Tumor Escape Mechanisms Research Articles

Abstract P2-20-09: Enhancement of anti-tumor immunity in ER+ breast cancer through dual inhibition of MCL1 and caspases

Abstract Breast cancer is one of the most commonly diagnosed cancer types and a leading cause of cancer death in women in the United States. However, it is difficult to treat with immunotherapy because of its immunological “cold” status. This underscores the importance of a novel clinical approach to increase efficacy of immunotherapy including acute upregulation of Type I Interferon (IFN) response. One strategy to enhance Type I IFN response is by release of damage-associated molecular patterns (DAMPs) from mitochondria, including mitochondrial DNA, RNA and oxidized proteins that are recognized by pattern recognition receptors (PRRs) in cytoplasm and activate downstream IFN signaling. Because many cancers including breast cancer experience increased and dysregulated mitochondrial biogenesis at baseline, they contain significantly higher levels of DAMPs compared to normal cells, but these substrates are sequestered in mitochondria and not accessible to PRRs. We set out to develop a strategy to exploit increased levels of DAMPs in cancer cell mitochondria to enhance Type I IFN response. While DAMPs are released from mitochondria upon apoptosis caused by mitochondrial outer membrane permeabilization (MOMP), Type I IFN response is still silenced due to cleavage of PRRs by activated caspases. One way to induce MOMP is by inhibition of Myeloid Cell Leukemia-1 (Mcl-1), an anti-apoptotic family protein that is frequently upregulated in breast cancer. Interestingly, breast cancer cell lines showed the highest dependency on Mcl-1 among solid cancers and inhibitors of Mcl-1 have shown efficacy in breast cancer early clinical trials. Based on our preliminary observations showing that Mcl-1 inhibition activates a robust Type I IFN response in MCF7 breast cancer cells deficient for caspase 3, we hypothesized that combining Mcl-1 inhibitors with caspase inhibitors would greatly enhance Type I IFN response in a robust and specific manner by facilitating release on DAMPs into the cytoplasm while preventing inactivation of PRRs. Strikingly, in contrast with Mcl-1 inhibitor treatment alone, combination treatment led to dramatic increase in expression levels of IFN-β and interferon-stimulated genes (ISGs) in MCF7 and ZR-75-1 ER+ breast cancer cell lines with minimal effects on cytotoxicity. In addition, T47d, a breast cancer line highly resistant to Mcl-1 inhibition due to compensation by other Bcl-2 family proteins, showed greatly enhanced Type I IFN response upon combined Mcl-1, Bcl-2 and caspase inhibition. Because reduced expression of MHC class I molecules on cell surface is one of the most important immune escape mechanisms in tumors including breast cancer, we examined whether the proposed combinatorial treatment would overcome this by enhancing expression of MHC class I. Immunofluorescence (IF) staining confirmed that the combination treatment dramatically increased total HLA-ABC protein expression on plasma membrane of ZR-75-1. Furthermore, combinatorial treatment upregulated transcription of most genes responsible for antigen presentation, a multistep process consisting of antigen peptide generation and loading of MHC class I molecules. Lastly, we sought to examine how elevated chemokine expression may impact patients with breast cancer. We examined association of cytokines highly upregulated by the combination treatment with immune cell infiltration using a TCGA dataset that included over a thousand breast cancer samples. This analysis was performed by xCell algorithm and demonstrated strong association of that cytokine expression with CD8+ T, B and DC infiltrate. Our results demonstrate that the combination of Mcl-1 and caspase inhibitors greatly enhances Type I IFN response and may improve anti-tumor immunity. We plan to further test this drug combination in immunocompetent mouse models, laying the foundation for a clinical trial assessing the combination treatment in patients with metastatic ER+ breast cancer. Citation Format: Jae Kyo Yi, Alexander Spektor. Enhancement of anti-tumor immunity in ER+ breast cancer through dual inhibition of MCL1 and caspases. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-09.

Clustering by antigen-presenting genes reveals immune landscapes and predicts response to checkpoint immunotherapy

Immune checkpoint blockade (ICB) has demonstrated efficacy by reinvigorating immune cytotoxicity against tumors. However, the mechanisms underlying how ICB induces responses in a subset of patients remain unclear. Using bulk and single-cell transcriptomic cohorts of melanoma patients receiving ICB, we proposed a clustering model based on the expression of an antigen-presenting machinery (APM) signature consisting of 23 genes in a forward-selection manner. We characterized four APM clusters associated with distinct immune characteristics, cancer hallmarks, and patient prognosis in melanoma. The model predicts differential regulation of APM genes during ICB, which shaped ICB responsiveness. Surprisingly, while immunogenically hot tumors with high baseline APM expression prior to treatment are correlated with a better response to ICB than cold tumors with low APM expression, a subset of hot tumors with the highest pre-ICB APM expression fail to upregulate APM expression during treatment. In addition, they undergo immunoediting and display infiltration of exhausted T cells. In comparison, tumors associated with the best patient prognosis demonstrate significant APM upregulation and immune infiltration following ICB. They also show infiltration of tissue-resident memory T cells, shaping prolonged antitumor immunity. Using only pre-treatment transcriptomic data, our model predicts the dynamic APM-mediated tumor-immune interactions in response to ICB and provides insights into the immune escape mechanisms in hot tumors that compromise the ICB efficacy. We highlight the prognostic value of APM expression in predicting immune response in chronic diseases.

HLA expression as a risk factor for metastases of cutaneous squamous-cell carcinoma in organ- transplant recipients

BackgroundSolid organ-transplant recipients (SOTR) have an increased risk of cutaneous squamous-cell carcinoma (cSCC), metastasis and death from cSCC. In immunocompetent patients with mucosal SCC, downregulation of HLA class I is associated with poor prognosis. Since the degree of HLA expression on tumor cells could play a role in immunogenicity and pathophysiology of cSCC metastasis, we hypothesized that decreased HLA expression is associated with an increased risk of metastasis. MethodsWe compared HLA expression between primary metastasized cSCCs, their metastases, and non-metastasized cSCCs from the same patients. Samples were stained for HLA-A, HLA-B/-C and quantified by calculating the difference in immunoreactivity score (IRS) of the primary cSCC compared with all non-metastasized cSCCs. ResultsThe mean IRS score for HLA-B/C expression was 2.07 point higher in metastasized compared to non-metastasized cSCCs (p = 0.065, 95 % CI −0.18–4.32). 83.3 % of the primary metastasized cSCCs had an IRS score of 4 or higher, compared to 42.9 % in non-metastasized cSCCs. Moderately to poorly differentiated cSCCs had more HLA class I expression compared to well-differentiated cSCCs. ConclusionContrary to immunocompetent patients, HLA-B/C expression tends to be upregulated in metastasized cSCC compared to non-metastasized cSCC in SOTR, suggesting that different tumor escape mechanisms play a role in SOTR compared to immunocompetent patients.

γδ T Lymphocytes as a Double-Edged Sword—State of the Art in Gynecological Diseases

Human gamma-delta (γδ) T cells are a heterogeneous cell population that bridges the gap between innate and acquired immunity. They are involved in a variety of immunological processes, including tumor escape mechanisms. However, by being prolific cytokine producers, these lymphocytes also participate in antitumor cytotoxicity. Which one of the two possibilities takes place depends on the tumor microenvironment (TME) and the subpopulation of γδ T lymphocytes. The aim of this paper is to summarize existing knowledge about the phenotype and dual role of γδ T cells in cancers, including ovarian cancer (OC). OC is the third most common gynecological cancer and the most lethal gynecological malignancy. Anticancer immunity in OC is modulated by the TME, including by immunosuppressive cells, cytokines, and soluble factors. Immune cells are exposed in the TME to many signals that determine their immunophenotype and can manipulate their functions. The significance of γδ T cells in the pathophysiology of OC is enigmatic and remains to be investigated.

Combined Genetic Ablation of CD54 and CD58 in CAR Engineered Cytotoxic Lymphocytes Effectively Averts Allogeneic Immune Cell Rejection

Allogeneic cell therapies hold promise to be cost effective with scaled manufacturing for multi-dosing and on-demand off-the-shelf availability. A critical consideration for allogeneic cell products is their ability to persist, maintain function and avoid rejection by the patient's immune system. Genetic knockout (KO) of beta-2-microglobulin (B2M) leads to complete loss of cell-surface human leukocyte antigen (HLA) class I expression and efficiently abrogates CD8+ T-cell reactivity. However, loss of HLA class I triggers NK cell-mediated missing-self recognition and manipulation of B2M must therefore be combined with other immune-modulating strategies to limit recipient NK cell reactivity. We hypothesized that rejection by the patient's immune system can be diminished in primary CAR T cells, iPSC-derived T (iT) and NK (iNK) cells by reverse-engineering common tumor escape mechanisms. The adhesion molecules CD54 and CD58 are both present at the target cell side of the immune synapse, and loss of either of these molecules have previously been reported to elicit immune escape. Here, we show that the combined deletion of CD54 and CD58 in allogeneic immune effector cells makes them resistant to rejection by recipient immune cells through unidirectional reduced synapse formation (Figure 1A). HLA class I down-regulation by B2M silencing in primary T and NK cells triggered potent cytotoxicity by resting allogeneic NK cells. This response was mostly driven by educated NK cells expressing either NKG2A or killer cell immunoglobulin-like receptors (KIR) binding to HLA-E and HLA-C, respectively. However, over-expression of HLA-E or single HLA-C ligands in a K562 screening model only shut down the specific response of the NK cell subset carrying the cognate inhibitory receptor, resulting in only partial resistance to NK cells at the bulk level. Notably, the introduction of HLA-E was particularly detrimental in donors with expanded NKG2C+ NK cell subsets, due to its stimulatory effect through the activating NKG2C receptor. In contrast, combined deletion of CD54 and CD58 in target cells uniquely decreased the response of all tested NK cell subsets and showed universal reduction across NK cell populations from 18 healthy donors (Figure 1B). To delineate the mechanisms behind the increased resistance of target cells carrying these edits, we studied NK cell-target cell interactions at the single cell level by confocal microscopy in microchips. Allogeneic NK cells formed fewer conjugates and failed to form productive immune synapses with CD54-/-CD58-/- target cells, supporting the notion that they are more resistant to NK-cell mediated killing by unidirectional altered adhesion. We next introduced these edits in primary B2M-/- T cells engineered to express a second generation CAR19 from the TRAC locus. Corroborating the K562 screen, CD54-/-CD58-/-B2M-/- CAR-T cells had a selective survival advantage over B2M-/- CAR T cells and HLA-E-over-expressing B2M-/- CAR T cells in conventional mixed lymphocyte reaction (MLR) assays in vitro. Furthermore, we established an in vivo model to probe the effect of different genetic edits on the persistence of allogeneic cell therapy products. To this end, a mixed population of B2M-/- CAR T cells additionally bearing either CD54 and/or CD58 KO, HLA-E over-expression, or no further edits were infused into mice harboring allogeneic healthy donor PBMC. We found that CD54-/-CD58-/-B2M-/- CAR T cells had significantly better in vivo persistence compared to both B2M-/- CAR T cells and HLA-E+B2M-/- CAR T cells in the presence of PBMC from healthy donors (Figure 1B). Although multiplexed editing is feasible in primary CAR T cells, the iPSC platform has an unmatched capacity for homogenously introducing multiple immune-evasion strategies for off-the-shelf cell therapy. Similar to primary CAR T cells, multiplexed edited CD54-/-CD58-/-B2M-/-CIITA-/- iNK cells showed normal growth kinetics and were resistant to rejection by activated allogeneic NK cells in MLR assays. Together, these data demonstrate that reverse-engineering of common tumor escape mechanisms, which render target cells less susceptible to immune synapse formation, is an effective strategy to avert immune rejection of allogeneic CAR T and iPSC-derived CAR NK cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transcriptomic Comparison of Non-Hodgkin Lymphomas in Relapsed/Refractory Versus Newly Diagnosed Patients Using Single FFPE Slides

Background Patients newly diagnosed with non-Hodgkin Lymphomas (NHLs) have been historically treated with B-cell depleting antibodies and chemotherapy. Standard of care (SoC) for patients with refractory or relapsed (R/R) disease has rapidly evolved over the past decade to incorporate chimeric antigen receptor T-cell therapy (CAR T), lenalidomide, antibody-directed chemotherapies and stem cell transplantation. This diverse treatment landscape can transform the tumor microenvironment and drive heterogenous tumor escape mechanisms. Understanding this varied tumor contexture will help inform efficacy results from NHL therapeutic studies. Core needle biopsies are used to sample tumor from R/R NHL patients; the bulk of tissue may be consumed for histopathology testing, leaving insufficient sample for transcriptomic profiling. We have shown that HTG EdgeSeq is useful for single-slide analysis of formalin fixed, paraffin embedded (FFPE) tissues (Loya et. al., JCOASCO Suppl. 2022). Here, we use this technique to compare tumors of R/R and newly diagnosed patients with Diffuse Large B-cell Lymphoma (DLBCL) and Follicular Lymphoma (FL). MethodsSamples and Transcriptomics: NHL tumor samples from newly diagnosed, treatment naïve patients (DLBCL n = 99, FL n = 19) and lymph node controls (n = 18) were sourced commercially. Tumor samples from a Phase 1/2a trial of epcoritamab in R/R patients (NCT03625037) were collected prior to treatment (DLBCL n = 38, FL n = 5). Single FFPE slides were processed using the EdgeSeq platform (19,000 gene HT panel, HTG Molecular). Tumors originated from lymphoid organs, gastrointestinal tract, testes, and the pleural cavity. Immunohistochemistry imaging for CD3, PAX5, CD20 was also performed (CellCarta, Belgium). Bioinformatics: Differential expression analysis was performed using linear models in R. Gene set enrichment analysis (GSEA) was performed with GSVA in R (Hänzelmann et. al., BMC Bioinformatics. 2013) using published signature sets (mSigDB: Liberzon et al., Cell Syst. 2015, XCell: Aran, et. al., Genome Biol. 2017). Results Single 5μm slides were analyzed with an average tissue size of 8mm2 for core needle biopsies and 40mm2 for resections. The number of detected genes was comparable to RNAseq, suggesting the HTG platform provides comprehensive data using core needle biopsies obtained during routine histopathology. We compared DLBCL tumor to normal lymph nodes and derived a gene set that was highly correlated to pathologist derived tumor purity. This quantitative proxy allows automated identification of samples that may contain source organ tissue. We applied differential expression analysis to identify gene, pathway and immune cell signatures contrasting newly diagnosed and R/R DLBCL. Top genes upregulated in R/R DLBCL tumors included B-cell markers as expected, T-cell function (CD25/IL2RA), and an intriguing epigenetic mechanism (YTHDC1; a N6-methyladenosine reader with a role in lymphoma proliferation [Liu et al., Front Onco 2022]). MYC pathway signaling was enriched in R/R tumors; consistent with known mutational risk factors for progression (Bisso et. al., Immunol Rev. 2019). Th2 polarized CD4+ T-cell signature was higher in treatment naïve DLBCL compared to R/R and normal lymph nodes. Fibroblast gene signatures were higher in R/R DLBCL, potentially a result of microenvironment remodeling due to chemotherapy or tumor driven adaptation (Dumontet et. al., Front Immunol. 2021). Comparison of DLBCL to FL was also performed in both R/R and untreated patient cohorts. Immune cell signatures for Tregs and CD4+ naïve T-cells were enriched in FL, whereas M1 macrophage signatures were enriched in DLBCL (findings were similar in both R/R and treatment naïve groups). Conclusions The EdgeSeq platform enabled whole-transcriptome profiling from very limited tissue samples in lymphoma clinical trials. This study highlights the importance of characterizing the transcriptional heterogeneity of NHL in relation to genetic alterations and T-cell immunity. Ongoing analysis with large patient cohorts in epcoritamab clinical trials will add statistical power to uncover associations with International Prognostic Score, Cell of Origin, effects of different treatment combinations, and genomic alterations on clinical outcomes.

Development of a High-Affinity Antibody against the Tumor-Specific and Hyperactive 611-p95HER2 Isoform.

Simple SummaryIn the present study, we addressed the unmet need for a molecular antibody (mAb) with high affinity and specificity against a truncated hyperactive isoform of human epidermal growth factor receptor 2 (HER2), called 611-carboxy terminal fragment (CTF)-p95HER2. Patients with p95HER2+ breast cancer are at risk of developing metastatic breast cancer with a poor prognosis and resistance to therapies targeting full-length HER2. We have generated a mAb named Oslo-2, which react specifically with 611-CTF-p95HER2 and has a high affinity. We also characterized the antigenic determinant (epitope) on the p95HER2 protein and the antigen-binding site (paratope) on the Oslo-2 mAb. The antibody can be used to develop antibody- or cell-based therapies targeting p95HER2, as well as a diagnostic assay to identify p95HER2+ disease.The expression of human epidermal growth factor receptor 2 (HER2) is a key classification factor in breast cancer. Many breast cancers express isoforms of HER2 with truncated carboxy-terminal fragments (CTF), collectively known as p95HER2. A common p95HER2 isoform, 611-CTF, is a biomarker for aggressive disease and confers resistance to therapy. Contrary to full-length HER2, 611-p95HER2 has negligible normal tissue expression. There is currently no approved diagnostic assay to identify this subgroup and no therapy targeting this mechanism of tumor escape. The purpose of this study was to develop a monoclonal antibody (mAb) against 611-CTF-p95HER2. Hybridomas were generated from rats immunized with cells expressing 611-CTF. A hybridoma producing a highly specific Ab was identified and cloned further as a mAb. This mAb, called Oslo-2, gave strong staining for 611-CTF and no binding to full-length HER2, as assessed in cell lines and tissues by flow cytometry, immunohistochemistry and immunofluorescence. No cross-reactivity against HER2 negative controls was detected. Surface plasmon resonance analysis demonstrated a high binding affinity (equilibrium dissociation constant 2 nM). The target epitope was identified at the N-terminal end, using experimental alanine scanning. Further, the mAb paratope was identified and characterized with hydrogen-deuterium-exchange, and a molecular model for the (Oslo-2 mAb:611-CTF-p95HER2) complex was generated by an experimental-information-driven docking approach. We conclude that the Oslo-2 mAb has a high affinity and is highly specific for 611-CTF-p95HER2. The Ab may be used to develop potent and safe therapies, overcoming p95HER2-mediated tumor escape, as well as for developing diagnostic assays.

1216P Prognostic potential of liquid biopsy in advanced HER2 positive esophagogastric adenocarcinoma under HER2 blockade and immune checkpoint therapy

Immune therapy and immune checkpoint inhibition (ICI) have drastically improved survival for an increasing number of entities and thereby revolutionized modern oncology. However, even in combined multi-agent approaches, therapy resistance presents a major limitation and imposes the need for tight and sensitive monitoring of the disease status. Liquid biopsy holds the potential to track both the tumor and the patient’s immune response and therefore to monitor even hardly accessible solid tumors as well as to predict therapy outcomes. In the work presented here, we explored the prognostic value of liquid biopsy in patients with advanced HER2+ esophagogastric adenocarcinoma (EGA) receiving first-line treatment of trastuzumab (αHER2) and nivolumab (αPD-1) in combination with ipilimumab (αCTLA-4) or FOLFOX as translational part of the INTEGA trial (NCT03409848). From blood sampled at baseline, after the first treatment cycle (2-3 weeks) and at the end of treatment, we isolated cell-free DNA (cfDNA) to quantify tumor burden and to derive the mutational landscape of the tumor from the contained circulating tumor DNA. Furthermore, we analyzed genomic DNA of blood cells via next-generation sequencing for imprints of the T cell receptor repertoire. We identified a potential tumor driver mutation from cfDNA at baseline in 88 % of cases implying that liquid biopsy is suitable to track malignant lesions in the setting of advanced HER2+ EGA. In some progressive patients, we found truncating or epitope-loss HER2 resistance mutations acquired on therapy, representing a new tumor escape mechanism under ICI with HER2 blockade. Analysis of the T cell immune repertoire in blood showed a significant correlation between high T cell richness and longer survival already after 2-3 weeks, which might represent a read-out for restored T cell activity upon ICI. Moreover, the crude amount of cfDNA per ml blood plasma after 2-3 weeks significantly correlated with response to treatment. Thus, the quantification of cfDNA could offer a relevant prognostic parameter to be implemented into the clinical praxis without the need for time-consuming and cost-intensive analyses.

Current clinical landscape of oncolytic viruses as novel cancer immunotherapeutic and recent preclinical advancements.

Oncolytic viruses (OVs) have been gaining attention in the pharmaceutical industry as a novel immunotherapeutic and therapeutic adjuvant due to their ability to induce and boost antitumor immunity through multiple mechanisms. First, intrinsic mechanisms of OVs that enable exploitation of the host immune system (e.g., evading immune detection) can nullify the immune escape mechanism of tumors. Second, many types of OVs have been shown to cause direct lysis of tumor cells, resulting in an induction of tumor-specific T cell response mediated by release of tumor-associated antigens and danger signal molecules. Third, armed OV-expressing immune stimulatory therapeutic genes could be highly expressed in tumor tissues to further improve antitumor immunity. Last, these OVs can inflame cold tumors and their microenvironment to be more immunologically favorable for other immunotherapeutics. Due to these unique characteristics, OVs have been tested as an adjuvant of choice in a variety of therapeutics. In light of these promising attributes of OVs in the immune-oncology field, the present review will examine OVs in clinical development and discuss various strategies that are being explored in preclinical stages for the next generation of OVs that are optimized for immunotherapy applications.

Immunosenescence, inflammaging, and cancer immunotherapy efficacy

ABSTRACT Introduction Immunosenescence is a progressive remodeling of immune functions associated with a decreased ability of the immune system to set up an efficient immune response, both innate and adaptive, with an increase of highly differentiated T cells at the expense of naive T cells. The incidence and prevalence of most cancers increase with age, which can partly be explained by tumor escape mechanisms and decreased immunosurveillance. Aging is also associated with inflammaging, a low-grade proinflammatory state characterized by an increase in inflammatory mediators. Anti-cancer immunotherapy has profoundly changed the landscape of oncology therapy in the last 10 years. Modern T-cell targeted therapies such as bispecific T cell engagers, CAR-T cells, or immune checkpoint blockers may be theoretically affected by immunosenescence or inflammaging. Areas covered A bibliographic review through PubMed and Embase was carried out using the following search terms: ‘immunosenescence,’ ‘immunotherapy,’ ‘inflammaging,’ ‘bispecific antibodies,’ ‘CAR-T cells,’ ‘immune checkpoint blockers,’ and ‘older patients.’ Expert opinion This review explores the potential impact of immunosenescence and inflammaging on anti-cancer immunotherapy and therapeutic strategies that could counter immune senescence. A more dedicated research on immunosenescence biomarkers in future clinical trials is warranted for the development of new, more effective and safer therapies.

Immune response to primary glioblastoma

Immunotherapy is a promising and rapidly developing method of therapy patients with different oncological pathology. Considering the demonstrated efficacy in the treatment of patients with some solid tumors, as well as low survival rates and the absence of a significant effect in the standard treatment of most patients with glioblastomas, the question of the use of immunotherapy for malignant gliomas of the brain was raised. However, to decision this problem, it is necessary to consider the interaction of the immune system with tumors of this group. The modern view of the interaction of immunity and glioblastoma is presented. Special attention is paid to the mechanisms of tumor escape and suppression of the functional activity of immune system. The current immunotherapeutic approaches in the treatment of patients with glioblastoma are presented. Interaction of glioblastoma with immune system at all the stages of tumor growth is a complex process. While planning the immunotherapy of this pathology its necessary to take into consideration all mechanisms used by the tumor cells to avoid the immune response and suppress it. However, the clinical studies of this type of therapy proved to be less successful than expected. Further detailed studies of immune-resistance and escape of gliobloastoma must contribute to working out more effective immunotherapy tactics.

Strategies for Tracking Immune Surveillance of Tumor Milieu during Angiogenesis

Cancer is a multi-stage, multi-mechanistic, multifactorial complex process that has excessive potential for excessive proliferation with no relation to the physiological organ. Inherited genetic inclinations contribute extensively to about 10 % of breast cancers and about 13 % of colon cancers incidences. In the industrialized countries, 7% of most cancer deaths result from viral infections; 4% from occupational hazards; 2% from sunlight; 2% from air, water, and soil pollution; and less than 1% from diet and lifestyle. Formation of new blood vessels, angiogenesis, is elicited by tissue hypoxia and is essential for normal course of development of every tissue and organ. Angiogenesis unequivocally promotes tumor growth and metastasis. Tumors exhibit different rates of pathological angiogenesis and involve not only abnormally proliferating cancer cells, but also various tumor- infiltrating leukocytes and stromal cells. Local milieu of the cancers polarizes the leukocytes to support the tumor growth further. Although conventional knowledge reveals that immune surveillance helps to suppress tumor development, unresolved immune mechanisms including chronic inflammation can promote growth and progression of tumors. In this review, we outline the immune cells and their derived factors, including immunosuppressive and inflammatory cytokines that either can promote or inhibit cancer development, and the role of tumor microenvironment in this process of regulation. In the present review, the role of T-lymphocytese, NK cells, antibody dependent cell cytotoxicity, tumor escape mechanisms are presented.

P871: ISB 1442, A FIRST-IN-CLASS CD38 AND CD47 BISPECIFIC ANTIBODY INNATE CELL MODULATOR FOR THE TREATMENT OF CD38 POSITIVE HEMATOLOGICAL MALIGNANCIES

Background: ISB 1442 is a first in class 2 + 1 biparatopic bispecific antibody targeting CD38 x CD47 using the BEAT® 2.0 (Bispecific Engagement by Antibodies based on the TCR) platform. The CD38 binding arm consists of bi-paratopic Fabs that strongly bind to CD38 in two different epitopes on tumor cells and do not functionally compete with daratumumab. The anti-CD47 arm comprises a single Fab arm designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes (including macrophages, monocytes and dendritic cells). With this design, the high affinity anti-CD38 Fab arms preferentially drive binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Aims: ISB 1442 was developed for the treatment of hematologic malignancies that express CD38, including multiple myeloma (MM), acute myeloid leukemia (AML), and T-acute lymphoblastic leukemia (T-ALL), with the intention of overcoming mechanisms of resistance to CD38-targeted therapies such as daratumumab and isatuximab, and minimizing hemagglutination/hemolysis on red blood cell (RBC) observed with anti-CD47 monoclonal antibodies (mAb) such as magrolimab. Methods: ISB 1442 was tested for its capacity in vitro to induce ADCP, ADCC and CDC relative to daratumumab and magrolimab across a broad range of MM, AML and T-ALL cell lines expressing different levels of CD38 and CD47. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells (ADCC), autologous macrophages (ADCP), and complement from human serum (CDC). In vivo, ISB 1442 was assessed in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice compared to daratumumab and magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. Results: In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing MM, AML and T-ALL tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. In the CDC, ADCC and MMoAK assays, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab in MM cell lines. In vivo, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. In monkeys, ISB 1442 showed more pronounced binding on RBC than magrolimab due to higher expression of CD38, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Summary/Conclusion: In summary, we report a novel approach for the treatment for CD38 positive hematologic malignancies by co-targeting CD38 and CD47 using a first in class multispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance.

Abstract 253: Analyses of tumor-specific T cell dynamics and tumor immune contexture in microsatellite instability-high cancers in response to first-line therapy

Abstract Microsatellite instability-high (MSI-H) phenotype arises from defective DNA repair mechanisms and is found in many malignancies, including colorectal and endometrial cancers. Due to high neoantigen loads derived from tumor-specific mutations, evidence of tumor-infiltrating T cells and high response rates to checkpoint blockade (30-60%), MSI-H cancers can be leveraged to study the features of neoantigen-induced anti-tumor immunity in response to therapy and disease outcome. Previously we identified a set of recurrent frameshift (fs) mutations in MSI-H cancers that yielded immunogenic polyepitope neoantigens (neoAgs) eliciting CD8+ T cell responses. Here we investigate whether these shared fs-neoAgs engendered spontaneous T cell responses in MSI-H colorectal and endometrial cancer patients. Using peripheral blood mononuclear cells (PBMCs), we evaluated the frequency of the fs-neoAg-specific T cells by ex vivo IFN-γ ELISPOT upon fs-peptide stimulation. Additionally, we isolated naïve and memory T cells from MSI-H patient PBMCs and expanded fs-neoAg-specific T cells within each subset. Fs-neoAg-induced effector cytokine (IFN-γ, TNF-α) production was measured by flow cytometry and was detected only in the memory subset, supporting the hypothesis that shared fs-neoAgs prime CD8+ T cells in vivo to generate clonally-expanded memory T cells in MSI-H patients. To further evaluate the clonal expansion of fs-neoAg-specific T cells in MSI-H cancer patients, we identified fs-neoAg-specific T cell receptors (TCRs). T cell lines enriched for fs-neoAg-specific T cells were generated from patient PBMCs and sequenced for variable β chains of TCRs. These fs-neoAg-specific TCRs were monitored in the blood and tumor tissues, including those from primary and recurrent sites. Finally, to determine the spatial relationships between the infiltrating immune cells and the malignant cells at single cell resolution, we performed multiplexed immunohistochemistry using CODEX technology. A panel of 35 markers was used to evaluate the myeloid (HLA-DR, CD11c, CD14, CD68, CD206) and T (CD4, CD8, Foxp3) cell subsets, the activation (CD107a, CD69, Ki67) and exhaustion (PD-1, TIM3, LAG3) status of T cells, T cell states (TCF-1, TOX, PD-1) and the presence of tertiary lymphoid structures/antigen presenting cell niches (CD19, CD20, HLA-DR) in the tumor microenvironment of primary or recurrent MS-stable and MSI-H tumors from endometrial cancer patients. We also investigated potential immune escape mechanisms in MSI-H tumors, such as the loss of B2M and MHC-I expression. This study will provide insights into the quality and quantity of shared anti-tumor T cells within the periphery and tumor tissues and will help identify the immune correlates of response or resistance to therapy in MSI-H cancer patients. Citation Format: Cansu Cimen Bozkus, Mona Saleh, Rachel Brody, Stephanie V. Blank, Nina Bhardwaj. Analyses of tumor-specific T cell dynamics and tumor immune contexture in microsatellite instability-high cancers in response to first-line therapy [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 253.

Abstract 2903: ISB 1442, a first-in-class CD38 and CD47 bispecific antibody innate cell modulator for the treatment of CD38+ malignancies

Abstract ISB 1442 is a fully human bispecific antibody (BsAb) using BEAT (Bispecific Engagement by Antibodies based on the T-cell receptor) platform to target CD38 and CD47 as treatment for CD38+ malignancies, including multiple myeloma (MM). ISB 1442 is designed with a bi-paratopic anti-CD38 arm that strongly binds to CD38+ tumor cells and an anti-CD47 arm made of a single Fab designed to block interaction between CD47 and the signal-regulatory protein alpha (SIRPα) with low affinity. This approach is expected to induce minimal unintended effects on red blood cells (RBC) compared to anti-CD47 monoclonal antibody (mAb) magrolimab as it enables the CD47 binding only upon avidity induced CD38 crosslinking. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells, autologous macrophages, as well as complement from human serum. In the MMoAK assay, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab. In vivo, in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower RBC hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. On the contrary, due to the higher expression of CD38 in monkey RBC compared to human, ISB 1442 showed more pronounced binding on RBC than magrolimab, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Finally, by integrating in vitro pharmacology data along with available clinical information on benchmark antibodies, a quantitative systems pharmacology model was developed to simulate potential efficacious dose range in MM. In summary, we report a novel approach for the treatment for CD38+ cancers by co-targeting CD38 and CD47. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity by overcoming known primary and acquired tumor escape mechanisms of resistance relative to daratumumab. Citation Format: Stefano Sammicheli, Camille Grandclement, Elie Dheilly, Maria Panagopoulou, Evangelia Martini, Perrine Suere, Blandine Pouleau, Carole Estoppey, Julia Frei, Jeremy Loyau, Thierry Monney, Adam Drake, Alain Rubod, Marie Agnes Doucey, Vinu Menon, Venkatesha Udupa, Sunitha GN, Daniel Rasmussen, Jeppe Koch Olsen, Roberto Gionannini, Girish Gudi, Ankita Srivastava, Cyril Konto, Mario Perro. ISB 1442, a first-in-class CD38 and CD47 bispecific antibody innate cell modulator for the treatment of CD38+ malignancies [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 2903.

Reprogramming T cells to target glycans and overcome glycan‐mediated immunosuppression for cancer therapy

T‐lymphocytes (or T‐cells) exhibit rapid proliferation and cytotoxicity after immune recognition of foreign or non‐self antigens. Cancerous cells can often display mutated peptides that can be recognized as non‐self by the immune system, but a frequent mechanism of tumor escape is loss of the antigen display complexes. Chimeric antigen receptor (CAR) technology allows T‐cells to be genetically engineered with a novel immune receptor (as well as additional pro‐inflammatory or effector molecules) that redirect T‐cell specificity based on the targets of monoclonal antibodies. This genetic engineering allows the manufacturing of billions of T‐cells specific for one tumor‐associated antigen.Previously, we designed a CAR T‐cell therapy that targets the glycopeptide epitope Tn‐MUC1 and demonstrated anti‐tumor efficacy against a variety of tumor histotypes, including T‐cell lymphoma, pancreatic cancer, breast cancer, prostate cancer, and other cancer histotypes. Now, we extend the targeting of Tn‐glycopeptides to an oncofetal form of fibronectin, recognized by the antibody FDC‐6. Tn‐fibronectin‐targeting CAR T‐cells exhibit similar efficacy against PC3 prostate tumors as CAR T‐cells targeting cell surface antigens; however, the mechanism of action of cytotoxicity appears to be different, suggesting that T‐cells targeting extracellular antigens may represent a novel platform for inducing anti‐tumor efficacy. Additionally, CAR T‐cell activity against tumors can be improved through additional engineering approaches, such as the use of enzymatic activity, the generation of immunomodulatory fusion proteins, and secretion of large molecules, such as cytokines and antibodies.

Programmed Cell Death-1 and Its Ligands as Targets for Therapy of Multiple Myeloma Patients.

PurposeAmong hematological malignancies, the expression profile of programmed cell death-1 (PD-1) and its ligands in multiple myeloma (MM) is still debated by numerous research groups. In current study, we characterized the expression of PD-1 and its ligands both on RNA and protein levels in MM patients. We have also attempted to analyze whether daratumumab therapy might overcome CD38-mediated immunosuppression that inhibits in particular CD8+ T-cell function.Patients and MethodsThis study included 149 newly diagnosed MM patients and 15 relapsed/refractory MM patients before and after daratumumab treatment. The mRNA levels of PDCD1, PDCD1LG1, PDCD1LG2 and their splicing variants was assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Flow cytometry was used to characterize the surface expression of PD-1 and its ligands on plasma cells, B and T cells. The surface expression of PD-1 on T cells was assessed by flow cytometry before and after daratumumab treatment.ResultsThe mRNA expression of PDCD1LG1, PDCD1LG2 and their splicing variants were higher in plasma cells as compared to bone marrow mononuclear cells (BMMCs). Our results show that the percentage of plasma cells expressing PD-L1 was significantly higher than plasma cells expressing PD-L2 (p<0.0001) in bone marrow (BM) of MM patients. There was no significant difference between the percentage of plasma cells expressing PD-1 and B cells expressing PD-1 in BM of MM patients (11.19% vs 8.91%). We also found that the percentage of CD8+PD-1+ T cells was significantly higher than CD4+PD-1+T cells in BM (p<0.0001) of MM patients. Here, we observed no change in PD-1 expression on CD4+ and CD8+ T cells after the daratumumab treatment.ConclusionThe PD-1 and its ligands might represent an interesting target for MM immunotherapy, as one would target both malignant plasma cells as well as the immune cells that play a key role in tumor escape mechanisms.

A Sampling of Highlights from the Literature.

Lactic acid is a metabolic checkpoint for Treg cells (from Jynto via Wikimedia Commons)The intratumoral ratio of CD8+ T cells to regulatory T (Treg) cells impacts responsiveness to PD-1 blockade. Kumagai et al. find the ratio of CD8+ T cells to Treg cells is low in highly glycolytic tumors, including MYC-amplified tumors and liver metastases, and that expression of PD-1 is lower in CD8+ T cells than Treg cells. This effect is mediated by tumor-derived lactic acid and leads to resistance to PD-1 blockade in mouse models of Myc-overexpressing colon cancer and intrahepatic tumors. Blocking lactic-acid production or its uptake by Treg cells overcomes this resistance, suggesting potential new targets for cancer immunotherapy.Kumagai S, …, Nishikawa H.Cancer Cell2022 Feb 14;40:201–18.e9.Pancreatic cancer can be protected from CAR T cells by N-glycans (from Neelima Shah and Edna Cukierman via NCI Visuals Online)Chimeric antigen receptor (CAR) T-cell therapy has limited efficacy against solid tumors. Greco et al. identify expression of N-linked glycans as a targetable barrier to killing of solid tumors by CAR T cells. Knockout of mannoside acetyl-glucosaminyltransferase 5 and exposure to 2-deoxy-D-glucose (2DG), both of which disrupt N-glycan expression, render T3M-4 human pancreatic cancer cells sensitive to killing by CD44v6-targeted CAR T cells in vitro. Treatment with 2DG enhances tumor-cell killing by CAR T cells in xenograft models of pancreatic, ovarian, bladder, and lung cancer, suggesting a new approach to improve CAR T-cell therapy for solid tumors.Greco B, …, Casucci M.Sci Transl Med2022 Jan 19;14:eabg3072.Blocking “don’t eat me” signals increases macrophage phagocytosis (from QuidsUp)Modulation of macrophage phagocytosis is a tumor escape mechanism. Li et al. find a role for signaling lymphocytic activation molecule (SLAM) family receptors (SFR) in limiting macrophage phagocytosis by hematopoietic tumors. Specifically, SLAMF3 and SLAMF4 are identified as “don’t eat me” receptors on macrophages. Deficiency in SFRs, with and without CD47 deficiency, increases phagocytosis of hematopoietic cells (i.e., “self”) and malignant cells, highlighting a novel signaling pathway that can modulate the function of macrophages in the tumor microenvironment.Li D, …, Dong Z.Sci Immunol2022 Jan 21;7:eabj5501.HLA-A*03 is associated with poor response to immune checkpoint blockade (from Pdeitiker via Wikimedia Commons)Human leukocyte antigens (HLA) play a vital role in immunity, and thus, could impact immunotherapy efficacy. Using multiple large-scale datasets, Naranbhai et al. identify HLA-A*03 as a biomarker of response to immunotherapy but not response to other types of cancer treatment. Expression of this HLA associates with worse progression-free survival and lower responses to immune checkpoint inhibitors, which was validated in several independent cohorts of patients with various tumor types.Naranbhai V, …, Carrington M. Lancet Oncol 2022 Jan 1;23:172–84.Understanding of the NSCLC immune landscape continues to deepen (from Librepath via Wikimedia Commons)Deepening understanding of the immune landscape of non–small cell lung cancer (NSCLC) can help identify biomarkers of response to immunotherapy. Using single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), Leader et al. find a strong correlation between PDCD1+CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages; they refer to these cells collectively as the lung cancer activation module (LCAM). The LCAMhi phenotype correlates with tumor mutational burden, TP53 mutations, and response to anti–PD-L1 therapy but not chemotherapy, suggesting that it could be a new biomarker of NSCLC response to immunotherapy.Leader AM, …, Merad M. Cancer Cell 2021 Dec 13;39:1594–609.e12.TRIB3 regulates CD8+ T-cell exclusion in the tumor microenvironment (from Max Pixel)Colorectal cancer resistance to immune checkpoint therapy is partly attributable to a lack of tumor-infiltrating T cells. Shang et al. report that TRIB3 expression in tumor cells correlates with CD8+ T-cell exclusion in mouse models of colorectal cancer and human samples. TRIB3 expression inhibits expression of the T cell–recruiting chemokine CXCL10 by activating STAT3, which inhibits transcription of STAT1 and subsequently CXCL10. Inhibition of the P300 acetylase, which acetylates TRIB3 and prevents E3 ligase–mediated degradation, improves CD8+ T-cell infiltration and sensitizes colorectal tumors to checkpoint blockade in vivo, suggesting a therapeutic strategy to improve immunotherapy efficacy in immunologically “cold” colorectal tumors.Shang S, …, Hua F. Sci Transl Med 2022 Jan 5;14:eabf0992.

Abstract P4-02-03: HER1-4 protein up-regulation following short-term neoadjuvant endocrine therapy in patients with hormone receptor-positive HER2-negative breast cancer

Abstract Background: Patients with hormone receptor-positive (HR+) breast cancer (BC) experience significant improvement in long-term outcomes with endocrine therapy (ET). However, metastatic recurrences of endocrine resistant disease develop in about 20-25% of patients and remain a major cause of BC mortality. Up-regulation of HER family growth factor receptor tyrosine kinases (RTKs) represent early escape mechanisms in tumors treated with ET. Neoadjuvant endocrine therapy (NET) offers a unique opportunity to identify responsiveness of HR+ BCs and detect tumors that display up-regulation of HER RTKs. Methods: A single arm, interventional, exploratory clinical trial evaluating four weeks of NET in early stage HR+/HER2-negative BC patients was conducted at our institution (NCT03219476). The primary objective was to assess changes in HER protein expression (HER1-4) from diagnostic core biopsies to surgically resected tumors treated with NET. Secondary objectives included assessment of other molecular markers, tumor proliferation and volumetric responses. Optimized protocols for immunohistochemistry (IHC) were used for HER1-4, and HER2 status was further assessed by fluorescence in situ hybridization (FISH) if IHC (2+) equivocal. Up-regulation was defined as an increase of ≥ 1 in IHC scores (ordinal 0,1,2,3) or gene amplification by FISH. Chi-square tests of independence and one sample difference in proportion tests were performed. Linear regression was used to evaluate association of variables of interest with the primary outcome. A significance level of 0.05 was used throughout. Results: Thirty-seven patients with cT1-T3, cN0 HR+/HER2-negative BC were enrolled. One patient had multifocal disease resulting in 38 tumors. Patient and tumor characteristics are shown in Table 1. There was no significant change in ER-positivity between pre- and post-treatment specimens. However, a trend towards decrease in PR-positivity was seen in post-treatment tumors consistent with functional ER pathway disruption (p=0.08). HER1-4 proteins were significantly up-regulated in 44.7% tumors (17/38) with HER2 being the most frequently up-regulated HER RTK (p=0.035). HER3 and HER4 were down-regulated in 50% tumors (19/38). Three patients converted to HER2-positive status (IHC 3+ or FISH amplified) at surgery and received adjuvant trastuzumab-based treatment. Ki67 decreased in 71% tumors [median 3.35 (range 0.06-25.5)] and increased in 26.3% tumors [median 1 (range 0.08-3.78)]. No significant associations were identified between any clinic-pathologic covariates and change in HER1-4 protein expression on multivariate analysis. Conclusions: HER1-4 proteins were significantly up-regulated in 44.7% of tumors after short-term NET in patients with early stage HR+/HER2-negative BC, with HER2 up-regulation being the most predominant and statistically significant. Down-regulation in HER3 and HER4 were seen in 50% of tumors, however the biological and clinical significance of this finding is unclear. Short-term NET is a promising strategy to identify HR+ tumors that up-regulate HER2 as an early escape pathway and endocrine resistance mechanism. Patients with such HER2 up-regulated tumors after NET may benefit from HER2-directed therapies upon disease recurrence or as adjuvant combination therapy. These findings need to be further explored in larger randomized clinical trials. Table 1.Median Age (range)64yrs (42-81)Median BMI (range)28.3 (19.3-55.1)Post-menopausal, N (%)31 (83.8)Median clinical tumor size (range)1.3cm (0.5-7.7)Invasive ductal, N (%)26 (70)Grade, N (%)Low16 (43.2)Intermediate18 (47.4)High4 (10.5)Median tumor size at surgery (range)1.2cm (0.09-4)pN stage at surgery, N (%)N030 (81)N17 (19)NET, N (%)Aromatase inhibitors28 (75.6)Tamoxifen9 (24.4) Citation Format: Lubna Naaz Chaudhary, Julie M Jorns, Yee Chung Cheng, Sailaja Kamaraju, John Burfeind, Mary Beth Gonyo, Amanda Kong, Caitlin Patten, Tina Yen, Chandler Cortina, Ebony Carson, Nedra Johnson, Carmen Bergom, Anjishnu Banerjee, Yu Wang, Yunguang Sun, Christopher R Chitambar, Hallgeir Rui. HER1-4 protein up-regulation following short-term neoadjuvant endocrine therapy in patients with hormone receptor-positive HER2-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-03.

Abstract P4-02-08: Amcenestrant in combination with CDK4/6 inhibitor palbociclib demonstrates synergistic anti-tumor activity in ER+ endocrine-resistant breast cancer xenograft models

Abstract Nearly 70% of newly diagnosed cases of breast cancer (BC) are estrogen receptor positive (ER+) where endocrine therapy is a primary treatment. Despite initial efficacy seen with endocrine therapies, approximately 40% of patients develop acquired resistance which ultimately limits the use of these agents even as most of these tumors continue to require ERα. The strategies to combat these cancers with resistance are not yet defined and represent the next major clinical challenge in ER+ breast cancer. Amcenestrant is an optimized oral SERD (selective estrogen receptor degrader) with potent antagonist and degradation activity irrespective of ESR1 mutational status, and is currently being evaluated as monotherapy and in combination with CDK4/6 inhibitors and other targeted therapies for ER+ breast cancer patients in clinical trials. Here, we report that in preclinical models, amcenestrant achieved significant anti-tumor efficacy/regression as a monotherapy and strong synergistic activity when administered together with the CDK4/6 inhibitor, palbociclib, in subcutaneously xenografted endocrine resistant ER+ BC mouse models: ESR1-Y537S mutant-driven MCF7 tumor and HCI-013 PDX (patient-derived xenograft). Transcriptional profile analysis of xenograft tumor biopsies revealed the modulation of different pathways by SERD and palbociclib and highlighted crosstalk of ER and CDK4 signaling pathways. Interestingly, CDK4/6 inhibition by palbociclib induces partial activation of ER pathways as potential mechanism of tumor escape which is completely abolished by the combination treatment of amcenestrant with palbociclib. Early clinical data also showed that amcenestrant in combination with palbociclib, exhibited a favorable safety profile and encouraging antitumor activity in patients with endocrine resistant, ER+/HER2-, advanced breast cancer, which supports its potential to become a new endocrine backbone therapy for ER+ HER2- breast cancer. In conclusion, amcenestrant is an oral SERD, which exhibits significant anti-tumor activity as a single agent and shows synergistic activity with CDK4/6 inhibitor palbociclib in ER+ BC xenografts preclinically and in early clinical development. Citation Format: Maysoun Shomali, Zhuyan Guo, Jane Cheng, Amy Sullivan, Youssef El-Ahmad, Fangxian Sun, Sukhvinder Sidhu, Joon Sang Lee, Hui Cai, Jack Pollard, Laurent Debussche, Chris Soria, Monsif Bouaboula. Amcenestrant in combination with CDK4/6 inhibitor palbociclib demonstrates synergistic anti-tumor activity in ER+ endocrine-resistant breast cancer xenograft models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-08.