AACR Annual Meeting 2020

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Check out this preview of what you’ll see from our scientific staff at the AACR Annual Meeting 2020. To access previously published posters, visit www.charlesriveroncology.com.

 

Antibody Technologies

Session: Experimental and Molecular Therapeutics | Abstract 523
Advanced Engineering Using CAR-T Display Libraries: Ultra-High Throughput Functional Screen

Distributed Bio In Collaboration With Charles River

Experimental and Molecular Therapeutics

Session: Targeting Tumor Microenvironment, Protein Kinases/Phosphatases, and Other Strategies | Abstract 3041
Co-administration of iRGD and therapeutic EGFR-antibodies Cetuximab and Panitumumab enhances drug penetration in NSCLC PDX based 3D multicellular spheroids

Julia Schueler, Charles River

Immunology

Session: Immunomodulatory Agents and Interventions 3 | Abstract 6705
An integrated in vitro and in vivo approach providing insights in immune modulation

Shilina Roman, Charles River

Session: Immune Checkpoints 2 | Abstract 2229
A panel of NSCLC patient derived xenografts displays a distinct sensitivity profile towards checkpoint inhibitor treatment in vitro and in vivo

Robert Nunan, Charles River

Session: Novel Animal Models | Abstract 5635
Enhanced anti-tumor lymphocyte function and frequencies measured by multichromatic flow cytometry in human CTLA-4 knock-in mice in a colorectal carcinoma model after treatment with ipilimumab

Elizabeth Reap, Charles River

Session: Novel Animal Models | Abstract 5625
Evaluation of in vivo anti-tumor response of solid tumors in a novel immune cell-humanized NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl mouse model

Jenny Rowe, Charles River

Session: Immune Checkpoints 1 | Abstract 942
HPK1, Hematopoietic Progenitor Kinase 1, is a Promising Therapeutic Target for Cancer Immunotherapy

Nimbus In Collaboration With Charles River

Session: Adoptive Cell Therapy 3 | Abstract 3232
In vitro cell based cytotoxicity and T cell activation assays to assess safety and efficacy of engineered T cell therapies

Sanne Holt, Charles River

Session: Novel Animal Models | Abstract 5634
The single mouse trial format predicts the sensitivity towards checkpoint inhibitor treatment in NSCLC PDX models in HuCD34NCG/CRL mice, an innovative triple immune deficient mouse strain

Julia Schuler, Charles River

Tumor Biology

Session: Mouse Models of Human Cancer in Imaging and Therapeutics | Abstract 2760
A mouse orthotopic model of pancreatic cancer and response to treatment

Elizabeth Rainbolt, Charles River

Session: Tumor Microenvironment 2 | Abstract 5085
A preclinical model using perfusion air culture of tumor tissue slices for personalized medicine

IKP In Collaboration With Charles River

Session: Immune Cells in the Tumor Microenvironment 3 | Abstract 3865
A translational immuno-oncology platform to model T cell activation and Tex recovery in the tumor microenvironment

Robert Nunan, Charles River

Session: Tumor-immune System Interactions 2 | Abstract 2824
In vitro tumor killing assays for predicting cellular therapeutic efficacy

Robert Nunan, Charles River

Session: New Imaging-based Approaches to Plan, Monitor, and Augment Cancer Treatment | Abstract 2774
Longitudinal characterization of patient-derived orthotopic xenograft brain tumor models

Julia Schuler, Charles River

Session: Patient-derived Organoid and Xenograft Models | Abstract 2813
Tumor models driven by EGFR: Optimizing the preclinical profiling of EGFR-targeting agents

Thomas Metz, Charles River

Session: Model Organisms for Cancer Research | Abstract 6125
Zebrafish patient tumor-derived xenograft models synergize with mouse-PDX models for understanding variation in anti-cancer drug responses

BioReperia In Collaboration With Charles River

Session: Model Organisms for Cancer Research | Abstract 6126
Zebrafish patient tumor-derived xenograft models used for pre-clinical evaluation of CAN04 for lung and pancreatic cancer

BioReperia In Collaboration With Charles River

Check out this preview of what you’ll see from our scientific staff at the AACR Annual Meeting 2020. To access previously published posters, visit www.charlesriveroncology.com.

 

Antibody Technologies

Session: Experimental and Molecular Therapeutics | Abstract 523
Advanced Engineering Using CAR-T Display Libraries: Ultra-High Throughput Functional Screen

Distributed Bio In Collaboration With Charles River

CAR-T-cell therapies represent a new area of synthetic biology success in cancer immunotherapy. However, the complexity of their adverse events related to their activity are still a major challenge. Using our in-house fully human computationally optimized phage display library, we established a novel cell-based ultra-high throughput panning and screening CAR-T platform. Our proprietary CAR-T vector is designed as a series of cassettes incorporating our SuperHuman phage library with an optimal backbone. This allows us to design CAR-T with desired properties, including: Range of Affinities, Humanness, Epitope Diversity, Thermostability, Immunogenicity, Tonic activation, Toxicity, and Functional Activity. Our library’s encoding ensures a single CAR-T construct per one reporter cell. Moreover, to confirm CAR-T-specific-activation, we incorporated three different markers that will be upregulated simultaneously. We used CD19 and BCMA to validate our platform selecting target-specific functionally active scFv candidates against both targets, which are selective to activation conditions such as tonic signaling, and tissue specific silencing. Collectively we show that our platform can be readily used to characterize CAR-T in high-speed and - throughput fashion.

Experimental and Molecular Therapeutics

Session: Targeting Tumor Microenvironment, Protein Kinases/Phosphatases, and Other Strategies | Abstract 3041
Co-administration of iRGD and therapeutic EGFR-antibodies Cetuximab and Panitumumab enhances drug penetration in NSCLC PDX based 3D multicellular spheroids

Julia Schueler, Charles River

Three-dimensional (3D) cell culture platforms are increasingly being used in cancer research and drug development. Using patient derived xenograft (PDX) as tumor cell source the clinical relevance of those platforms increases significantly. In combination with live cell imaging capabilities a powerful tool for dynamic studies of drug-cell interactions arises. In the current study we determined the penetration of anti-EGFR antibodies cetuximab and panitumumab in NSCLC spheroids in different culture conditions (w/wo human dermal fibroblasts, HDF) and in combination with the permeabilization-enhancing peptide iRGD . Six different NSCLC PDX models were selected based on their EGFR protein expression level (ranging from high to low determined by immunohistochemistry on PDX tissue). Cetuximab and panitumumab, were labelled with an Alexa-Fluor488 dye and their real-time uptake kinetics were quantified in an IncuCyte S3 device (1 image/well/h, Sartorius). Apart from the absolute fluorescence within the spheroid, we developed a mathematical description, fitting intensity levels in separate regions of the spheroids to estimate spatial dependent penetration of the compound in a time-resolved manner. The accumulation of EGFR antibodies over time was positively correlated with the target expression on the corresponding PDX. In addition, the invasion of the antibodies was significantly influenced by the compactness of the spheroids. The compactness of the tumor spheroid was model immanent and dependent on the molecular make-up of the respective tumor line. Enhancing the compactness by adding HDF to the tumor spheroid reduced the infiltration of both tested antibodies markedly. The simultaneous treatment of anti-EGFR antibodies with iRGD enhanced the penetration capacity in a dose dependent manner. The data of the current study highlight the importance of the tumor microenvironment (TME) for the sensitivity of tumor cells towards anticancer treatment. The modulation of the TME will be an important approach to increase antitumoral activity by enhancing the compound concentration in the tumor tissue. In general, this screening approach can be applied to fluorescence-labelled large molecules as well as compounds with intrinsic fluorescence characteristics. The combination of life cell imaging and image analysis is a promising tool in preclinical drug development to optimize compounds with regard to tissue penetration and binding efficiency.

Immunology

Session: Immunomodulatory Agents and Interventions 3 | Abstract 6705
An integrated in vitro and in vivo approach providing insights in immune modulation

Shilina Roman, Charles River

In order to exploit the complex tumour microenvironment, immunoreactivity in a multitude of cell-based assays is required to predict mode of action (MOA), pharmacodynamics, and efficacy downstream. We have developed an integrated approach starting with single cell and co-culture cell-based assays in 2D and 3D formats, followed by an in vivo set-up based on humanized mice to monitor an immunotherapeutic’s interaction with specific cell types such as T cells including CAR-T cells, natural killer cells, macrophages, dendritic cells, neutrophils, and fibroblasts. Rituximab, a monoclonal antibody against CD20, is mainstay in the therapy for a broad variety of B-cell malignancies. Its MOA is not fully understood as direct signalling, complement dependent cellular cytotoxicity and antibody dependent cellular cytotoxicity (ADCC) all seem to have an impact. To elucidate the share and effect each of those mechanism has, we tested Rituximab in our immuno-oncology translational platform. Furthermore, our immuno-oncology platform was utilized to assess efficacy, bystander effects and off-target toxicity of a CD19 CAR-T cell therapy. In vitro assays for efficacy and bystander effect involved co-culture of the CAR-T cells with a mixture of non-target expressing cells and mid to high target antigen expressing cells, where T cell activation and cell death of both target cell types was quantified. Beyond efficacy the assay platforms were used to examine the influence of the therapy, antibodies or CAR-T cells, on normal human cells. These in vitro assays utilizing primary human cells from healthy tissue and/or differentiated iPCS-derived cells were used to assess cytotoxicity. The primary tissues were selected based on their potential safety risk by either expressing low levels of the target antigen or being major organs to de-risk for off-target effects. The results from our in vitro screen were validated in a xenograft in vivo system in the presence and absence of human immune cells. Four cell line derived xenografts were tested in vivo in immunocompromised mice subcutaneously and disseminated. The presence of human immune cells enhanced the antitumoral activity of Rituximab markedly, although the targeted effect of Rituximab induced already a significant prolongation in the overall survival (p< 0.005; Log-rank (Mantel-Cox) test).Over the last 30 years IO has progressed considerably with approvals for the use of various IO therapeutics including vaccines, cytokines, tumor-directed monoclonal antibodies, immune checkpoint inhibitors as well as Chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cell therapies. Comprehensive characterization of novel IO compounds using a multitude of such in vitro and in vivo assays is essential to provide insights and understanding of the detailed mechanism of action and identify potential toxicity issues to ultimately improve translation to the clinic.

Session: Immune Checkpoints 2 | Abstract 2229
A panel of NSCLC patient derived xenografts displays a distinct sensitivity profile towards checkpoint inhibitor treatment in vitro and in vivo

Robert Nunan, Charles River

Robert Nunan, Charles River Session Title: Immune Checkpoints 2 Permanent Abstract Number: 2229 Abstract: The field of cancer immunology is rapidly moving towards innovative therapeutic strategies. As a consequence there is a need for robust and predictive preclinical platforms for assessing therapies. The current project aims to establish a drug screening workflow bridging between innovative 3D in vitro assays and humanized mouse models based on the same PDX model panel. A total of six different lung cancer patient derived xenograft models (NSCLC PDX) were transduced to express nuclear RFP, cultivated as 3D spheroids and co-cultured with human peripheral blood monocytes (PBMC). Spheroid fluorescent intensity was monitored every 4 hours over a total of 120h. Efficacy of different checkpoint inhibitors was determined using a live cell imaging technology (IncuCyte S3, Sartorius). In vivo a total of 14 different NSCLC PDX were screened for their sensitivity towards α-CTLA-4, α-PD-1 or the combination thereof. With n=1 per treatment arm and model and the study design followed the screening approach of the single mouse trial (SMT). Human immune cell infiltrates of tumor (=TILs), peripheral blood, spleen and bone marrow (determined by flow cytometry, FC, and IHC) and secretion of human and mouse cytokines in murine serum (determined by cytokine array & Luminex based technology) were determined in all models.In the spheroid assay as well as in vivo all three treatment arms displayed a discrete activity pattern throughout the PDX panel. The activity pattern of the 14 PDX mirrored the clinical diversity of tumor responses to checkpoint inhibitor treatment ranging from complete remission to resistance. The sensitivity towards checkpoint inhibitors was stable in a distinct PDX model across the two drug development platforms. Tumor models with high tumor infiltrating lymphocyte (TIL) rates (>5%) in the untreated control group were more susceptible towards checkpoint inhibitor treatment than models with low TIL rates. In either case, numbers of TILs were markedly increased in the treatment groups as compared to control vehicle. The analysis of human and mouse cytokines in the serum of tumor bearing mice led to the identification of a cytokine pattern specific for PDX models sensitive towards checkpoint inhibitor treatment. 11 human cytokines were upregulated in the sensitive tumor models (e.g. GM-CSF, IL-4 & CX3CL1). The use of PDX based innovative 3D in vitro models in combination with humanized mouse models enables screening campaigns in the immune-oncology field using clinically relevant tumor models. The predictivity of the 3D spheroid platform towards the in vivo humanized mouse assay is a critical feature as it allows the optimal selection of promising drug candidates to be profiled in more detail.

Session: Novel Animal Models | Abstract 5635
Enhanced anti-tumor lymphocyte function and frequencies measured by multichromatic flow cytometry in human CTLA-4 knock-in mice in a colorectal carcinoma model after treatment with ipilimumab

Elizabeth Reap, Charles River

Targeting CTLA-4 has shown remarkable long-term benefits and thus remains a valuable approach for combating cancers of many types. A number of preclinical models have been developed over the years to evaluate the efficacy of immune checkpoint blockade in promoting antitumor immunity. In particular, knock-in (KI) humanized mouse models offer the possibility to study clinical grade immune checkpoint inhibitors (ICI) in the context of a fully functional immune system. Here we show the response to ipilimumab in a newly developed hCTLA-4 KI humanized mouse model. Our results demonstrate significant tumor growth inhibition as well as complete tumor regressions in the MC38 colorectal cancer model following treatment with ipilimumab. We have extended these studies by re-challenging the tumor-free surviving animals with tumors cells implanted opposite to the original tumor site. We established that all re-challenged hCTLA-4 KI mice remained tumor free suggesting potent T cell memory was maintained. To identify the immune cells and cytokines that could be responsible for the durable responses, we used multiparameter flow cytometry to characterize and compare blood, spleen and tumors tissues from the control, ipilimumab and mCTLA-4 treated mice. Comprehensive multichromatic phenotyping and functional intracellular cytokine staining (ICS) using validated 18-color flow cytometry panels showed a significant increase in CD8+ T cell frequencies when mice were treated with anti-hCTLA-4 but not with the mouse counterpart or the isotype control. Importantly, hCTLA-4 blockade reduced the regulatory T cell (FoxP3+ Treg) frequency and increased leukocyte infiltration into the tumor in the hCTLA-4 treated mice but not in the other two treated groups. Although equivalent numbers of CD4+ and CD8+ total T lymphocytes where found in the blood of all groups after each treatment, the quality of the T cell responses was found to be significantly different. CD45+ lymphocytes from the blood analyzed from the hCTLA-4 treated mice showed significant increases in both central memory (CM) CD8+ and CD4+ T cells consistent with increased anti-tumor efficacy seen in these mice. Furthermore, treatment of MC38 tumor-bearing mice with ipilimumab enhanced significantly the secretion of IFNγ from CD8+ and CD4+ T cells and the proliferation of both T cell populations as shown by the upregulation of Ki67 staining. These results indicate that immune cells were actively proliferating and had the appropriate effector functions to impact tumor growth. Altogether, the data presented here demonstrates that hCTLA-4 KI humanized mice are a robust model for evaluating the immune-modulatory effects and the activity of clinical grade ICI against tumors.

Session: Novel Animal Models | Abstract 5625
Evaluation of in vivo anti-tumor response of solid tumors in a novel immune cell-humanized NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl mouse model

Jenny Rowe, Charles River

Checkpoint blockade inhibitors targeting PD-1 and CTLA-4 pathways are clinically approved therapies for multiple cancer types. The performance of targeted interventions has been effective, but clinical response rates vary. In vivo models of human immunity in human tumor bearing mice (TBM) is an important tool for studying mechanisms of targeted therapies and developing new and effective treatments. The NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl), a recently developed triple immunodeficient mouse strain, is a stable host for both human immune cells and tumors for the study of immuno-oncology-based therapeutics.

We evaluated the anti-tumor effects of immune checkpoint inhibitors (anti-human PD-1, including Pembrolizumab, and CTLA-4) on colon epithelial carcinoma (RKO) and basal lung cell adenocarcinoma (A549) cell lines in a human donor immune cell-humanized NCG/CRL mouse model (HuCD34NCG). In separate studies Charles River humanized NCG (HuCD34NCG) mice were implanted subcutaneously on the flank with either RKO or A549 tumors. Group randomization occurred when the average tumor size reached a volume of ~100mm3 (A549) or 30-60mm3 (RKO). Control mice were treated with isotype control IgG antibodies. RKO TBM were treated with anti-PD-1 antibody alone, while A549 TBM were dosed with anti-PD1-1 and anti-CTLA-4 antibodies independently and in combination therapy.

Human immune cell engraftment levels were confirmed in the peripheral blood, spleen and tumor (hCD45, hCD3, hCD4, hCD8, hCD19, NK, myeloid, macrophages; markers vary based on study) of HuCD34NCG humanized mice. Tumor growth kinetics were monitored throughout the study. Inhibition of RKO and A549 tumor growth upon anti-PD-1 monotherapy was significant. Human T-cell infiltration was observed in A549 and RKO tumors with the majority of live T-cells responsive post infiltration. Human cytokines (IFN-γ, IL-2 and TNF-α) were released by tumor-infiltrating total T-cells (CD3+) and subsets (CD4+ and CD8+), as demonstrated by intracellular cytokine staining following PMA/Ionomycin stimulation. Polyfunctional T-cell responses were detected in all treatment groups at study termination.

The results from these studies demonstrate significant immunomodulatory anti-tumor response to immune checkpoint inhibitors. The newly developed HuCD34NCG humanized mouse model showed robust and sustained engraftment of human immune cell populations and demonstrated infiltration of T-cells into tissues and tumors making this mouse model ideal for immuno-oncology studies.

Session: Immune Checkpoints 1 | Abstract 942
HPK1, Hematopoietic Progenitor Kinase 1, is a Promising Therapeutic Target for Cancer Immunotherapy

Nimbus In Collaboration With Charles River

Introduction: HPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in regulating signal transduction cascades in cells of hematopoietic lineage. Recent data from HPK1 knockout animals and kinase-inactive knock-in animals underscores the role of HPK1 in negatively regulating lymphocyte activation. This negative-feedback role of HPK1 downstream of lymphocyte activation and function combined with its restricted expression in cells of hematopoietic origin make it an ideal drug target for enhancing anti-tumor immunity.

A structure-based drug design approach was used to identify potent and selective inhibitors of HPK1. Various biochemical and biophysical assays, as well as a primary in vitro T cell activation assay, were utilized for multiple rounds of structure-activity relationship (SAR) studies. In vivo target engagement and pharmacodynamic data were generated using an anti-CD3 mouse model.

In vitro, HPK1 small molecule inhibition resulted in enhanced IL-2 production in primary mouse T cells and in purified human T cells stimulated with a suboptimal dose of anti-CD3/anti-CD28. Increased selectivity of HPK1 inhibitors relative to T cell-specific kinases and within the MAP4K family was responsible for further enhancing the IL-2 response in activated T cells. In vivo, qd oral dosing of an HPK1 inhibitor completely abrogated phosphorylated SLP-76, induced by administration of anti-CD3. Furthermore, inflammatory cytokine production was enhanced in vivo upon HPK1 inhibition.

Pharmacological blockade of HPK1 kinase activity represents a novel and powerful immunomodulatory approach for anti-tumor immunity.

Session: Adoptive Cell Therapy 3 | Abstract 3232
In vitro cell based cytotoxicity and T cell activation assays to assess safety and efficacy of engineered T cell therapies

Sanne Holt, Charles River

Chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cells are part of a big wave of immunotherapies showing great promise in cancer clinical trials. With the first T cell based therapies targeting hematological malignances now approved, their next challenge is solid tumors. Solid tumors create an additional challenge due to the lack of tumor specific target antigens, posing significant safety risks, i.e. on-target on-tumor, on-target off-tumor and off-target toxicities. Toxic effects previously reported vary from mild-severe cytokine release syndrome (CRS) to neurotoxicity and death.

We have developed in vitro assays utilizing primary human cells from healthy tissue and/or differentiated iPCS-derived cells to assess on-target off-tumor and/or off-target cytotoxicity for engineered T cell therapies. The presence of CAR-T cell mediated cytotoxicity was measured through co-culture with healthy human primary cells to assess unwanted CAR-T reactivity as well as a high target antigen expressing control cancer cell line to confirm CAR-T functionality. The human primary cell type was selected based on its potential safety risk by establishing low level protein expression of the target antigen. Readouts included IFNγ production determined by MesoScale Discovery platform as a measure of T cell activation and Hoechst/PI staining of target cells by flow cytometry.

Our study generated high quality data of the CAR-T cell, confirming functionality by showing consistent T cell activation and killing against a positive control cell line. Moreover, we were able establish a clear absence of activity against the primary human cells thus providing insight into the safety of the CAR-T therapy.

The developed safety assays provide a robust and rapid platform to assess on-target off-tumor and off-target effects within immuno-oncology therapies, either TCR or CAR-T cells, in both early stage development or late stage testing of the therapeutic product. Through inclusion of a wide range of human primary cells, both high risk tissues and major organs at risk of off-target toxicity, a clear safety profile can be generate in vitro for these novel T cell therapies. Safety risks associated with cell based IO-therapies is the biggest challenge for the success of these therapies, performing a thorough safety screen on healthy primary human tissues is therefore crucial.

Session: Novel Animal Models | Abstract 5634
The single mouse trial format predicts the sensitivity towards checkpoint inhibitor treatment in NSCLC PDX models in HuCD34NCG/CRL mice, an innovative triple immune deficient mouse strain

Julia Schuler, Charles River

Immunotherapy is revolutionizing cancer treatment. As a consequence, there is an urgent need for predictive preclinical models to support compound development with the aim of bringing optimal drug candidates into clinical trials. Patient-derived xenografts (PDX) are the gold-standard in cancer drug development. One major drawback of PDX is the lack of an immunological competent host. To overcome this hurdle triple immunodeficient mice are humanized with hematopoietic stem cells (HSC) to enable the interaction between human immune cells and human tumor cells in a rodent host. The current study uses HSC humanized NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl) mice, HuCD34NCG, to evaluate the sensitivity of five NSCLC PDX models towards anti CTLA-4 (Ipilimumab), anti-PD-1 (Nivolumab) and the combination thereof. We identified one predicted responder and one non-responder and in a single mouse trial approach (SMT) and confirmed the results in a conventional experimental set-up with n=5 per treatment arm. Infiltration of human immune cells was detected by flow cytometry in tumor tissue (TIL) and hematopoietic organs. Serum of tumor bearing animals was analyzed for human and mouse cytokine secretion pre- and post-treatment. The NCG mouse strain was genetically characterized by whole exome sequencing (WES) and the mutational profile compared to other common mouse strains. The mutational profile showed the highest overlap with the genome data from other triple immune compromised mice and with NOD/SCID indicating their close hereditary relationship. Non-tumor bearing HuCD34NCG mice depicted engraftment of human CD45+ cells in peripheral blood of >25% and >60% in bone marrow and spleen after 8-12 wks post injection. Thus, they fulfilled enrollment criteria for conducting a drug screen for immune modulating compounds. The five NSCLC PDX models showed a distinct sensitivity profile ranging from partial remission (tumor growth inhibition, TGI, of 87.5% compared to untreated control) to progression (no TGI). In all models the combination treatment was the most active therapy (TGI: 60%), followed by Ipilimumab (TGI: 55%) and Nivolumab (TGI: 48%). Nevertheless, in selected models anti-CTLA-4 treatment was the most efficacious therapy. The two models examined in more detail LXFE 397 (=responder, TGI 55% in both formats) and LXFA 400 (non-responder, TGI 2%SMTvs 5%, n=5/group) displayed the same sensitivity pattern in the conventional set-up as compared to the SMT screening approach. The TIL analysis revealed a pronounced rise in the CD3+ subset and increased CD4+/CD8+ ratio in the most effective treatment arms. Subsequent serum analyses will help identify possible biomarkers predicting drug response towards checkpoint inhibition. Taken together our study proves that the HuCD34NCG mouse is a fully functional drug development tool. In combination with highly predictive PDX tumor models this in vivo platform provides a further step to support the development of new drugs targeting the host immune response.

Tumor Biology

Session: Mouse Models of Human Cancer in Imaging and Therapeutics | Abstract 2760
A mouse orthotopic model of pancreatic cancer and response to treatment

Elizabeth Rainbolt, Charles River

The mouse pancreatic ductal adenocarcinoma PAN02 was produced in C57BL/6 mice exposed to 3-methyl-cholanthrene and initially reported and characterized by Corbett et al (1). PAN02, also designated Panc 02, is generally resistant to standard cancer therapeutics, thus providing an appropriately rigorous model for the development of clinically effective agents to treat aggressive and intractable pancreatic ductal adenocarcinoma. Subcutaneous implants of syngeneic PAN02 provide the dual advantages of being able to closely monitor changes in tumor growth without compromising the complex pro- and anti-tumor immune responses of the host that collectively determine the outcome of tumor progression (reviewed in 2). Unfortunately, subcutaneous PAN02 tends to grow slowly and tumor ulcerations are common which may result in tumor regression or loss of animals from study. Here we report the growth properties and response to therapy of orthotopically implanted PAN02 designed to provide a more clinically relevant model for evaluation of novel therapies. To establish the orthotopic model, we implanted cells by direct injection into the pancreas through an incision in the abdominal wall at two implant densities, 3x105 and 3x106 PAN02 cells in 30 μl injection volume. Both cell inoculums demonstrated progressive growth with median survival of thirty-seven and thirty days post implant respectively. We chose 3x106 PAN02 cells to evaluate efficacy of selected cytotoxic agents gemcitabine and oxaliplatin. Dosing was initiated 7 days after implant. Gemcitabine was dosed at 120 mg/kg, by intraperitoneal injection (IP) every third day for four doses. Oxaliplatin was dosed at 10 mg/kg IP once a week for 3 weeks. Gemcitabine and oxaliplatin increased life span by 9.0 days and 20.5 days respectively over vehicle control. The morphology of the tumors revealed by histological staining is also presented.

Session: Tumor Microenvironment 2 | Abstract 5085
A preclinical model using perfusion air culture of tumor tissue slices for personalized medicine

IKP In Collaboration With Charles River

For personalized medicine it is crucial that a preclinical model captures the complex tumor biology in vitro in order to individually predict in vivo therapy of tumors. Precision-cut tumor slices maintain tissue heterogeneity with regard to different cell types and preserved native microenvironment. To enable the use of tumor slices as preclinical model that fulfills these criteria we developed a perfusion air culture (PAC) system with continuous and precisely controlled oxygen, medium and drug supply. In the PAC system, precision-cut tumor slices are kept in-between two organotypic supports fixed in a special chamber and placed inside of a 50 mL tube with air exchange capacity housed in a standard CO2-incubator.

To analyze therapy response, Cisplatin was applied to phOVT tumor slices for 3 days. Cisplatin treatment was accompanied by minor increase of ɤ-H2AX in both MF and PAC systems while only in the PAC system strongly enhanced cleavage of caspase-3 was observed, indicating that the PAC system is suitable to assess functional response to drug treatment. To test whether the PAC system is also suitable for the detection of immune response in tumor slices, we analyzed the immune cells of tissue slices before and after cultivation. The patient specific immune cells and their composition is preserved throughout the culture period in the PAC system.

In conclusion, cultivation of tumor tissue slices in the PAC system provides an ex vivo model that preserves tumor heterogeneity and native microenvironment. Because the PAC system facilitates homogenous and precisely controlled supply of oxygen, nutrients and drugs, it allows long-term culture of tumor tissue and analysis of therapy response - including immune therapy. We conclude that the newly developed PAC system is suitable to perform patient specific ex vivo tests and thus allows personalized therapy adaption.

Session: Immune Cells in the Tumor Microenvironment 3 | Abstract 3865
A translational immuno-oncology platform to model T cell activation and Tex recovery in the tumor microenvironment

Robert Nunan, Charles River

The tumor microenvironment (TME) is a complex network, consisting of malignant tumor cells, stromal and immune cells, blood vessels, extracellular matrix and soluble factors. The immune system plays an important role in combating tumor growth, and multiple studies associate immune infiltrate with beneficial outcome. Various tractable immuno-oncology targets have been identified that influence the behavior of immune cells in the TME. These include Cytotoxic CD8+ T cells; which can directly kill tumor cells and produce pro-inflammatory cytokines such as IFNγ. However, CD8+ T cell activity and survival can be suppressed by the TME and chronic exposure leads to the generation of exhausted T cells (Tex) which have a lowered proliferative capacity and diminished capacity for pro-inflammatory cytokine production. Conversely, a regulatory subset of CD4+ T cells (Tregs) act to skew the TME towards immunosuppression. Here we describe human and murine models to assess both relative T cell subset activation and whether T cell exhaustion can be broken. Screening assays using T cell stimulants such as SEB or IL-2 can be utilized to select candidate therapeutics based on functional target engagement. SEB stimulation of PBMC reveals a robust additive effect on T cell activation by established and emerging checkpoint inhibitors. Use of more complicated flow based readouts allows the discrimination of immune subset responses. IL-2, a key lymphocyte activator, drives pSTAT5 expression with differing potency in PBMC subsets (regulatory T cells, conventional CD4 cells, CD8 T cells, NKT cells and NK cells). The ability to target specific subset activation could expand anti-tumour responses. Persistent antigenic stimulation, such as chronic exposure to the TME (MHC I restricted peptides), can lead to a CD8 Tex cell phenotype. Tex cell generation and reversal can be modelled using CEFT peptide pool and monitored via the expression of markers such as LAG-3, TIM-3, TOX and IFNγ. We show here that CD8 Tex cells respond positively to PD-1 blockade treatment, as seen by a restoration in cellular proliferation and increased IFNγ production. Therapeutic candidates identified through in vitro lead optimization can be assessed for their ability to activate T cells via a murine pharmacodynamic (PD) model. Transgenic OT-I CD8 T cells expressing TCR recognising OVA peptide (SIINFEKL) were transferred into syngeneic mice with SIINFEKL challenge in the presence of test therapeutic. Flow cytometry was used to determine OT-I T cell proliferation, activation and cytokine release. As seen in vitro, OT-I cell activation was increased by CPI treatment. We show here the powerful combination of using sophisticated in vitro and in vivo models to determine the effect of therapeutic intervention in an immuno-oncology context.

Session: Tumor-immune System Interactions 2 | Abstract 2824
In vitro tumor killing assays for predicting cellular therapeutic efficacy

Robert Nunan, Charles River

Cellular immunotherapy is emerging as a fifth pillar of cancer treatment. Clinical efficacy has been proven for a variety of cancers; however, its true potential has yet to be unlocked. Development of reliable and mechanistically appropriate in vitro screening assays that recapitulate in vivo immune-tumor interactions have been outpaced by in vivo living cell therapy efficacy models. Here we describe a range of in vitro primary human assays to model tumor killing by immune cells. This platform addresses multiple anti-tumor immune effector pathways and has been validated with standard of care therapeutics. Fluorescently labelled target cells (PDX-cell lines, tumor cell lines or human primary/iPSC derived cells) screened for relevant antigen expression were cultured in monolayer or spheroid forms and co-cultured with the cellular therapeutic. On target killing by effectors is driven by binding of the TCR or CAR to its cognate antigen (MHC/peptide or protein, respectively) with readouts of cell killing utilising live cell imaging (IncuCyte) and flow cytometry based approaches to measure target cell growth and apoptosis. Additional analysis of soluble mediators can determine the extent of effector cell activation. To discriminate between ‘on’ and ‘off’ target killing by cellular therapeutics, primary/iPSC-derived normal cell types representative of different tissue types were screened for expression of an HLA subtype capable of binding exogenously applied tumor-associated antigen (TAA) prior to co-culture. Under these conditions in the case of a TCR based cellular therapeutic, efficacy can be screened for, or in the absence of the putative TAA expression, the extent of off-target killing determined. Enhancement of tumor targeting and killing by immune cells is an attractive therapeutic modality partly due to the clinical success of checkpoint inhibitors which can enhance the activation and effector function of endogenous T/NK cells or cellular therapeutics. The presented data highlight the importance of assays which can recapitulate tumor-immune interactions and include immune effector pathways known to potentiate anti-tumor immune responses. These assays represent valuable screening tools capable of informing cellular therapeutic target selection and refinement before moving to more complex in vivo models.

Session: New Imaging-based Approaches to Plan, Monitor, and Augment Cancer Treatment | Abstract 2774
Longitudinal characterization of patient-derived orthotopic xenograft brain tumor models

Julia Schuler, Charles River

Gliomas are the most common primary brain tumors and currently the prognosis is still poor. Due to this, it is one of the main areas in oncological research and drug development programs. Innovative therapies are vital to improve treatment outcomes but must be developed to enable trafficking across the blood brain barrier (BBB). For this advent, animal models provide important information prior to clinical studies. Among the different in vivo models, orthotopic PDX models represent best the tumor microenvironment and natural variability of tumors, hence providing the most reliable results over species.

In the brain tumor field, imaging has a central role in clinical diagnosis and as a prognostic factor to monitor therapy response in patients. Magnetic resonance imaging (MRI) is widely used for clinical diagnosis and disease follow up as it offers unprecedented soft tissue contrast and high spatial resolution non-invasive way. In addition to imaging, molecular profiling, e.g. circulating immune biomarkers and local oncometabolites in the tumor environment may facilitate as important translational biomarkers in development of immunotherapy for gliomas in addition to traditional histopathological readouts.

The purpose of this work was to analyze possible heterogeneity of tumors in vivo, growth rate and volume in stereotactically implanted orthotopic PDX brain tumor models using MRI/MRS imaging. In addition to imaging, neurological scoring was performed to monitor general animal welfare, cytokine profiles from plasma to observe immunological responses over time and determination of oncometabolites in plasma and brain tissues combined with traditional histopathological changes were performed. The data from orthotopic models was also compared to readouts in subcutaneous models.

As a conclusion, translational in vivo imaging techniques were applied to study orthotopic tumor progression combined with circulating immune biomarkers, and general welfare scoring. These readouts provide a powerful and translational research tool together with oncological disease animal models allowing comprehensive evaluation of disease progression and treatment interventions for in vivo studies.

Session: Patient-derived Organoid and Xenograft Models | Abstract 2813
Tumor models driven by EGFR: Optimizing the preclinical profiling of EGFR-targeting agents

Thomas Metz, Charles River

The epidermal growth factor receptor (EGFR, HER1, ERBB1) is a driver of many human cancers. Standard of care treatment for colon, head and neck and non-small cell lung cancer includes drugs targeting EGFR. Numerous molecular alterations activating the oncogenic potential of the EGFR gene have been described including activating point mutations, point mutations causing resistance against EGFR-targeting drugs, activating deletions and truncations as well as overexpression of EGFR and its ligands, occasionally induced by gene amplification. In other situations, EGFR has been identified as a driver in the absence of any obvious molecular alterations of the EGFR gene. The discovery and development of EGFR-targeting agents depends on the availability of relevant tumor models. We provide an overview of our collection of EGFR-driven tumor models, including PDXs along with PDX-derived cell lines and human tumor cell lines. We compare sensitivity profiles of tumor models for EGFR-targeting agents obtained in vivo and in vitro and demonstrate that for EGFR-targeting agents, data obtained in 2D and 3D assays are predictive for the in vivo situation. We propose an optimized strategy for the preclinical profiling of EGFR-targeting anti-cancer agents.

Session: Model Organisms for Cancer Research | Abstract 6125
Zebrafish patient tumor-derived xenograft models synergize with mouse-PDX models for understanding variation in anti-cancer drug responses

BioReperia In Collaboration With Charles River

Lung cancer accounts for the 2nd most common cancer among men and women, representing 24% of cancer deaths worldwide. Standard-of-care treatments vary considerably depending on the tumor type and staging. Identifying which patients will benefit from treatment with a certain drug remains one of the major challenges in the clinic.

Genetic analyses are widely used but have low applicability as only ~10% of the patients have mutations coupled to available targeted therapies and relatively low sensitivity as therapeutic effects are absent in ~50% of the predicted responders. Mouse-PDX models can accurately determine drug response rates for 50-60% of the patients, but are not well suited for evaluating metastatic risk. As metastasis is a major cause of disease-associated mortality and no drugs that target metastasis exist today, there is considerable need to develop new drugs able to impair metastatic dissemination in lung (and other) cancers. To meet this need, zebrafish-PDX (ZTX) models are ideally positioned as a synergistic complement to mouse-PDX models allowing evaluation of drug responses, in a non-rodent in vivo system with the turnaround time and scalability of an in vitro platform.

Here we generated zebrafish- and mouse-PDX models based on 20 patient NSCLC samples and compared the efficacy of standard-of-care treatment (erlotinib and paclitaxel) on primary tumor growth/regression as well as metastatic dissemination in the zebrafish-PDX models. The ZTX models exhibited variable sensitivity to the drugs tested with 11 of 20 and 16 of 20 models being sensitive to erlotinib and paclitaxel respectively. The efficacy of erlotinib and/or paclitaxel was identical in 9 of 11 mouse- and zebrafish-PDX models where these drugs were compared head-to-head. The models metastasized within three days of tumor implantation in the zebrafish larvae, seeding an average of 2 - 8 metastatic lesions per model in the caudal hematopoietic plexus. Paclitaxel and erlotinib inhibited metastasis in 7 of 20 and 6 of 20 models respectively. The anti-metastatic activity did not correlate with the activity against the primary tumor. Investigations as to what extent this correlates with invasive phenotypes observed in histological preparations of the mouse-PDX models, and clinical data on metastasis in the patients, are currently ongoing.

In conclusion we provide evidence of the accuracy of the ZTX models in predicting anti-tumor responses to commonly used drugs in NSCLC compared to mouse-PDX models and demonstrate that ZTX models provide a sensitive method for determining metastatic risk and the anti-metastatic efficacy of NSCLC relevant drugs.

Session: Model Organisms for Cancer Research | Abstract 6126
Zebrafish patient tumor-derived xenograft models used for pre-clinical evaluation of CAN04 for lung and pancreatic cancer

BioReperia In Collaboration With Charles River

Lung cancer is one of the most deadly diseases accounting for 24% of all cancer deaths worldwide. One reason for this high mortality is the high interindividual heterogeneity but generally poor efficacy of current treatments, leading to an urgent need for new and more effective drugs. Understanding the individual variability in the efficacy of new treatment candidates, delineating whether they should be combined with existing chemotherapeutics and to what extent they affect metastatic dissemination of the tumor cells are key preclinical indicators needed to increase the chance of success in clinical trials. Developing such data, however, requires animal models that recapitulate individual differences of different lung cancer patients, include insights into metastatic activity and allow analysis of a large amounts of treatment combinations for each patient model. As such, an in vivo screening system which has higher throughput than mouse models and at the same time allows analysis of metastatic activity would be very valuable in mimicking human disease.

Here we conducted zebrafish patient tumor derived xenograft (PDX)-studies based on cisplatin sensitive and -resistant lung cancer PDX material, to test the efficacy of a a novel antibody, CAN04, under development for this indication. CAN04 targets Interleukin-1 Accessory Protein (IL1RAP) and has shown synergistic effects with cisplatin in murine models of cancer. CAN04 is currently in phase II development in combination with chemotherapy in lung cancer and pancreatic cancer. CAN04 was given either alone or with cisplatin at three different concentrations, and the effects on primary tumor growth and metastasis three days after tumor implantation was evaluated. We show that CAN04 was able to synergize with cisplatin in causing almost complete (85%-98%) tumor regression even of cisplatin-resistant tumors, compared to non-treated controls. The effects were concentration- and model-dependent. Interestingly, in the cisplatin-resistant model, the antibody and cisplatin co-treatment led to robust inhibition of metastatic dissemination, which was not seen in either group alone. This substantiates the beneficial therapeutic efficacy of combining CAN04 to cisplatin treatment in lung cancer.

In conclusion, zebrafish-PDX (ZTX) models are powerful tools for evaluating individual differences in drug sensitivity on both primary tumor growth and metastasis and are suitable for screening various drug concentrations and/or combinations in multiple models with results being generated within one or a few weeks. We further conclude that CAN04 is inducing cisplatin sensitivity and synergize with cisplatin to inhibit metastasis, at least in some cisplatin resistant lung cancers.

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In the research community, we know our work doesn’t stop when crisis hits. Keep yours going strong with this collection of insights, tools, and resources prepared by our team.

On-Demand Webinars

medically accurate 3d illustration of cancer cells.

When Immune Cells Invade a Tumor: Using the TME to Drive Your Oncology Research
Presented by: Robert Nunan, PhD, Group Leader, Charles River

 

micro electron image of breast cancer cells.

Emergent Humanized Mouse Models Are Driving Immuno-Oncology Strategy
Presented by: Paula Milinani de Marval, PhD, Associate Research Director, Discovery Services, Charles River

 

composition of tumor microenvironment

Microdialysis: A Novel Tool to Sample the Tumor Microenvironment
Presented by: Nadege Morisot, PhD, Senior Scientist, In Vivo Pharmacology Charles River

 

Glioblastoma Model imagaing

Preclinical Imaging: A Translational Approach to Assess New Cancer Treatments
Presented by: Tuulia Huhtala, PhD, Head of Biomarkers and Molecular Imaging, Charles River

 

Computer generated abstract fractal illustration of a virus in human body.

How to Design & Execute Successful CAR-T Cell Therapy Programs (Available in The Source℠)
Presented by: David Harris, PhD, Research Director, Oncology, Charles River

 

3D illustration of DNA helix connected by nodes and connections.

Bioinformatics: Mining Big Data to Discover Precision Oncology Therapies
Presented by: Max Bylesjo, PhD, MSc, Technical Director, Fios Genomics

 

Podcast

Tumor Cell.

Sounds of Science
Episode 20: A Tumor's Lair