Cell Checkpoint Inhibitors Research Articles

TET2 promotes tumor antigen presentation and T cell IFN-γ which is enhanced by vitamin C.

Immune evasion by tumors is promoted by low T cell infiltration, ineffective T cell activity directed against the tumor and reduced tumor antigen presentation. The TET2 DNA dioxygenase gene is frequently mutated in hematopoietic malignancies and loss of TET enzymatic activity is found in a variety of solid tumors. We showed previously that vitamin C (VC), a co-factor of TET2, enhances tumor-associated T cell recruitment and checkpoint inhibitor therapy responses in a TET2-dependent manner. Using single-cell RNA sequencing (scRNA-seq) analysis performed on B16-OVA melanoma tumors, we have shown here that an additional function for TET2 in tumors is to promote expression of certain antigen presentation machinery genes, which is potently enhanced by VC. Consistently, VC promoted antigen presentation in cell-based and tumor assays in a TET2-dependent manner. Quantifying intercellular signaling from the scRNA-seq dataset showed that T cell-derived IFNγ-induced signaling within the tumor and tumor microenvironment requires tumor-associated TET2 expression which is enhanced by VC treatment. Analysis of patient tumor samples indicated that TET activity directly correlates with antigen-presentation gene expression and with patient outcomes. Our results demonstrate the importance of tumor-associated TET2 activity as a critical mediator of tumor immunity which is augmented by high-dose VC therapy.

Revolutionizing Cancer Treatment: Recent Advances in Immunotherapy.

Cancer immunotherapy has emerged as a transformative approach in oncology, utilizing the body's immune system to specifically target and destroy malignant cells. This review explores the scope and impact of various immunotherapeutic strategies, including monoclonal antibodies, chimeric antigen receptor (CAR)-T cell therapy, checkpoint inhibitors, cytokine therapy, and therapeutic vaccines. Monoclonal antibodies, such as Rituximab and Trastuzumab, have revolutionized treatment paradigms for lymphoma and breast cancer by offering targeted interventions that reduce off-target effects. CAR-T cell therapy presents a potentially curative option for refractory hematologic malignancies, although challenges remain in effectively treating solid tumors. Checkpoint inhibitors have redefined the management of cancers like melanoma and lung cancer; however, managing immune-related adverse events and ensuring durable responses are critical areas of focus. Cytokine therapy continues to play a vital role in modulating the immune response, with advancements in cytokine engineering improving specificity and reducing systemic toxicity. Therapeutic vaccines, particularly mRNA-based vaccines, represent a frontier in personalized cancer treatment, aiming to generate robust, long-lasting immune responses against tumor-specific antigens. Despite these advancements, the field faces significant challenges, including immune resistance, tumor heterogeneity, and the immunosuppressive tumor microenvironment. Future research should address these obstacles through emerging technologies, such as next-generation antibodies, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-based gene editing, and AI-driven drug discovery. By integrating these novel approaches, cancer immunotherapy holds the promise of offering more durable, less toxic, and highly personalized treatment options, ultimately improving patient outcomes and survival rates.

ADVANCEMENTS IN CANCER PHARMACOTHERAPY: TARGETED THERAPIES AND IMMUNOTHERAPY STRATEGIES, A REVIEW

The development of immunotherapy and targeted medicines has fundamentally changed and revolutionized the field of cancer treatment. These cutting-edge methods, which include cancer vaccines, CAR-T cell therapy, checkpoint inhibitors, monoclonal antibodies, and small molecule inhibitors, are not only therapeutic interventions but also revolutionary instruments. They are carefully crafted to interfere with particular molecular pathways that propel tumors' growth and spread and use patients' immune systems to fight cancerous cells. This research aims to thoroughly investigate the mechanisms, clinical uses, efficacy, obstacles, current developments, and prospects of these noteworthy achievements in cancer pharmacotherapy. The paradigm changes in cancer, combining immunotherapy and targeted medicines, provide individualized treatment plans that consider each patient's particular needs, offering a sense of reassurance and confidence in the treatment process.

Practical management of renal cell carcinoma: integrating current approaches with advances in bone metastasis treatment.

Renal cell carcinoma (RCC) is a common type of tumor that can develop in the kidney. It is responsible for around one-third of all cases of neoplasms. RCC manifests itself in a variety of distinct subtypes. The most frequent of which is clear cell RCC, followed by papillary and chromophobe RCC. RCC has the potential for metastasis to a variety of organs; nevertheless, bone metastases are one of the most common and potentially fatal complications. These bone metastases are characterized by osteolytic lesions that can result in pathological fractures, hypercalcemia, and other complications, which can ultimately lead to a deterioration in quality of life and an increase morbidity. While nephrectomy remains a foundational treatment for RCC, emerging evidence suggests that targeted therapies, including tyrosine kinase inhibitors and T cell checkpoint inhibitors, may offer effective alternatives, potentially obviating the need for adjuvant nephrectomy in certain cases of metastatic RCC Bone metastases continue to be a difficult complication of RCC, which is why more research is required to enhance patient outcome.

Abstract SoA1-03: Current overview of immunotherapy in breast cancer

Abstract T cell checkpoint inhibitors are now standard of care for patients with triple negative breast cancer. Currently these agents are combined with combination cytotoxic chemotherapy. In this talk we will cover the composition of the immune infiltrate, rationale for T cell therapies, more or less chemotherapy and biomarkers of response and resistance. Citation Format: S. Loi. Current overview of immunotherapy in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr SoA1-03.

Revolutionizing cancer treatment: comprehensive insights into immunotherapeutic strategies.

Cancer, characterized by the uncontrolled proliferation of aberrant cells, underscores the imperative for innovative therapeutic approaches. Immunotherapy has emerged as a pivotal constituent in cancer treatment, offering improved prognostic outcomes for a substantial patient cohort. Noteworthy for its precision, immunotherapy encompasses strategies such as adoptive cell therapy and checkpoint inhibitors, orchestrating the immune system to recognize and selectively target malignant cells. Exploiting the specificity of the immune response renders immunotherapy efficacious, as it selectively targets the body's immune milieu. Diverse mechanisms underlie cancer immunotherapies, leading to distinct toxicity profiles compared to conventional treatments. A remarkable clinical stride in the anticancer resources is immunotherapy. Remarkably, certain recalcitrant cancers like skin malignancies exhibit resistance to radiation or chemotherapy, yet respond favorably to immunotherapeutic interventions. Notably, combination therapies involving chemotherapy and immunotherapy have exhibited synergistic effects, enhancing overall therapeutic efficacy. Understanding the pivotal role of immunotherapy elucidates its complementary value, bolstering the therapeutic landscape. In this review, we elucidate the taxonomy of cancer immunotherapy, encompassing adoptive cell therapy and checkpoint inhibitors, while scrutinizing their distinct adverse event profiles. Furthermore, we expound on the unprecedented potential of immunogenic vaccines to bolster the anticancer immune response. This comprehensive analysis underscores the significance of immunotherapy in modern oncology, unveiling novel prospects for tailored therapeutic regimens.

Structural basis for the activity and specificity of the immune checkpoint inhibitor lirilumab

The clinical success of immune checkpoint inhibitors has underscored the key role of the immune system in controlling cancer. Current FDA-approved immune checkpoint inhibitors target the regulatory receptor pathways of cytotoxic T-cells to enhance their anticancer responses. Despite an abundance of evidence that natural killer (NK) cells can also mediate potent anticancer activities, there are no FDA-approved inhibitors targeting NK cell specific checkpoint pathways. Lirilumab, the most clinically advanced NK cell checkpoint inhibitor, targets inhibitory killer immunoglobulin-like receptors (KIRs), however it has yet to conclusively demonstrate clinical efficacy. Here we describe the crystal structure of lirilumab in complex with the inhibitory KIR2DL3, revealing the precise epitope of lirilumab and the molecular mechanisms underlying KIR checkpoint blockade. Notably, the epitope includes several key amino acids that vary across the human population, and binding studies demonstrate the importance of these amino acids for lirilumab binding. These studies reveal how KIR variations in patients could influence the clinical efficacy of lirilumab and reveal general concepts for the development of immune checkpoint inhibitors targeting NK cells.

Existing methods to improve immunotherapy's limitations: CAR-T cell therapy and checkpoint inhibitors

Immunotherapy is a form of treatment for cancer that works by provoking the immune system of the body into tracking down and destroying cancer cells. CAR-T-CT is a treatment that makes use of CAR-T cells (CARs), which are T cells (T-Cs) that have been modified in some way by the incorporation of engineered synthetic receptors. This therapy is designed to destroy cells that express a specific target antigen, and its primary objective is to do so by locating and killing those cells. Due to the fact that Immune CPI inhibits the production of specific proteins (checkpoint protein) on immune cells as well as on cancer cells thereby preventing the immune system from combating cancer, Immune CPI are used to treat cancer. By inhibiting the activity of these proteins, CPI is able to contribute to the process of stimulating the immune system to specifically target and destroy cancer cells. However, there are still some limitations that exist, and these have the potential to reduce the effectiveness of the treatment or produce some side effects that are life-threatening. This article makes a few suggestions for innovative steps that could be taken in order to both improve the current state of affairs and find answers to any problems that may arise in the near or distant future. The article makes these suggestions in order to improve the current state of affairs and find answers to any problems that may arise in the future.

CAR-modified immune cells as a rapidly evolving approach in the context of cancer immunotherapies

Nowadays, one of the main challenges clinicians face is malignancies. Through the progression of technology in recent years, tumor nature and tumor microenvironment (TME) can be better understood. Because of immune system involvement in tumorigenesis and immune cell dysfunction in the tumor microenvironment, clinicians encounter significant challenges in patient treatment and normal function recovery. The tumor microenvironment can stop the development of tumor antigen-specific helper and cytotoxic T cells in the tumor invasion process. Tumors stimulate the production of proinflammatory and immunosuppressive factors and cells that inhibit immune responses. Despite the more successful outcomes, the current cancer therapeutic approaches, including surgery, chemotherapy, and radiotherapy, have not been effectiveenough for tumor eradication. Hence, developing new treatment strategies such as monoclonal antibodies, adaptive cell therapies, cancer vaccines, checkpoint inhibitors, and cytokines helps improve cancer treatment. Among adoptive cell therapies, theinteraction between the immune system and malignancies and using molecular biology led tothe development of chimeric antigen receptor (CAR) T cell therapy. CAR-modified immune cells are one of the modern cancer therapeutic methods with encouraging outcomes in most hematological and solid cancers. The current study aimed to discuss the structure, formation, subtypes, and application of CAR immune cells in hematologic malignancies and solid tumors.

Abstract 6354: A new tumor-bearing humanized mouse model to evaluate the efficacy of bispecific T cell engager and monoclonal checkpoint antibodies

Abstract T cell-based immunotherapies such as bispecific T- cell engagers (BiTE) and checkpoint inhibitors (CIs) have been developed rapidly for cancer treatment, but each has their own limitations in the clinic. Combination therapies are an emerging approach in ongoing clinical trials to enhance the drug efficacy of monotherapy. Here we described a new preclinical animal model using immunodeficient NSG™-SGM3-IL15 mice engrafted with human CD34+ hematopoietic stem cells (HSC) to evaluate the combination treatment of EGFRXCD3 BiTE and two CIs (anti-PD-1 pembrolizumab and anti-PD-L1 avelumab). NSG™-SGM3-IL15 mice, which were created by crossing NSG™-SGM3 with NSG™-IL15, develop a more complete humane immune system after HSC engraftment. Humanized NSG™-IL15 mice have a higher peripheral NK cell frequency, and NK cells are functional with ADCC activity when compared to NSG™ (Aryee et al., 2022 FASEB). Here we show that humanized NSG™-SGM3-IL15 mice show improved human CD33+ myeloid progenitor cells and near physiological levels of NK cells and Tregs in peripheral blood when compared to humanized NSG™-SGM3. Triple negative breast cancer MDA-MB-453 cell line is PD-L1 positive, expresses low levels of EGFR and has a low baseline tumor immune cell infiltration in xenograft using humanized NSG™ mice. Previously we have shown BiTE efficacy on this tumor model. Here tumor-bearing humanized NSG™-SGM3-IL15 mice from two donors were used to evaluate the efficacy and immune responses from the BiTE treatment in combination with CIs. For donor 0348, monotherapy BiTE and avelumab didn’t result in significant tumor growth reduction when compared to PBS control whereas the combination treatment significantly reduced tumor growth. BiTE treatment alone resulted in a significant increase in CD3+ T cell infiltration into tumor and higher frequency of CD69+ CD4 T and CD8 T cells. PD-1 expression on the tumor infiltrating lymphocytes and PD-L1 expression on tumor cells increased significantly. BiTE and avelumab combination treatment further increased human CD45+ and CD3+ T cell infiltrations with activated T cell phenotypes in tumor. For the second donor 0313, BiTE alone or in combination with the CIs didn’t show anti-tumor growth activity. Flow cytometry analysis showed that the immune response to BiTE treatment was less pronounced in this donor, especially there were little change in lymphocyte infiltration and CD4+ T cell population in tumor. In this study, we demonstrated donor-dependent response to combination treatment of BiTE and avelumab and found that the responding donor showed an increase in T cell infiltration and activation in tumor after the treatment. The data suggested beneficial effects from inhibition of PD-1/PD-L1 axis when used in combination with an immunotherapy targeting tumor antigen. The potential involvement of NK cells in this combination therapy remained to be examined. Citation Format: Li-Chin Yao, Danying Cai, Destanie Rose, Guoxiang Yang, Mingshan Cheng, Michael A. Brehm, Dale L. Greiner, Leonard D. Shultz, James G. Keck. A new tumor-bearing humanized mouse model to evaluate the efficacy of bispecific T cell engager and monoclonal checkpoint antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6354.

Recent advances in understanding and managing pituitary adenomas.

Pituitary adenomas (PAs) are common intracranial tumors. Despite their benign nature, PAs may cause a significant burden of disease, leading to either hormonal disturbances or local compression. A subset of PAs presents an aggressive behavior that remains difficult to predict, and in rare cases they metastasize. Therefore, early diagnosis and treatment are important. Advances in molecular pathology have improved the understanding of their pathogenesis and offer opportunities to identify and target novel pathways. Improved imaging and functional molecular techniques precisely detect even very small tumors and guide targeted treatment. Transsphenoidal surgery is the first-line treatment for the majority of PAs, and advances in the field of endoscopic neurosurgery offer excellent outcomes. Dopamine agonists (DAs) are traditionally the first-line treatment for prolactinomas. For patients with acromegaly, first- and second-generation somatostatin analogues (SSAs) are applied when surgery is not successful or not indicated. For Cushing's disease (CD), drugs targeting adrenal steroidogenesis, somatostatin receptors in the pituitary, and glucocorticoid receptors are used to treat hypercortisolism in patients with persistent or recurrent CD, for those who are not good surgical candidates, and as a bridge treatment for those who have undergone radiation treatment until cortisol levels are controlled. Temozolomide (TMZ) is the first-line chemotherapy for aggressive PAs, but new experimental therapies, like the anti-vascular endothelial growth factor (anti-VEGF) therapy, mechanistic target of rapamycin (mTOR) inhibitors, tyrosine kinase inhibitors, and cell cycle and checkpoint inhibitors, are now available. Radiotherapy is offered to patients with residual, recurrent, or progressive tumors. Modern techniques in radiotherapy planning and delivery are able to deliver high doses to the target tissue while sparing vital structures. As we familiarize ourselves with the biological behavior of PAs and our therapeutic armamentarium expands, the next goal is to tailor and personalize treatment to each individual patient so as to achieve the best outcome.

Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies.

Hepato-pancreatico-biliary (HPB) malignancies are difficult-to-treat and continue to to have a high mortality and significant therapeutic resistance to standard therapies. Immune oncology (IO) therapies have demonstrated efficacy in several solid malignancies when combined with chemotherapy, whereas response rates in pancreatic ductal adenocarcinoma (PDA) are poor. While promising in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), there remains an unmet need to fully leverage IO therapies to treat HPB tumors. We therefore defined T cell subsets in the tumor microenvironment of HPB patients utilizing a novel, multiparameter flow cytometry and bioinformatics analysis. Our findings quantify the T cell phenotypic states in relation to checkpoint receptor expression. We demonstrate the presence of CD103+ tissue resident memory T cells (TRM), CCR7+ central memory T cells, and CD57+ terminally differentiated effector cells across all HPB cancers, while the anti-tumor function was dampened by expression of multiple co-inhibitory checkpoint receptors. Terminally exhausted T cells lacking co-stimulatory receptors were more prevalent in PDA, whereas partially exhausted T cells expressing both co-inhibitory and co-stimulatory receptors were most prevalent in HCC, especially in early stage. HCC patients had significantly higher TRM with a phenotype that could confer restored activation in response to immune checkpoint therapies. Further, we found a lack of robust alteration in T cell activation state or checkpoint expression in response to chemotherapy in PDA patients. These results support that HCC patients might benefit most from combined checkpoint therapies, whereas efforts other than cytotoxic chemotherapy will likely be necessary to increase overall T cell activation in CCA and PDA for future clinical development.

PD-1 and TIM-3 differentially regulate subsets of mouse IL-17A-producing γδ T cells.

IL-17A-producing γδ T cells in mice consist primarily of Vγ6+ tissue-resident cells and Vγ4+ circulating cells. How these γδ T cell subsets are regulated during homeostasis and cancer remains poorly understood. Using single-cell RNA sequencing and flow cytommetry, we show that lung Vγ4+ and Vγ6+ cells from tumor-free and tumor-bearing mice express contrasting cell surface molecules as well as distinct co-inhibitory molecules, which function to suppress their expansion. Vγ6+ cells express constitutively high levels of PD-1, whereas Vγ4+ cells upregulate TIM-3 in response to tumor-derived IL-1β and IL-23. Inhibition of either PD-1 or TIM-3 in mammary tumor-bearing mice increased Vγ6+ and Vγ4+ cell numbers, respectively. We found that genetic deletion of γδ T cells elicits responsiveness to anti-PD-1 and anti-TIM-3 immunotherapy in a mammary tumor model that is refractory to T cell checkpoint inhibitors, indicating that IL-17A-producing γδ T cells instigate resistance to immunotherapy. Together, these data demonstrate how lung IL-17A-producing γδ T cell subsets are differentially controlled by PD-1 and TIM-3 in steady-state and cancer.

SHP-2 Regulates Myeloid Cell Differentiation, Anti-Tumor Responses and Innate Immune Memory

PD-1 is a T cell checkpoint inhibitor and the most extensively exploited therapeutic target of cancer immunotherapy. PD-1-mediated T cell inactivation is attributed to the function of SHP-2 phosphatase, which is activated by recruitment to PD-1 cytoplasmic tail. However, T cell-specific SHP-2 deletion did not improve anti-tumor immunity or altered responses to anti-PD-1 immunotherapy. Previously, we determined that PD-1 is also expressed in common myeloid progenitors and granulocyte/macrophage progenitors, which accumulate during cancer-driven emergency myelopoiesis and give rise to immunosuppressive myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs). In tumor-bearing mice with myeloid-specific PD-1 ablation, accumulation of MDSCs was prevented, while output of differentiated effector myeloid cells with monocytic lineage dominance was increased. However, the molecular mechanism remains elusive. It is also unknown whether a canonical PD-1:SHP-2 axis is operative in myeloid cells. Temporal activation of SHP-2 is critical for myeloid cell fate. Gain of function SHP-2 mutations, resulting in constitutive phosphatase activation, prevent myeloid differentiation and lead to the accumulation of immature myelocytes and development of leukemia. HOXA proteins, members of the HOX family of transcription factors involved in normal hematopoiesis, are targets of SHP-2, which dephosphorylates HOXA and prevents derepressing of HOXA target genes and myeloid cell differentiation. In addition, SHP-2 regulates the function of IFN regulatory factor (IRF)-8, a transcription factor that has a decisive role in myeloid progenitor differentiation toward monocytes and DCs. IRF8 phosphorylation is mandatory for its nuclear translocation and initiation of gene transcription. The cytoplasmic-nuclear shuttling of IRF8 is regulated by SHP-2, which dephosphorylates IRF8 and prevents its nuclear localization and transcriptional activation. We generated mice with conditional targeting of the Ptpn11 gene (encoding for Shp-2) in T cells (Shp2f/fLckCre) or myeloid cells (Shp2f/fLysMCre), and found that Shp2f/fLysMCre but not Shp2f/fLckCre mice had diminished tumor growth. As determined by RNA-sequencing, this was paralleled by the presence of inflammatory neutrophils and tumor-associated macrophages (TAMs) with molecular signatures of enhanced differentiation, phagocytosis and antigen-processing and presentation. SHP-2 deficient TAMs also had increased monocyte and dendritic cells (DCs) specification transcription factors, chemokine and cytokine production, and expression of immunostimulatory molecules that promote T cell recruitment and activation. Moreover, monocytes from tumor-bearing Shp2f/fLysMCre mice suppressed tumor growth after transfer to naïve recipients indicating development of innate immune memory, named trained immunity. In bone marrow myelocytes, SHP-2 deletion enhanced GM-CSF-mediated phosphorylation of the transcription factors HOXA10 and IRF8 that promote myeloid differentiation and monocytic/moDC lineage commitment, respectively. In contrast, in WT bone marrow myelocytes GM-CSF induced PD-1 expression, phosphorylation and interaction with SHP-2, the Src family kinase Lyn, and GM-CSF receptor beta chain, indicating that the PD-1:SHP-2 axis targets a key pathway of myelocyte differentiation. Our results reveal a previously unidentified mechanistic role of SHP-2 and the PD-1:SHP-2 axis in regulating myeloid cell differentiation in the context of cancer and provide evidence that SHP-2 poses a signaling restrain to myeloid differentiation and monocyte lineage commitment resulting in a myeloid landscape that suppresses anti-tumor immunity. This process is hijacked by the PD-1/PD-L1 pathway.

409P Tertiary lymphoid structures are scarce but associated with BCR clonal expansion, B cell activity and checkpoint inhibitor response in advanced osteosarcoma

Tertiary lymphoid structures (TLS) have been associated with tumor response to checkpoint inhibitors (ICIs) in soft tissue sarcoma as well as several epithelial cancers. However, its role in bone sarcoma has not been thoroughly investigated to date.

Expression of nectin-4 in papillary renal cell carcinoma

BackgroundNectin-4 contributes to tumor proliferation, lymphangiogenesis and angiogenesis in malignant tumors and is an emerging target in tumor therapy. In renal cell carcinoma (RCC) VEGF-directed tyrosine kinase inhibitors and checkpoint inhibitors are currently treatments of choice. Enfortumab vedotin-ejf (EV) is an antibody drug conjugate that targets Nectin-4. The aim of our study was to investigate the expression of Nectin-4 in a large cohort of papillary RCC specimens.Patients and methodsSpecimens were derived from the PANZAR consortium (Erlangen, Heidelberg, Herne, Homburg, Mainz, Mannheim, Marburg, Muenster, LMU Munich, TU Munich, and Regensburg). Clinical data and tissue samples from n = 190 and n = 107 patients with type 1 and 2 pRCC, respectively, were available. Expression of Nectin-4 was determined by immunohistochemistry (IHC).ResultsIn total, Nectin-4 staining was moderately or strongly positive in of 92 (48.4%) of type 1 and 39 (36.4%) type 2 of pRCC cases. No associations between Nectin-4 expression and age at diagnosis, gender, grading, and TNM stage was found. 5 year overall survival rate was not statistically different in patients with Nectin-4 negative versus Nectin-4 positive tumors for the overall cohort and the pRCC type 2 subgroup, but higher in patient with Nectin-4 positive pRCC type 1 tumors compared to Nectin-4 negative tumors (81.3% vs. 67.8%, p = 0.042).ConclusionNectin-4 could not be confirmed as a prognostic marker in pRCC in general. Due to its high abundance on pRCC specimens Nectin-4 is an interesting target for therapeutical approaches e.g. with EV. Clinical trials are warranted to elucidate its role in the pRCC treatment landscape.

Abstract 1650: Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune cell engagers

Abstract Background The preclinical evaluation of novel immune therapies demands humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By transplantation of cell-line-derived (CDX) or patient-derived (PDX) tumor xenografts on humanized mice, we successfully generated a full human tumor-immune-cell model for different tumor entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies and immune cell engagers. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+stem cells to immunodeficient NOG mice. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities (i.e. lymphoma, neuroblastoma, and breast cancer) were transplanted on those humanized mice. These models were used to evaluate novel immune therapies. Blood and tumor samples were analysed by FACS for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. 14 weeks after HSC inoculation up to 20% of the human immune cells in the blood were functional T-cells, characterized by a high PD-1 expression. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Checkpoint inhibitor treatments induced tumor growth delay in selected models. FACS analysis of tumors revealed an increased percentage of tumor infiltrating T-cells. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies. Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been employed for preclinical evaluation of novel checkpoint inhibitors, cell therapies and immune cell engagers. Our models allow preclinical, translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Wolfgang Walther, Jens Hoffmann. Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune cell engagers [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 1650.

Abstract 601: IO-108, A fully human therapeutic antibody blocking the myeloid checkpoint LILRB2/ILT4, promotes innate and adaptive anti-cancer immunity in preclinical studies

Abstract Tumor-associated myeloid cells inhibit anti-cancer immune responses systemically and in the tumor microenvironment (TME), thereby limiting the efficacy of T cell checkpoint inhibitors. However, the plasticity of myeloid cells may enable therapeutic intervention. The immune inhibitory receptor LILRB2 (also known as ILT4) is expressed primarily by myeloid cells (monocytes, macrophages, dendritic cells and neutrophils), and has emerged as a key myeloid checkpoint contributing to the tolerogenic activity of myeloid cells associated with cancer. LILRB2 has several ligands (classical and non-classical MHC-I, ANGPTL2/5, SEMA4A and CD1) and most of these are known to contribute to immune suppression in the TME. Using computational biology approaches, we found that high LILRB2 expression is associated with macrophage infiltration in many solid tumor types from TCGA. Therefore, LILRB2 is a compelling target to overcome immune suppressive activity of cancer-associated myeloid cells. IO-108 is a fully human IgG4 therapeutic antibody that binds to LILRB2 with high affinity and specificity. IO-108 binds to all myeloid cells in the solid TME and periphery. In vitro studies support that blockade of LILRB2 interaction with its various ligands is the anticipated mechanism of action of IO-108. The LILRB2 antagonist activity of IO-108 produces the desired pro-inflammatory (re)polarization of myeloid cells. IO-108 treatment results in increased pro-inflammatory responses and an enhanced antigen-presenting cell (APC) phenotype to multiple stimuli (e.g., T cell activators, STING and TLR agonists) in ex vivo assays. In addition, IO-108 precludes the anti-inflammatory myeloid cell phenotype resulting from “tumor conditioning” and promotes the differentiation of monocytes and immature dendritic cells (DC) into pro-inflammatory DC. IO-108 enhances the effect of PD-1 blocking antibodies in allogeneic mixed leukocyte reactions of CD4+ T cells and macrophages. Moreover, IO-108 polarized primary myeloid cells isolated from solid tumor patient blood and ovarian cancer-associated ascites towards a proinflammatory phenotype and attenuated their suppressive effect on autologous T cell proliferation and production of proinflammatory cytokines. In vivo, IO-108 inhibits tumor growth in mouse models, which is associated with immune cell activation. Importantly, IO-108 presents a favorable pharmacokinetic and safety profile in preclinical models. Collectively, the preclinical characterization of IO-108 enabled a comprehensive clinical biomarker plan and lends rationale to the clinical study (NCT05054348) of IO-108 as a novel immunotherapy for multiple solid tumor types, including those relapsed/refractory to standard of care therapies. Citation Format: Jing-Tyan Ma, Xiaoye Liu, Ryan Stafford, Krista McCutcheon, Heyu Chen, Tao Huang, Hongyu Tian, Kyu Hong, ChengCheng Zhang, Luke Chung, An Song, Paul Woodard, XCharlene Liao, MariaJose Costa. IO-108, A fully human therapeutic antibody blocking the myeloid checkpoint LILRB2/ILT4, promotes innate and adaptive anti-cancer immunity in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 601.

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

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

Abstract 2162: Inhibition of GPR65 counteracts low pH induced immunosuppressive polarization of macrophages: In vitro and in vivo characterization of potent, selective and orally bioavailable small molecule GPR65 antagonists

Abstract The acidic tumour microenvironment (TME) and the abundance of tumour associated macrophages (TAMs) are key features of solid tumours that drive immune suppression, support tumour growth and limit the efficacy of approved therapies. We identified the pH sensing GPCR, GPR65, as a key determinant of low pH induced immune suppression in human cancers, particularly in TAMs, based on the following observations: 1) cancer patients who are homozygous for a hypomorphic coding variant in GPR65 (I231L) show a statistically significant increase in survival and altered expression of key immune system genes compared to other genotypes; 2) single cell RNA sequencing (scRNAseq) data from multiple solid tumors show that GPR65 and downstream pathway genes are ubiquitously expressed in myeloid and other innate immune cells in human cancers; and 3) low pH acting via GPR65 profoundly alters gene expression in human macrophages in vitro, bringing about a pronounced suppression of proinflammatory cytokines and a marked upregulation of tissue repair genes. These findings identify GPR65 as a novel innate immune check point, and suggest that GPR65 inhibition could be highly beneficial in cancer. Indeed, previous work has shown that genetic ablation of the GPR65 signaling pathway in B16.F10 tumour bearing mice upregulates immunostimulatory genes in TAMs and significantly reduces tumor growth1. Pathios has identified potent and selective antagonists of human GPR65 with excellent oral bioavailability and pharmacokinetics. In line with their potencies in recombinant cell systems, lead molecules are able to inhibit low pH induced cAMP elevations in primary human macrophages with IC50 values in the single digit nM range. In macrophages subjected to acidic conditions, the inhibitory effects on cAMP are accompanied by a reduction in the expression of anti inflammatory and tissue repair genes, and the enhancement of immunostimulatory genes. In particular, GPR65 inhibition counteracts the pronounced low pH induced suppression of key Type I/II interferon (IFN) response genes and chemokines such as CXCL9, CXCL10 and CXCL11. Following oral administration in tumour bearing mice, lead compounds up-regulate the expression of anti-tumor immune response genes at systemic exposures that significantly suppress GPR65 signalling in vitro. Results from tumour growth inhibition studies in mice with our GPR65 inhibitors will be also be presented. In summary, ‘Macrophage Conditioning’ via GPR65 inhibition holds substantial promise as a novel immunoncology strategy to counteract the immunosuppressive effects of the acidic TME on TAMs, and could be deployed either as monotherapy or in combination with T cell checkpoint inhibitors or other standard of care treatments. 1Nat. Immunol. 19:1319 Citation Format: Barbara Cipriani, David Miller, Alan Naylor, Gavin Milne, Barbara Young, Rupert Satchell, Suorav Sarkar, Zoe Smith, Rhoanne McPherson, Anastasia Nika, Preeti Singh, Toszka Bohn, Tobias Bopp, Tom McCarthy, Stuart Hughes. Inhibition of GPR65 counteracts low pH induced immunosuppressive polarization of macrophages: In vitro and in vivo characterization of potent, selective and orally bioavailable small molecule GPR65 antagonists [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 2162.