeloma patients with lower survival

Abstract The molecular pathogenesis of many cancer types, including multiple myeloma (MM) and mantle cell lymphoma (MCL), involves alterations in the PI3K/Akt/mTOR and cyclin/CDK/CDKI/Rb (Rb) pathways. Previously, we showed that the combination of an HDAC inhibitor (HDACi) with rapamycin synergistically inhibited proliferation in 88% of human MM cell lines tested, and effectively controlled tumor growth in preclinical studies. To gain an initial understanding of the molecular mechanism of the synergistic action of the drug combination, we used an unbiased systems-level approach to analyze our gene expression profile (GEP) data with weighted gene co-expression network analysis (WGCNA). This analysis delineated the contribution of HDACi and rapamycin, singly and in combination, to the overall gene expression change of the combination by considering not only measures of fold-change and significance testing, but also the degree of gene expression inter-connectedness. WGCNA identified five gene modules, each representing a particular gene expression effect of the combination. Each gene module was individually tested for functional and clinical enrichment using gene set enrichment analysis (GSEA) and survival analyses with Cox regression. Of particular interest, the module containing genes cooperatively affected by both compounds was highly enriched (p<0.001) for genes involved in cell cycle (especially mitotic processes), immune recognition, and DNA damage/repair, which we investigated further. Genes down-regulated by the drug combination were most significantly correlated with genes over-expressed in MM patients. Furthermore, analysis of the cooperative drug signature in publicly available patient GEP datasets with survival annotation found it predictive of increased survival (p<0.01), thus linking the drug combination-induced transcriptional changes to predictions for enhanced survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4734. doi:1538-7445.AM2012-4734

Highlights of This Issue

This phase I multicenter study enrolled 42 patients with advanced cancer who were treated with a combination of the mTOR inhibitor temsirolimus with cixutumumab. The combination was well tolerated. Clinical benefits were observed in Ewing sarcoma and adrenocortical carcinoma. Pharmacodynamic data suggest that cixutumumab alone or combined with temsirolimus increased plasma insulin-like growth factor (IGF)-1 and IGF binding protein 3. Assessment via 2[18F]fluoro-2-deoxy-D-glucose positron emission tomography combined with x-ray computed tomography showed the odds of achieving stable disease decreased by 58% (P = 0.1213) with a 1-unit increase in absolute change of standard uptake value from baseline to day 3.Pancreatic cancers are resistant to most chemotherapeutic agents, and novel treatments are needed. Du and colleagues assessed the activity of an antimesothelin immunotoxin on pancreatic cancer cells and found that these cells are resistant to immunotoxin due to low levels of the proapoptotic protein Bak. Combining TRAIL or an anti-TRAIL receptor 2 antibody with immunotoxin caused synergistic cell death and reduced tumor volume in a xenograft mouse model. These data demonstrate that cancer cells with low BAK are resistant to immunotoxin treatment, and that combining immunotoxin with TRAIL or a TRAIL agonist antibody is an effective way to overcome such resistance.Fas ligand (FasL)-844T/C polymorphism has a demonstrated association with tumor progression via immune escape. To explore the role of FasL haplotype on the outcome of resected non–small cell lung cancer (NSCLC), Sung and colleagues followed the survival and relapse of 385 NSCLC patients and determined their FasL-844 genotype. The FasL-844CC genotype was highly prevalent in patients with advanced tumors, who are prone to disease relapse. Additionally, patients with the FasL-844CC genotype had worse overall survival and relapse-free survival than did those with the FasL-844TT+TC genotype. Therefore, FasL-844 polymorphism evaluated by PCR restriction fragment length polymorphism may potentially predict disease relapse and prognosis in resected NSCLC.Gemcitabine and doxorubicin (GEM/DOX) induced tumor suppressor sphingolipid C18-ceramide generation and inhibited head and neck squamous cell carcinoma (HNSCC) tumor growth in preclinical studies. To evaluate the clinical efficacy and relevance of this strategy, Saddoughi and colleagues conducted a phase II trial in patients with recurrent HNSCC. In 17 patients, the median progression-free survival was 1.6 months [95% confidence interval (CI): 1.4, 4.2], with a median survival of 5.6 months (95% CI: 3.8, 18.2). C18-ceramide elevation was observed in the serum of patients with response and stable disease, compared with progressive disease, suggesting that GEM/DOX-induced C18-ceramide may be a novel serum biomarker for monitoring therapeutic response.

481 Serum levels of placental growth factor correlate with survival in patients with neuroendocrine tumors of the pancreas

Calixarene 0118 is a potent anti-angiogenic agent that effectively inhibited tumor growth in preclinical studies, and is currently being evaluated in a phase I clinical trial. We have designed two close mimetics of calixarene 0118 containing a terminal alkynyl-functional group, and developed an optimized semi-automated procedure for radiolabeling with 2-[18F]fluoroethylazide using click chemistry. Following semi-preparative HPLC purification and formulation, the lower-rim modified analog [18F]6 and the equatorially labeled [18F]13 were obtained in >97% radiochemical purity and overall decay-corrected isolated radiochemical yields of 18.7 ± 2.7% (n = 4) and 10.2 ± 5.0% (n = 4), respectively, in a total synthesis time of about 2 h. Preliminary in vivo studies in nude mice bearing human tumor xenografts revealed highest accumulation of both tracers in the liver, followed by spleen, kidney, lung and bone, with no substantial uptake in the tumor. Still, these first-in-class radiotracers are a valuable tool for pharmacokinetic profiling and improvement of calixarene-based anti-angiogenic therapeutics in the future, as similar radiolabeling strategies may be applied to other compounds in the calixarene series. The cold reference compounds of the radiotracers were characterized in terms of cytotoxicity and anti-proliferative effects on HUVEC cells and on MA148 human ovarian carcinoma cells, along with the respective precursors, a small series of 0118 analogs modified with short-chain linear alkyl substituents, and a PEG3-spaced calixarene dimer. While all of the new analogs proved at least equipotent to parent 0118, some of them inhibited HUVEC and MA148 cell growth almost 4- and 10-fold more effectively, rendering these analogs promising candidates for further evaluation in anti-angiogenic cancer therapy.

Human monoclonal antibodies to the insulin-like growth factor 1 receptor inhibit receptor activation and tumor growth in preclinical studies

The insulin-like growth factor type 1 (IGF-1) receptor contributes importantly to transformation and survival of tumor cells both in vitro and in vivo, and selective antagonists of the IGF-1 receptor (IGF-1R) activity represent an attractive experimental approach for human cancer therapy. Using a phage display library, we identified several high-affinity fully human monoclonal antibodies with inhibitory activity against both human and rodent IGF.1Rs. These candidate therapeutic antibodies recognized several distinct epitopes and effectively blocked ligand-mediated receptor signal transduction and cellular proliferation in vitro. They also induced IGF-1R downregulation and catabolism following antibody-mediated endocytosis. These antibodies exhibited activity against human, primate, and rodent IGF-1Rs, and dose-dependently inhibited the growth of established human tumors in nude mice. These fully human antibodies therefore have the potential to provide an effective anti-tumor biological therapy in the human clinical setting.

Abstract B235: The utility of TRC093; A humanized monoclonal antibody directed against cleaved collagen in the detection of patients at risk of ovarian and breast cancer

Abstract Background: TRC093 is a humanized monoclonal antibody that specifically binds cleaved collagen and has been shown to inhibit angiogenesis and tumor growth in preclinical studies. TRC093 is currently being evaluated in a phase I clinical study for the treatment of metastatic human tumors (Gordon et al. EJC Supp 6, abs #414, pp130, 2008). Interestingly, one of the patients in this study with granulosa cell carcinoma of the ovary with progressive disease had a mixed response in the liver after 2 months of treatment. Given these encouraging results, we began to examine the biological relevance of the cryptic epitope recognized by TRC093 in ovarian carcinoma and whether a soluble form of this cryptic collagen epitope may represent a clinically useful marker for patients at risk for ovarian and breast cancer. To this end, we have adapted an ELISA assay for the detection of the shed collagen cryptic epitope that is defined by this antibody to examine a patient population at risk for ovarian and breast cancer. Methods: To begin to assess the relevance of the cryptic collagen epitope recognized by TRC093 in ovarian tumor growth, human SKOV3 ovarian carcinoma cells were injected subcutaneously into nude mice. Six days later when detectable tumors were observed, mice were treated (i.p.) 3x per week with TRC093 over a dose range up to 250 µg/injection for a period of 28 days. To assess the relevance of a shed soluble form of the cryptic collagen epitope in a patient population at risk for ovarian and breast cancer, serum samples from a group of high-risk women volunteers prospectively enrolled at the NYU School of Medicine were analyzed for TRC093 epitope concentration by ELISA as previously described (Ng et al., Clin Can Res, 14:6253). Results: TRC093 significantly (p< 0.05) inhibited SKOV3 tumor growth at 100 µg and 250 µg/injection as compared to control. These findings suggest that theTRC093 cryptic collagen epitope may represent a relevant therapeutic target for ovarian carcinoma. In a group of 23 high-risk women identified with respect to their clinical status, a mean ± SEM of 42.9 ± 9.8 µg/ml was determined for the patients with ovarian/ breast cancer history compared with a mean of 15.3 ± 2.9 µg/ml for normal women in this study population (p = 0.0254). Conclusions: The TRC093 shed serum epitope can distinguish between a high-risk population of women with breast or ovarian cancer and normal clinical status. We are expanding the sample size with more ovarian surgical patients. The xenograft studies provide further support for the potential use of the SKOV3 human ovarian carcinoma model to examine the effect of TRC093 in combination with cisplatin and or bevacizumab. The TRC093 epitope may represent a key therapeutic target in ovarian cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B235.

Measuring Brain Tumor Growth: Combined Bioluminescence Imaging–Magnetic Resonance Imaging Strategy

Small-animal tumor models are essential for developing translational therapeutic strategies in oncology research, with imaging having an increasingly important role. Magnetic resonance imaging (MRI) offers tumor localization, volumetric measurement, and the potential for advanced physiologic imaging but is less well suited to high-throughput studies and has limited capacity to assess early tumor growth. Bioluminescence imaging (BLI) identifies tumors early, monitors tumor growth, and efficiently measures response to therapeutic intervention. Generally, BLI signals have been found to correlate well with magnetic resonance measurements of tumor volume. However, in our studies of small-animal models of malignant brain tumors, we have observed specific instances in which BLI data do not correlate with corresponding MRIs. These observations led us to hypothesize that use of BLI and MRI together, rather than in isolation, would allow more effective and efficient measures of tumor growth in preclinical studies. Herein we describe combining BLI and MRI studies to characterize tumor growth in a mouse model of glioblastoma. The results led us to suggest a cost-effective, multimodality strategy for selecting cohorts of animals with similar tumor growth patterns that improves the accuracy of longitudinal in vivo measurements of tumor growth and treatment response in preclinical therapeutic studies.