huNOG Mice Research Articles

Clinically Relevant Humanized Mouse Models of Metastatic Prostate Cancer Facilitate Therapeutic Evaluation.

There is tremendous need for improved prostate cancer models. Anatomically and developmentally, the mouse prostate differs from the human prostate and does not form tumors spontaneously. Genetically engineered mouse models lack the heterogeneity of human cancer and rarely establish metastatic growth. Human xenografts are an alternative but must rely on an immunocompromised host. Therefore, we generated prostate cancer murine xenograft models with an intact human immune system (huNOG and huNOG-EXL mice) to test whether humanizing tumor-immune interactions would improve modeling of metastatic prostate cancer and the impact of androgen receptor-targeted and immunotherapies. These mice maintain multiple human immune cell lineages, including functional human T-cells and myeloid cells. Implications: To the best of our knowledge, results illustrate the first model of human prostate cancer that has an intact human immune system, metastasizes to clinically relevant locations, responds appropriately to standard-of-care hormonal therapies, and can model both an immunosuppressive and checkpoint-inhibition responsive immune microenvironment.

Epstein-barr virus infections induce aberrant osteoclastogenesis in immune system-humanized NOD/Shi-scid/IL-2RγCnull mice

Epstein-Barr virus (EBV) and other viral infections are possible triggers of autoimmune diseases, such as rheumatoid arthritis (RA). To analyze the causative relationship between EBV infections and RA development, we performed experiment on humanized NOD/Shi-scid/IL-2RγCnull (hu-NOG) mice reconstituted human immune system components and infected with EBV. In EBV-infected hu-NOG mice, breakdown of knee joint bones was found to be accompanied by the accumulation of receptor activator of nuclear factor-κB (NF-κB) (RANK) ligand (RANKL), a key factor in osteoclastogenesis, human CD19 and EBV-encoded small RNA (EBER)-bearing cells. Accumulation of these cells expanded in the bone marrow adjacent to the bone breakage, showing a histological feature like to that in bone marrow edema. On the other hand, human RANK/human matrix metalloprotease-9 (MMP-9) positive, osteoclast-like cells were found at broken bone portion of EBV-infected mouse knee joint. In addition, human macrophage-colony stimulating factor (M-CSF), an essential factor in development of osteoclasts, evidently expressed in spleen and bone marrow of EBV-infected humanized mice. Furthermore, RANKL and M-CSF were identified at certain period of EBV-transformed B lymphoblastoid cells (BLBCs) derived from umbilical cord blood lymphocytes. Co-culturing bone marrow cells of hu-NOG mice with EBV-transformed BLBCs resulted in the induction of a multinucleated cell population positive for tartrate-resistant acid phosphatase and human MMP-9 which indicating human osteoclast-like cells. These findings suggest that EBV-infected BLBCs induce human aberrant osteoclastogenesis, which cause erosive arthritis in the joints.

Abstract 5338: Evaluation of checkpoint inhibitor efficacy in a humanized immune system mouse model lacking murine Fc gamma receptors

Abstract Humanized immune system (HIS) mice are critical immuno-oncology tools for evaluating antibody-based therapeutic target engagement and efficacy in a human-like context. Most antibody-based therapeutics have an IgG backbone and thus engage Fc gamma receptors. Traditional HIS mice retain murine immune cells such as neutrophils and macrophages which can interact with human IgG-based therapeutics and confound preclinical results. To determine whether knockout of murine Fc gamma receptors (FcγRs) in a super-immunodeficient mouse model would alter anti-PD1 efficacy compared to the parent strain, we studied tumor growth kinetics, human reconstitution, and tumor infiltrating leukocytes (TILs) in each strain engrafted with HCC827 lung adenocarcinoma tumor cells treated with pembrolizumab or vehicle. Methods: HIS NOG (huNOG) or HIS FcγR knockout NOG mice (FcResolv™ huNOG) were created using identical protocols with CD34+ cells from three human donors shared across both strains. Baseline reconstitution was evaluated in naïve animals via flow cytometry. HCC827 cells were inoculated in remaining animals. Animals were randomized on day 7 post-tumor implantation into 12 groups. Body weight, clinical observations, and tumor growth were measured. Mice received treatment from D7, dosed twice weekly for four weeks, and were then euthanized for FACS analysis, collecting blood, spleen, and tumor samples. Results: For a given donor, no significant differences were seen between strains in human immune reconstitution and kinetics. Pembrolizumab treatment showed significant tumor growth inhibition in one donor in FcResolv huNOG, but not in donor-matched huNOG mice. Evaluation of human TILs in pembrolizumab-treated animals showed significant differences between the strains across all donors, with FcResolv huNOG mice showing increased CD8+ T cells and decreased tumor-associated macrophages compared to vehicle-treated mice, and no significant differences in huNOG. Evaluation of murine TILs revealed differences in murine macrophage populations, regardless of treatment, with Ly6Clo dominant in FcResolv huNOG and Ly6Chi dominant in huNOG. In the presence of pembrolizumab, the murine myeloid and granulocyte population within the spleen is significantly decreased in FcResolv huNOG mice whereas no change was seen in huNOG. Conclusions: Our study demonstrates when treated with anti-PD1, FcResolv huNOG mice show expected pharmacodynamic changes and donor-dependent efficacy whereas, despite identical donors and creation protocols, pembrolizumab-treated huNOG mice showed neither. These differences are due to the presence or absence of murine FcγRs and their impact on antibody IgG-based therapeutics. Citation Format: Nicholas Smith, Monika Buczek, Megan MacBride, Esther Andino, Emily Sack, Debra Freer, Michelle Vedder, Kathleen Bott, Louise Baskin, Damien France, Francis Bichat, Caroline Mignard, Janell Richardson. Evaluation of checkpoint inhibitor efficacy in a humanized immune system mouse model lacking murine Fc gamma receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5338.

Kidney Immune Cell Characterization of Humanized Mouse Models.

Experimental studies often fail to translate to clinical practice. Humanized mouse models are an important tool to close this gap. We immunophenotyped the kidneys of NOG (EXL) and NSG mouse strains engrafted with human CD34 + hematopoietic stem cells or PBMCs and compared with immune cell composition of normal human kidney. Human CD34 + hematopoietic stem cell engraftment results in steady renal immune cell populations in mouse kidney with key similarities in composition compared with human kidney. Successful translation of experimental mouse data to human diseases is limited because of biological differences and imperfect disease models. Humanized mouse models are being used to bring murine models closer to humans. However, data for application in renal immune cell-mediated diseases are rare. We therefore studied immune cell composition of three different humanized mouse kidneys and compared them with human kidney. NOG and NOGEXL mice engrafted with human CD34 + hematopoietic stem cells were compared with NSG mice engrafted with human PBMCs. Engraftment was confirmed with flow cytometry, and immune cell composition in kidney, blood, spleen, and bone marrow was analyzed in different models. The results from immunophenotyping of kidneys from different humanized mouse strains were compared with normal portions of human kidneys. We found significant engraftment of human immune cells in blood and kidney of all tested models. huNSG mice showed highest frequencies of hTCR + cells compared with huNOG and huNOGEXL in blood. huNOGEXL was found to have the highest hCD4 + frequency among all tested models. Non-T cells such as hCD20 + and hCD11c + cells were decreased in huNSG mice compared with huNOG and huNOGEXL. Compared with normal human kidney, huNOG and huNOGEXL mice showed representative immune cell composition, rather than huNSG mice. In summary, humanization results in immune cell infiltration in the kidney with variable immune cell composition of tested humanized mouse models and partially reflects normal human kidneys, suggesting potential use for translational studies.

OR05-05 A Novel Model Of Prostate Cancer Suggests Enzalutamide Functions Through The Immune System To Diminish Castration Resistant And Metastatic Growth

Abstract Disclosure: S. Kregel: None. Background: There exists a tremendous need for establishing prostate cancer (PCa) murine models capable of recapitulating the progress of human PCa. By itself, the murine prostate does not form sporadic tumors and is anatomically and developmentally different from the human prostate. Engineered mouse models, however, lack the heterogeneity of human disease, are often driven in a contrived manner, and rarely (if ever) establish metastatic growth. Human xenografts represent an alternative, but they rely on tumor growth in an immunocompromised host, thus preventing the study of tumor-immune interactions and immunotherapies. Accordingly, we generated PCa xenograft models in a murine system with an intact human immune system (huNOG mice) to test the hypothesis that humanizing tumor-immune interactions would improve modeling of metastatic PCa as well as our ability to model the impact of hormonal and immunotherapies. Methods: Male huNOG and huNOG-EXL mice were produced at Taconic Biosciences by engrafting juvenile NOG mice with human CD34+ hematopoietic stem cells. These mice stably maintained multiple human cell lineages, including functional human T-cells. We utilized two human PCa xenograft cell line models transduced with luciferase to assay organ-specific metastatic growth. First, castrated and intact control mice were injected subcutaneously with 22Rv1 cells. When tumors reached >100 mm3, half of the castrated mice were treated with enzalutamide (enza), and tumor growth was then monitored to endpoint. Additionally, VCaP tumor-bearing mice were castrated when the tumors reached ∼200 mm3, and once the tumor grew back, they were randomized and treated with enza and/or the anti-PD-1 antibody, pembrolizumab (pembro) as well as with vehicle controls. At sacrifice, organs were ex vivo analyzed for metastatic growth, tumor infiltrating lymphocytes, and splenic immune reconstitution. Results: In 22Rv1 tumor-bearing mice, subcutaneous tumor size was not significantly altered across conditions. However, the extent and growth at the secondary sites differed markedly in castrate huNOG vs conventional NOG mice treated with enza. VCaP xenograft tumors showed marked decreases in growth with enza and pembro treatments in huNOG mice, while no effect was observed with either of these treatments in NOG mice. Furthermore, immune responses to enza treatment in huNOG and NOG mice were distinct, as enza treatment only increased intra-tumor CD3+ T-cells as well as CD3+ T-cell activation in huNOG mice. Conclusions: These results illustrate, to the best of our knowledge, the first model of human PCa that metastasizes to clinically relevant locations, has an intact human immune system, and responds to standard-of-care hormonal therapies in a manner similar to responses observed under clinical settings. Presentation: Thursday, June 15, 2023

Lysine N-methyltransferase SETD7 promotes bladder cancer progression and immune escape via STAT3/PD-L1 cascade.

Background: The immunotherapy sensitivity of patients with bladder cancer (BCa) remains low. As the role of protein methylation in tumorigenesis and development becomes clearer, the role of lysine N-methyltransferase SET domain containing 7 (SETD7) in the progression and immune escape of BCa is worth studying. Methods: The correlation between lysine methyltransferase family and prognosis or immunotheray sensitivity of BCa patients were analyzed, and SETD7 was screened out because of the significant correlation between its expression and survival data or immunotherapy sensitivity. The expression of SETD7 in BCa tissues and cell lines were explored. The functions of SETD7 were investigated by proliferation and migration assays. The role of SETD7 in BCa immune escape was validated by analyzing the correlation between SETD7 expression and tumor microenvironment (TME)-related indicators. The results were further confirmed by conducting BCa cell-CD8+ T cell co-culture assays and tumorigenesis experiment in human immune reconstitution NOG mice (HuNOG mice). Bioinformatic prediction, CO-IP, qRT-PCR, and western blot were used to validate the SETD7/STAT3/PD-L1 cascade. Results: SETD7 was highly expressed in BCa, and it was positively associated with high histological grade and worse prognosis. SETD7 promoted the proliferation and migration of BCa cells. The results of bioinformatics, in vitro co-culture, and in vivo tumorigenesis assays showed that SETD7 could inhibit the chemotoxis and cytotoxicity of CD8+ T cells in BCa TME. Mechanistically, bioinformatics analysis, CO-IP assay, qRT-PCR, and western blot results indicated that SETD7 could increase the expression of PD-L1 via binding and promoting STAT3. Conclusions: Taken together, SETD7 indicated poor prognosis and promoted the progression and immune escape of BCa cells. It has great potential to act as a new indicator for BCa diagnosis and treatment, especially immunotherapy.

Abstract 6026: A novel humanized mouse model based on NOG-FcgR-/-mice recapitulates anti-tumor immune reactions by human immune systems in response to anti-PD-1 antibody (Nivolumab)

Abstract Humanized mice with human hematopoietic and immune systems have been frequently used for examining in vivo anti-cancer effects of various drugs, including immune checkpoint inhibitors (ICIs) like anti-PD-1 or anti-CTLA-4 antibodies, yet the reliability of the mouse models to be improved. We have recently developed NOD-scid, IL-2Rγ KO (NOG) mice deficient for the mouse FcεRI and FcγIIb genes, thus lacking the expression of functional mouse FcγRs, (NOG-FcγR-/-) and the activity of endogenous antibody-dependent cellular cytotoxicity (ADCC). Reconstitution experiments with human hematopoietic stem cells (HSC) showed that NOG-FcγR-/- mice had higher engraftment capacity for human leucocytes than NOG mice, namely the numbers of HSC-derived human CD19+ B cells and CD33+ myeloid cells, but not CD3+ T cells, were higher in the various tissues in NOG-FcγR-/- mice than those in NOG mice. In tumor-bearing experiments using those reconstituted mice, it was of interest that humanized NOG-FcγR-/- mice (huNOG-FcγR-/-) could invoke strong tumor growth suppression or rejection in response to anti-PD-1 antibody (Nivolumab) treatment in several cancer cell lines, which was absent in conventional huNOG mice. Accordingly, Nivolumab treatment in huNOG-FcγR-/- mice induced a significant increase of human T cells in number in the spleen and infiltration of human T cells with Granzyme B and Perforin-expression into tumor. Furthermore, the composition of activated T cells was different between huNOG-FcγR-/- and huNOG mice. HuNOG-FcγR-/- mice had elevated numbers of activated T cells, both T central memory cells (TCM) and T effector memory cells (TEM), whereas huNOG mice showed an opposite trend, reduced TCM and TEM and increase of naïve T cells. Our results suggest that the novel NOG-FcγR-/- mice become a good model for elucidating the cellular and molecular mechanisms of human tumor rejection by human T cells, and that they will provide a superior in vivo platform for evaluation of anti-cancer drugs, especially, in combination with ICIs like Nivolumab. Citation Format: Ikumi Katano, Asami Hanazawa, Iyo Otsuka, Takuya Yamaguchi, Misa Mochiduki, Kenji Kawai, Ryoji Ito, Motohito Goto, Takahiro Kagawa, Takeshi Takahashi. A novel humanized mouse model based on NOG-FcgR-/-mice recapitulates anti-tumor immune reactions by human immune systems in response to anti-PD-1 antibody (Nivolumab) [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 6026.

Development of a novel humanized mouse model for improved evaluation of in vivo anti-cancer effects of anti-PD-1 antibody.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer in the clinic. Further discovery of novel drugs or therapeutic protocols that enhance efficacy requires reliable animal models that recapitulate human immune responses to ICI treatment in vivo. In this study, we utilized an immunodeficient NOG mouse substrain deficient for mouse FcγR genes, NOG-FcγR−/− mice, to evaluate the anti-cancer effects of nivolumab, an anti-programmed cell death-1 (PD-1) antibody. After reconstitution of human immune systems by human hematopoietic stem cell transplantation (huNOG-FcγR−/− mice), four different programmed death-ligand 1 (PD-L1)-positive human cancer cell lines were tested. Among them, the growth of three cell lines was strongly suppressed by nivolumab in huNOG-FcγR−/− mice, but not in conventional huNOG mice. Accordingly, immunohistochemistry demonstrated the enhanced infiltration of human T cells into tumor parenchyma in only nivolumab-treated huNOG-FcγR−/− mice. Consistently, the number of human T cells was increased in the spleen in huNOG-FcγR−/− mice by nivolumab but not in huNOG mice. Furthermore, human PD-L1 expression was strongly induced in the spleen of huNOG-FcγR−/− mice. Collectively, our results suggest that the anti-cancer effects of anti-PD-1 antibodies can be detected more clearly in NOG-FcγR−/− mice than in NOG mice.

Abstract 1806: Novel preclinical orthotopic and bone metastasis TNBC models for assessment of immunotherapy and chemotherapy responses

Abstract Triple-negative breast cancer (TNBC) is one of the most complicated types of breast cancer to treat. However, clinical trials continue to demonstrate that combining immunotherapy with chemotherapy can improve survival in patients with metastatic TNBC. In preclinical immunotherapy development, one of the challenges and limitations is the lack of representative TNBC models for advanced metastatic disease that could be used to study survival benefit of these compounds. The aim of this study was to develop local and bone metastatic TNBC models where survival could be followed as primary endpoint, and to assess effects of standard-of-care (Nab-paclitaxel, Abraxane) and anti-PD-1 (pembrolizumab, Keytruda). Age-matched immunodeficient female CIEA NOG® mice (NOG) and NOG mice engrafted with human CD34+ hematopoietic stem cells (huNOG) were used in the study. In the orthotopic model, the luciferase labelled human TNBC cells, MDA-MB-231(SA)-luc, were inoculated into mammary fat pad of the mice. In the intratibial model, MDA-MB-231(SA)-luc cells were inoculated into bone marrow cavity of tibia to model the growth of primary and bone metastatic TNBC. The treatment with vehicle or paclitaxel (10 mg/kg) in NOG mice or isotype control or pembrolizumab (5 mg/kg) in huNOG mice was started 3 days after cancer cell inoculation and continued twice a week until termination. The mice were sacrificed individually according to predefined sacrifice criteria or latest at study week 8. Nab-paclitaxel prolonged survival of immunodeficient mice with both primary and metastatic TNBC, but the effect was more profound in the orthotopic model. Pembrolizumab treatment in humanized mice with either tumor site did not result in survival benefit. In an earlier study, we have observed modest tumor growth inhibition with pembrolizumab in the orthotopic model suggesting donor variation effect. The findings are consistent with the clinical studies where pembrolizumab as monotherapy has demonstrated benefit for only some of the TNBC patients. In summary, novel preclinical models mimicking primary and bone metastatic TNBC were established that could be used to study the survival benefit of developing new treatments, including immunotherapies, against local and advanced TNBC. Citation Format: Justyna Zdrojewska, Ivan Gladwyn-Ng, Jenni H. Mäki-Jouppila, Katja M. Fagerlund, Mari I. Suominen, Ross Horn, Jukka Rissanen. Novel preclinical orthotopic and bone metastasis TNBC models for assessment of immunotherapy and chemotherapy responses [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 1806.

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice.

Many human viruses, including Epstein-Barr virus (EBV), do not infect mice, which is challenging for biomedical research. We have previously reported that EBV infection induces erosive arthritis, which histologically resembles rheumatoid arthritis, in humanized NOD/Shi-scid/IL-2Rγnull (hu-NOG) mice; however, the underlying mechanisms are not known. Osteoclast-like multinucleated cells were observed during bone erosion in this mouse model, and therefore, we aimed to determine whether the human or mouse immune system activated bone erosion and analyzed the characteristics and origin of the multinucleated cells in hu-NOG mice. Sections of the mice knee joint tissues were immunostained with anti-human antibodies against certain osteoclast markers, including cathepsin K and matrix metalloproteinase-9 (MMP-9). Multinucleated cells observed during bone erosion stained positively for human cathepsin K and MMP-9. These results indicate that human osteoclasts primarily induce erosive arthritis during EBV infections. Human osteoclast development from hematopoietic stem cells transplanted in hu-NOG mice remains unclear. To confirm their differentiation potential into human osteoclasts, we cultured bone marrow cells of EBV-infected hu-NOG mice and analyzed their characteristics. Multinucleated cells cultured from the bone marrow cells stained positive for human cathepsin K and human MMP-9, indicating that bone marrow cells of hu-NOG mice could differentiate from human osteoclast progenitor cells into human osteoclasts. These results indicate that the human immune response to EBV infection may induce human osteoclast activation and cause erosive arthritis in this mouse model. Moreover, this study is the first, to our knowledge, to demonstrate human osteoclastogenesis in humanized mice. We consider that this model is useful for studying associations of EBV infections with rheumatoid arthritis and human bone metabolism.

89Zr-pembrolizumab biodistribution is influenced by PD-1-mediated uptake in lymphoid organs

BackgroundTo better predict response to immune checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal antibodies is essential. Therefore, we aimed...

Abstract B143: Establishment of primary tumor models of TNBC and ER+/HER2- breast cancer in humanized mice and validation of pembrolizumab efficacy

Abstract Background: Immunotherapy is an emerging treatment option for different subtypes of breast cancer. Pembrolizumab (Keytruda®) targets PD-1 expressing T cells in tumors and mediates anti-tumor effects. Targeting PD-1 has shown promising results in advanced breast cancer and is being evaluated in clinical trials in triple-negative breast cancer (TNBC) and in estrogen receptor positive, human epidermal growth factor 2 negative (ER+/HER2-) breast cancer patients. Immunotherapy is not yet an established treatment option for early breast cancer. The aim of this study was to establish preclinical primary tumor models for TNBC and ER+/HER2- in humanized mice, and to evaluate efficacy of pembrolizumab in the models. Materials and methods: Immunodeficient CIEA NOG female mice engrafted with human CD34+ hematopoietic stem cells (huNOG mice) were inoculated orthotopically into mammary fat pad with either MDA-MB-231(SA) (TNBC) or MCF-7 (ER+/HER2-) human breast cancer cells. The mice receiving MCF-7 cells were implanted with estradiol (E2) releasing pellets before cancer cell inoculation to support tumor growth. The mice were treated with pembrolizumab (5 mg/kg) or isotype control in a Q5D schedule. The treatments were started 3 or 14 days after cancer cell inoculation in the TNBC and ER+/HER2- models, respectively. Tumor growth was monitored by caliper measurements, and the study was terminated at 3 weeks (TNBC model) or 7 weeks (ER+/HER2- model). Tumors were collected and analyzed immunohistochemically for tumor-infiltrating lymphocytes (TILs), and hematological analysis was performed from the mice in the ER+/HER2- model. Results: Tumors responded to pembrolizumab treatment in the TNBC model. Three out of eight mice responded to the treatment and one of them had a tumor rejection. Pembrolizumab treatment was associated with decrease in CD8+ TILs and granzyme B+ cells in the tumor. In the ER+/HER2- model, E2 supplementation was essential for tumor growth. However, E2 caused a decrease in the clinical condition, including decrease in white and red blood cells, leading to early sacrifices. Only modest responses for pembrolizumab were observed in a few mice and the results did not reach statistical significance. No major changes were observed in TILs due to pembrolizumab treatment. Conclusions: In the established breast cancer models in humanized mice, pembrolizumab decreased tumor growth in the TNBC model but induced only minor tumor responses in the ER+/HER2- model. Estrogen was essential for ER+/HER2- tumor growth but induced severe anemia and had immunomodulatory effects potentially influencing the lack of pembrolizumab efficacy in the model. Citation Format: Tiina E Kähkönen, Mari I Suominen, Jenni HE Mäki-Jouppila, Azusa Tanaka, Philip Dube, Michael Seiler, Jussi M Halleen, Jenni Bernoulli. Establishment of primary tumor models of TNBC and ER+/HER2- breast cancer in humanized mice and validation of pembrolizumab efficacy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B143. doi:10.1158/1535-7163.TARG-19-B143

Macrophage Transfer to HSCs Assigns Residence in Bone Marrow

Hematopoietic stem cells (HSCs), residing at the apex of the hematopoietic hierarchy, are a rare and heterogeneous cell population. Although HSC subsets with various repopulation capacities and lineage bias have been identified, there is no available information about whether each HSC has an equal chance of being mobilized or whether there are specific pools of mobilizable or non-mobilizable HSCs in bone marrow (BM). Here, we identify a BM resident HSC subset based on the expression of the macrophage marker F4/80. Flow cytometry analysis revealed that F4/80 was expressed on 75% of HSCs (Lin- Sca1+cKit+CD150+CD34-; hereafter referred to as HSCM) in the BM. In competitive transplantation assays, HSCM exhibited significantly lower engraftment potential, but no lineage bias, compared to F4/80- HSCs (HSCM: 19.7±4.8%; F4/80- HSC: 37.1±4.5%, P<0.05). We next analyzed the ability of HSCM and F4/80- HSCs to be mobilized after G-CSF treatment and found that F4/80- HSCs, but not HSCM, were exclusively mobilized (F4/80- HSC: 5042±912.5; HSCM: 6.6±6.6 per mL blood, P<0.001). Similar selective mobilization was observed after administration of AMD3100 or macrophage depletion using clodronate liposomes. To confirm this observation in a genetic model, we crossed transgenic mice expressing Cre recombinase knocked into the Cd169 locus, a marker of BM macrophages (MΦ), with ROSA26-loxP-stop-loxP-tdTomato (CD169/Tomato). We found CD169/Tomato labelled a large fraction of HSCM (31.7±8.4%), in contrast to F4/80- HSCs which were largely unlabelled (2.1±1.2%). Remarkably, CD169/Tomato+ HSCs were not mobilized into the circulation after G-CSF treatment (CD169/Tomato- HSC: 4573±1416; CD169/Tomato+ HSC: 90.6±90.6 per mL blood, P<0.01). Although HSCs are reported to express Csf1r, the expression of macrophage markers on HSCs (F4/80 and CD169) was unexpected. To ascertain the expression origin, we transplanted CD169/Tomato+ and CD169/Tomato- sorted HSCs into lethally irradiated CD45.1 congenic mice. If HSCs expressed CD169, we would expect that donor HSC tomato expression would be retained. However, to our surprise, we found that the proportion of Tomato+ HSCs was higher in the CD169/Tomato- group, suggesting that F4/80 and CD169 expression may be acquired, rather than expressed within HSCs. Indeed, co-culture of TdTomato+ BM MΦ with GFP+ HSCs consistently revealed the acquisition of tdTomato (GFP+ TdTomato+: 9.9±1.9%). Transwell experiments revealed that cell contact was important as GFP+ cells did not show any TdTomato expression in the absence of direct cell contact (GFP+TdTomato+: 0%). To investigate the mechanism of HSC retention, we analyzed CXCR4 expression on HSCM and F4/80- HSC subsets. These analyses revealed that CXCR4 was expressed at higher expression levels on HSCM compared to F4/80- HSCs (HSCM: 79.4±9.1% CXCR4+;F4/80- HSC: 45.6±8.5% CXCR4+, P<0.001). In accordance with their increased CXCR4 expression, HSCM exhibited a 4.7-fold increase in pERK1/2 after CXCL12 stimulation, while CXCL12 only triggered a modest increase (1.5-fold) in pERK1/2 in F4/80- HSCs, suggesting that HSCM have a higher signaling response to CXCL12, and thus exhibit enhanced BM retention. To further confirm that the cell transfer occurred in vivo, we used xenografted mice in which human HSCs were transplanted into NOG mice. We found a significant presence of murine F4/80 on human CD34+ HSCs in xenografted huNOG mice, confirming the F4/80 transfer from host MΦ into human HSCs in vivo. To investigate further the mechanism of cell transfer, we carried out co-culture experiments of GFP+ lineage-depleted BM cells with TdTomato+ MΦ in the presence of inhibitors known to prevent cell transfer through membrane and cytoskeleton remodeling. Of all conditions tested, only the presence of the GAP junction inhibitor carbenoxolone (CBX) could inhibit the transfer of TdTomato from MΦ to GFP+ cells (control: 21.4±3.0%; CBX: 12.6±2.1%, P<0.001). Furthermore, treatment of wild-type mice with CBX led to a marked suppression of F4/80 transfer in vivo, similar to that observed in vitro, suggesting that GAP junctions are responsible for the direct transfer of cellular content from BM MΦ to HSCs. Our results thus identify a novel mechanism by which macrophages can assign HSC residence in BM. Manipulation of macrophage-mediated transfer may enhance the mobilizable HSC pool and provide a new method to improve HSC yields after mobilization. Disclosures Frenette: Cygnal Therapeutics: Equity Ownership; Ironwood Pharmaceuticals: Research Funding; Albert Einstein College of Medicine, Inc: Patents & Royalties; Pfizer: Consultancy.

Abstract LB-031: A novel model of prostate cancer suggests enzalutamide functions through the immune system to diminish metastatic growth

Abstract Purpose: There is tremendous need for improved prostate cancer models. The murine prostate is anatomically and developmentally different from the human prostate, and unlike the human prostate, does not form sporadic tumors, limiting relevance of genetically engineered model systems. Furthermore, engineered models lack the heterogeneity of human disease, rarely (if ever) establish metastatic growth, and tend to be driven in a contrived manner, not at all related to human disease or the human drivers of disease progression. Human xenografts represent alternative models, but they rely on tumor growth in an immunocompromised murine host, preventing the study of tumor-immune interactions and immunotherapy interventions. Consequently, translational progress in prostate cancer research is hampered by the lack of human-derived models that can recapitulate the natural history of the disease—from initiation to metastatic spread—that will respond appropriately to the standard of care hormonal therapies. Accordingly, we generated a prostate cancer xenograft model in a murine system with an intact human immune system to test the hypothesis that humanizing tumor-immune interactions would improve modeling of metastatic prostate cancer, and further-enable improved modeling of hormonal and immune therapies. Procedure: Male huNOG mice were produced at Taconic Biosciences by engrafting juvenile NOG mice with human CD34+ hematopoietic stem cells. These mice stably develop and maintain multiple human cell lineages, including functional human T-cells. HuNOG and NOG control mice were surgically castrated. One week following castration, castrated and intact control mice were injected subcutaneously with luciferase-transduced 22Rv1 human prostate cancer cells to assay organ-specific metastatic growth. After tumors reached 100mm3, half of the castrated mice were treated with enzalutamide, and then tumor growth was monitored to endpoint. At sacrifice, organs were ex-vivo analyzed for metastatic growth, tumor infiltrating lymphocytes, and splenic immune reconstitution. Results: Primary tumor size was not significantly altered across conditions; however, the extent and growth at the secondary sites differed markedly in castrate huNOG vs conventional NOG mice treated with enzalutamide. Furthermore, enzalutamide responses in huNOG and NOG mice were distinct, and associated with increased CD3+ T-cells within tumors of enzalutamide treated huNOG mice, and increased CD3+ T-cell activation, accessed by intracellular interferon-γ. These results illustrate, to the best of our knowledge, the first model of human prostate cancer with metastases to clinically relevant locations, an intact human immune system, that responds appropriately to standard-of-care hormonal therapies. Citation Format: Steven Kregel, Jae Eun Choi, Kristen Juckette, Brooke Mccollum, Stephanie Simko, Parth Desi, Yuanyuan Qiao, Paul A. Volden, Arul M. Chinnaiyan. A novel model of prostate cancer suggests enzalutamide functions through the immune system to diminish metastatic growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-031.

Novel monoclonal antibody therapeutics for metastatic castration resistant prostate cancer.

e14222 Background: Therapeutic potential of innate immunity comprising Natural killer cell based targets are beginning to unravel the complexity of immune responses. NK cells recognize and induce cytotoxicity of wide range of target cells, such as, tumor cells without prior antigen sensitization. In this study, we have studied Lectin-like transcript 1 (LLT1), a member of the C-type lectin super family, is expressed on target cells and various immune cells. LLT1 isoform 1, is known to interact with CD161, a critical receptor on NK cells. CD161 is expressed on most of human NK cells, NK-T cells, γδ T cells and so on. Tumors exploit the CD161- LLT1 interaction to evade host defense mechanism (“DO NOT KILL” signal); indicating LLT1 as an attractive immunotherapeutic strategy. Methods: Prostate cancer cell lines and other tumor cell lines were used to evaluate novel anti LLT1 antibodies for therapeutic potential - IFNγ production assays and tumor cell death assays were carried out. In vivo efficacy of these antibodies were established using PC3 xenograft in humanized mouse model (HuNOG-EXL). Results: Human androgen independent prostate cancer cell line, PC3 was studied for LLT1 expression and interactions with immune cells, to understand role of LLT1 in metastatic castration resistant prostate cancer (mCRPC). Overexpression of LLT1 on tumor cells was influenced by cytokines and various TLRs. Inhibition of CD161-LLT1 interaction with novel anti LLT1 antibodies leads to IFNγ production and consequent NK cell mediated cytotoxicity – hall mark of anti-tumor responses. Disruption of LLT1 - CD161 innate immunity axis with anti LLT1 antibody releases the break on NK cell cytotoxicity and hence, established a new therapeutic option. PC3 xenograft on HuNOG mouse revealed in vivo efficacy of LLT1 antibody. Significant tumor growth reduction was observed with specific anti LLT1 antibodies alone and in combination with check point antibodies. Thus, synergistic tumor growth reduction was established by combinatorial application of anti LLT1 antibody and PD1/PDL1 axis inhibitors. Conclusions: PC3 xenograft study and other results point to therapeutic opportunities in metastatic castration resistant prostate cancer, a disease condition which needs improved patient outcomes. The ligation of CD161/LLT1 will serve as a new immuno-oncology pair regulating innate and adaptive immune responses; novel human antibodies against LLT1 described here will bring therapeutic benefit to patients in need.

Human Immune System Increases Breast Cancer-Induced Osteoblastic Bone Growth in a Humanized Mouse Model without Affecting Normal Bone.

Bone metastases are prevalent in many common cancers such as breast, prostate, and lung cancers, and novel therapies for treating bone metastases are needed. Human immune system-engrafted models are used in immuno-oncology (IO) studies for subcutaneous cancer cell or patient-derived xenograft implantations that mimic primary tumor growth. Novel efficacy models for IO compounds on bone metastases need to be established. The study was performed using CIEA NOG (NOG) mice engrafted with human CD34+ hematopoietic stem cells (huNOG) and age-matched immunodeficient NOG mice. Bone phenotyping was performed to evaluate baseline differences. BT-474 human breast cancer cells were inoculated into the tibia bone marrow, and cancer-induced bone changes were monitored by X-ray imaging. Bone content and volume were analyzed by dual X-ray absorptiometry and microcomputed tomography. Tumor-infiltrating lymphocytes (TILs) and the expression of immune checkpoint markers were analyzed by immunohistochemistry. Bone phenotyping showed no differences in bone architecture or volume of the healthy bones in huNOG and NOG mice, but the bone marrow fat was absent in huNOG mice. Fibrotic areas were observed in the bone marrow of some huNOG mice. BT-474 tumors induced osteoblastic bone growth. Bone lesions appeared earlier and were larger, and bone mineral density was higher in huNOG mice. huNOG mice had a high number of human CD3-, CD4-, and CD8-positive T cells and CD20-positive B cells in immune-related organs. A low number of TILs and PD-1-positive cells and low PD-L1 expression were observed in the BT-474 tumors at the endpoint. This study reports characterization of the first breast cancer bone growth model in huNOG mice. BT-474 tumors represent a “cold” tumor with a low number of TILs. This model can be used for evaluating the efficacy of combination treatments of IO therapies with immune-stimulatory compounds or therapeutic approaches on bone metastatic breast cancer.

Combination of immunogenic oncolytic adenovirus ONCOS-102 with anti-PD-1 pembrolizumab exhibits synergistic antitumor effect in humanized A2058 melanoma huNOG mouse model

ABSTRACTMalignant melanoma is an aggressive type of skin cancer whose incidence is increasing globally. Although surgery is effective in early stage melanoma, patients with advanced melanoma only have a 20% 5-year survival rate. Hence, combinations of existing and new immunotherapy technologies and immunotherapeutic agents are being evaluated. ONCOS-102 is an oncolytic adenovirus armed with human GM-CSF and an Ad5/3 chimeric capsid. It has shown to be well tolerated in phase I study (NCT01598129) wherein it induced antitumor immunity, infiltration of CD8 + T cells to tumors, and up-regulation of PD-L1. We propose that ONCOS-102 could serve as an immunosensitizer in combination therapies with checkpoint inhibitors. In this preclinical study, we investigated the cytotoxicity of ONCOS-102 and pembrolizumab, an anti-PD-1 antibody, in four human melanoma cell lines, A375, A2058, SK-Mel-2 and SK-Mel-28. Humanized mice engrafted with A2058 melanoma cells showed significant tumor volume reduction after ONCOS-102 treatment. Combination of pembrolizumab with ONCOS-102 reduced tumor volume to an even greater extent, while pembrolizumab (200 µg, or 400 µg) did not show any therapeutic benefit by itself. Body weight loss, and metastasis were not significantly affected by any treatment. These data support the scientific rationale for the ongoing clinical study of combination therapy of ONCOS-102 and pembrolizumab for the treatment of melanoma (NCT03003676).

Abstract 3028: PET imaging with the bispecific 89Zr-antibody ERY974 targeting CD3 and glypican 3 in tumor-bearing mouse models

Abstract BACKGROUND: ERY974, a modified monoclonal IgG4 bispecific antibody directed against human CD3 on T cells and glypican 3 (GPC3) on tumor cell, is currently in phase I clinical trial. The oncofetal protein GPC3 is overexpressed in several tumor types. Radiolabeling ERY974 with positron emission tomography (PET) isotope zirconium-89 (89Zr) enables non-invasive molecular imaging of tumor targeting and whole-body distribution. We aimed to evaluate 89Zr-ERY974 tumor targeting and effect of T cells on tumor uptake in mouse models, including a humanized mouse model. METHODS: ERY974 and two control molecules namely bispecific CD3xkeyhole limpet hemocyanin (KLH) and KLHxKLH antibodies were radiolabeled with 89Zr. Studies were performed in immunodeficient NOD/Shi-SCID/IL-2Rgnull (NOG) as well as human CD34+ hematopoietic stem cell engrafted NOG mice (huNOG), all subcutaneously inoculated with GPC3 overexpressing human hepatocellular carcinoma HepG2 cells. Mice received 10 µg 89Zr-ERY974, 89Zr-CD3xKLH or 89Zr-KLHxKLH intravenously, with subsequent µPET scanning at 24, 72, 120 and 168 h followed by ex vivo biodistribution. Organs of interest were quantified on µPET scans as mean standardized uptake value (SUVmean) and with ex vivo biodistribution as % injected dose/gram of tissue (%ID/g). Tumor, spleen and lymph nodes were analyzed with autoradiography and immunohistochemical CD3 staining. RESULTS: µPET imaging revealed increased tumor-to-blood ratio (TBR) of 89Zr-ERY974 in NOG over time with maximal TBR of 2.2±0.3 at 168 h post tracer injection (pi). At 168 h, tumor uptake was specific as 89Zr-CD3xKLH and 89Zr-KLHxKLH showed a TBR of only 0.6±0.2 and 0.8±0.3, respectively. In huNOG mice human CD3+ T cells were present in tumor, spleen and lymph nodes. In huNOG mice tumor uptake of 89Zr-ERY974 was higher than in NOG mice as measured on µPET scans (SUVmean at 168 h pi 6.9±2.6 vs 2.9±0.2; P<0.01) and with ex vivo biodistribution (60.9±26.2 %ID/g vs 16.7±2.3 %ID/g; P<0.001), whereas 89Zr-CD3xKLH tumor uptake in both mouse models was lower (P<0.05) but were similar in these mouse models. Autoradiography 168 h following 89Zr-ERY974 administration to huNOG mice showed 89Zr in extensive T cell infiltrate areas in the tumors of huNOG mice, whereas T cell infiltrate was lower in tumors of 89Zr-CD3xKLH and 89Zr-KLHxKLH injected huNOG mice. Spleens of huNOG mice showed CD3+ specific uptake as 89Zr-ERY974 and 89Zr-CD3xKLH uptake were higher than 89Zr-KLHxKLH uptake(P<0.05), whereas spleen uptake in NOG mice of the 3 tracers was similar. Moreover, in huNOG CD3+ mesenteric lymph nodes 89Zr-ERY974 uptake was higher than 89Zr-KLHxKLH uptake (P<0.05) CONCLUSION: 89Zr-ERY974 demonstrates specific tumor uptake in NOG and huNOG mice, while in huNOG mice tumor uptake colocalized with T cell rich infiltrate and also uptake in in spleen and lymph nodes was observed. Citation Format: Stijn J. Waaijer, Danique Giesen, Takahiro Ishiguro, Yuji Sano, Norihisa Ohishi, Athos Gianella-Borradori, Carolien P. Schröder, Elisabeth G. de Vries, Marjolijn N. Lub-de Hooge. PET imaging with the bispecific 89Zr-antibody ERY974 targeting CD3 and glypican 3 in tumor-bearing mouse 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 3028.

Abstract 1709: Enhanced proliferation and effector functions of human immune cells in response to immune checkpoint therapy of MDA-MB-231 tumor bearing huNOG mice engrafted with human CD34+ cells

Abstract Given the recent success of cancer immunotherapies, well characterized pre-clinical models are needed to enable drug discovery efforts. The recently developed humanized mouse models composed of various types of immune cells offer a unique tool to evaluate aspects of the human immune system in response to immunotherapy. MDA-MB231 cells express high levels of the T-inhibitory molecule PD-L1 making this breast cancer line an ideal candidate for testing the efficacy of the immune-checkpoint inhibitors pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4) in the CD34+ huNOG humanized mouse strain. We analyzed the distribution of CD4 and CD8 T lymphocytes, Treg cells, NK cells and B cells in blood, spleen and tumor samples from MDA-MB231 tumor bearing huNOG humanized treated-mice via flow cytometry. We found that combination therapy enhanced the effector functions of CD4+ and CD8+ T cells associated with increased expression of IFN-gamma and Granzyme B. Supporting this data, we observed an increase in Ki-67+ cells which correlates with a decrease in PD-1 expression in response to pembrolizumab therapy associated with activation of T lymphocytes. In summary, these results demonstrate the potential of humanized mouse models to evaluate the anti-tumor activity of human immune cells in a preclinical setting in response to human specific immune checkpoint targeted therapies. Citation Format: Thi Bui, Karyn McArtan Shinn, Emily G. O'Koren, Ashleigh Derrick, Beverly Godfrey, Ian Belle, Jason Davis, Aidan Synnott, Anya Avrutskaya, Paula L. Miliani De Marval. Enhanced proliferation and effector functions of human immune cells in response to immune checkpoint therapy of MDA-MB-231 tumor bearing huNOG mice engrafted with human CD34+ cells [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 1709.

Abstract 1677: Differential efficacy of PD-1 targeted immunomodulation in preclinical models of primary and bone metastatic triple-negative breast cancer

Abstract Immuno-oncology (IO) has provided groundbreaking results in cancer treatment. Triple-negative breast cancer (TNBC) tumors attract immune cells, and the presence of tumor-infiltrating lymphocytes (TILs) is linked to improved survival. Programmed cell death 1 (PD-1) is expressed by TILs and its ligand (PD-L1) by TNBC cells, and targeting of PD-1 has shown promising results in treatment of patients with primary TNBC. High frequency of bone metastases is typical in TNBC patients. As immune regulation is different in bone than in other organs it is essential to study the efficacy of IO therapies in bone microenvironment. The aim of this study was to assess the efficacy of anti-PD-1 therapy (pembrolizumab, Keytruda) in the growth of primary and bone metastatic TNBC in preclinical models. MDA-MB-231(SA)-luc human TNBC cells were inoculated into the mammary fat pad (orthotopic model) or tibia bone marrow (bone model) of female huNOG mice engrafted with CD34+ cells from two different donors. Treatments with pembrolizumab or human IgG4 isotype control (5 mg/kg, i.p., Q5D, n=8) were started 3 days after the inoculations. Tumor growth was monitored by bioluminescence imaging (BLI), orthotopic tumor volume by caliper, and tumor induced changes in bone by X-ray imaging for 21-24 days. Tumor samples were processed to immunohistochemical (IHC) analysis of TILs, PD-1 and PD-L1. Bone volume was analyzed ex vivo by micro-computed tomography, and serum TRACP5b values were determined as a marker of osteoclast number. Tumor growth at treatment start was confirmed by BLI. Pembrolizumab decreased tumor growth in the orthotopic model. 37.5% of the mice had partial response and 12.5% had tumor rejection. The mice exhibited donor-related differences in efficacy but the overall response was similar. IHC showed low to moderate PD-L1 expression in the TNBC tumors (tumor proportion score 1-49%) and moderate number of CD4+ and CD8+ TILs. No PD-1 expression was observed in pembrolizumab treated mice due to antibody blocking of the epitope. In the bone model, pembrolizumab had no effect in tumor growth or any bone parameters studied. PD-L1 expression was comparable in orthotopic and bone tumors. In bone tumors, CD4+ cells were dominant and CD8+ and PD-1+ cells were rarely observed. In orthotopic model the response rate was similar to what has been observed in TNBC patients, but the bone metastatic growth could not be inhibited by the PD-1 blockage. Bone marrow has a unique microenvironment and immune cell compartment compared to any other organ, and it is known to be involved in immunosurveillance and to favour tumor cells to become immune evasive. The lack of efficacy of immunotherapy in bone compared to orthotopic tumor may be due to these differences. These results highlight the importance of using both orthotopic and metastasis models in preclinical oncology studies. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Teppo Haapaniemi, Azusa Tanaka, Michael Seiler, Jenni Bernoulli. Differential efficacy of PD-1 targeted immunomodulation in preclinical models of primary and bone metastatic triple-negative breast cancer [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 1677.