Bispecific antibodies (bsAbs) directed against PD-1/PD-L1 and CTLA-4; a mini review.

Outcomes of Immunotherapy (ICI) Alone vs. Stereotactic Radiosurgery (SRS) Alone vs. ICI and SRS Combined in Melanoma Brain Metastasis

2597 Background: In order to improve the expected response rate (ORR) of less than 10% in cold tumors, several ICI combinations are being evaluated in clinical trials. However, most of these trials don’t require any biomarker and pts are included based solely in histology. We aimed to assess the benefit of ICI combinations in pts with unselected cold tumors included in early CT. Methods: ICI naïve pts with cold tumors treated from 2015 to 2021 with ICI combinations in early CT at VHIO were reviewed. Clinico-pathological data and anti-tumor activity were extracted from a prospective database. ORR was defined as per RECIST v1.1 and clinical benefit rate (CBR) as complete/partial response (CR/PR) + stable disease (SD) for ≥ 4 months (m). Kaplan Meier estimates of progression-free survival (PFS) and overall survival (OS) were calculated and a Cox model according to LIPI (Lung Immune Prognostic Index = baseline LDH and derived neutrophil to lymphocyte ratio) was constructed. Immune-related adverse events (irAE) were classified as per CTCAE v.4.03. Hyperprogressive disease (HPD) was evaluated using RECIST v1.1 (Matos et al, 2020). Results: Out of 97 pts, median age was 62y, 61% had ECOG 0 and 29.8% had LIPI 0 (good prognostic score). Most pts had microsatellite stable (MSS) colorectal cancer (60.8%) or ovarian cancer (14.4%). Regimens included anti-PD1/L1 + another ICI in 69% (most commonly anti-LAG3 [26,8%] and CD40 agonist [20.9%]), anti-PD1/L1 + other molecule in 21.7% (most commonly SHP2 inhibitor [33.3%] and anti p53-HDM2 [28.5%]) and bispecific antibodies in 9.3% (anti-PD1/L1 + anti-LAG3 or CD137 agonist). No patient achieved a response. CBR was 15.3% (11 pts with MSS colorectal cancer, 2 ovarian cancer, 1 olfactory neuroblastoma, 1 paraganglioma). 33 pts (34%) presented irAE, 15 pts (15.5%) had irAE ≥ G2, 4 pts (4.1%) had G3 irAE (dry mouth, hypertransaminasemia, myocarditis and neutrophils count decreased) and 1 patient (1%) had G4 hyperglicemia. 58 pts (59.7%) had progressive disease (PD) as best response, 19 of these pts (32.7%) presented irAE. Overall, 20 pts (20.6%) met definition of HPD, representing 34.4% of pts with PD as best response. Median PFS for overall and CBR population were 1.9 m (CI95% 1.7-2.0) and 5.9 m (5.4-NR), respectively. Median OS for overall population was 7.6 m (5.9-9.5), with a trend for improved OS if LIPI good score vs. others (12.6 m vs. 6.2 m, hazard ratio 1.9, (CI 95% 1.1-3.3), p = 0.02). Among hyperprogressors, median OS was 5.33 m (3.39 - NR) and significantly worse LIPI scores (intermediate [1] or poor [2]) were observed as compared to pts with CBR (75% vs 53.3% p = 0.001). Conclusions: ICI combinations demonstrated very limited activity in pts with unselected cold tumors. However, the risk for irAE and HPD remain substantial. Further drug-biomarker co-development strategies are urgently needed to increase the risk benefit ratio for these pts.

Immune checkpoint inhibitors (ICI) have revolutionized the therapeutic direction for lung cancer, yet their response rates remain unsatisfactory. Recently, the combination of ICI and low dose radiotherapy (LDR), a novel approach that effectively mobilizes innate and adaptive immunity, has gained interest among scientists. However, the underlying molecular mechanisms are not clearly elucidated. The in vivo anti-tumor effects of LDR and ICI were measured in murine tumor models. The immune response and alterations in the tumor microenvironment were measured using flow cytometry and enzyme-linked immunosorbent assay (ELISA). Cell viability and death were assessed using CCK-8 assays. Fluorescent probes and ELISA were used to assess ferroptosis induced by the combination therapy in vitro and in vivo. Western blotting and qPCR were performed to detect alterations in the Nrf2/HO-1/GPX4 pathway. Furthermore, a phase 1 clinical trial with a combined regimen of LDR and anti-PD-1 antibodies in patients with lung cancer was conducted. The combined LDR and ICI regimen exhibited considerable anti-tumor effects in murine tumor models, promoting immune response and increasing the IFN-γ levels. In vitro data showed that LDR plus ICI induced ferroptosis in cancer cells by increasing reactive oxygen species and MDA levels, promoting Fe2+ accumulation, and suppressing GSH. Furthermore, ferroptosis induced by combination therapy was associated with suppression of the Nrf2/HO-1/GPX4 antioxidant axis. Importantly, a phase 1 clinical trial of the combination therapy showed promising efficacy in patients with lung cancer with chemoimmunotherapy resistance. This study demonstrated that LDR plus ICI induces ferroptosis through the Nrf2/HO-1/GPX4 pathway, resulting in a significant anti-tumor effect and providing a combinatorial strategy to overcome lung cancer. However, this combined strategy merits further clinical investigation.

Development of a Two-part Strategy to Identify a Therapeutic Human Bispecific Antibody That Inhibits IgE Receptor Signaling

e21731 Background: Over the last decade six different immune-checkpoint inhibitors (ICI) have obtained approval in 42 tumor types, several of them are indeed competing for the same indication and there is a lack of direct comparative data to guide the medical oncologist. Network meta-analysis (NMA) is a technique developed as an extension of pairwise meta-analysis to allow comparisons of multiple treatments, using both direct and indirect evidence. This NMA evaluates the effectiveness of several ICIs, either in combination or as single agents, as frontline therapy for patients with advanced non-squamous non-small cell lung cancer (NSCLC). Methods: We conducted a systematic review of randomized controlled trials (RCT) through Pubmed, Embase, Cochrane Central register of Controlled trial databases, Clinicaltrial.gov database and ASCO abstracts (up to June 2019), to identify phase III RCTs on advanced non-squamous NSCLC in the first-line setting. Both the search and study screening were performed for two independent reviewers. NMA of survival outcomes in different subsets of PD-L1 expression groups was performed and ICIs indirect comparisons were performed. Results: 6 RCTs with 5860 patients were included for the NMA. ICIs were evaluated in combination with several chemotherapy regimens and in the case of Nivolumab in combination with ipilimumab. Atezolizumab was evaluated in one of the RCTs in combination with chemotherapy and bevacizumab. Both Pembrolizumab and Nivolumab were evaluated as monotherapy, either in a separate treatment arms within a trial or in a separate trial. Overall ICI in combination improved survival across PD-L1 expression level subgroups compared with chemotherapy (platinum doublets +/- bevacizumab). Indirect comparisons of ICIs in combination therapy for first-line treatment in advanced non-squamous NSCLC showed little evidence of differences between pembrolizumab or atezolizumab in combination with chemotherapy and nivolumab/ipilimumab. Evaluated as monotherapy pembrolizumab seems to have more evidence of effectiveness than nivolumab. Conclusions: ICIs in combination therapy improve survival in previously untreated advanced NSCLC across PD-L1 expression levels compared with standard first line chemotherapy regimens. There is little evidence of survival difference between pembrolizumab and atezolizumab in combination, and nivolumab requires a combination with ipilimumab to reach similar levels of effectiveness.

Immunotherapy has revolutionised the treatment of oncological patients, but its application in various endocrine tumours is rather limited and is mainly used when conventional therapies have failed. Immune checkpoint inhibitors (ICIs) have been employed in progressive adrenocortical carcinoma, primarily utilizing the anti-PD-L1 agent pembrolizumab, obtaining overall response rates ranging between 14% and 23%. In contrast, the response rate in phaeochromocytoma/paraganglioma was substantially less at 9%, considering the small number of patients treated. Similarly, the response rate in advanced differentiated thyroid carcinomas treated with pembrolizumab was also low at 9%, although the combination of ICIs with tyrosine kinase inhibitors showed higher efficacy. Low response rates to ICIs have also been observed in progressive medullary thyroid cancer, except in tumours with a high mutation burden (TMB). Pembrolizumab or spartalizumab can be utilized in patients with high TMB anaplastic thyroid cancer, obtaining better response rates, particularly in patients with high PD-L1 expression. Immunotherapy has also been used in a few cases of parathyroid carcinoma, showing limited antitumour effect. Pituitary carcinomas may exhibit a more favourable response to ICIs compared to aggressive pituitary tumours, particularly corticotroph tumours. Patients with advanced neuroendocrine tumours achieve an overall response rate of 15%, which varies according to the primary tumour site of origin, degree of differentiation, and therapeutic regimen utilised. Future research is needed to evaluate the potential role of immunohistochemical biomarkers, such as programmed death 1/programmed death ligand 1 and TMB, as predictors for the response to immunotherapy. Furthermore, randomised prospective studies could provide more robust data on the efficacy and side effects of ICIs.

e21057 Background: Immune checkpoint inhibitors (ICIs), which are currently used in the standard second-line treatment for non-small cell lung cancer (NSCLC), have largely improved the prognosis of advanced NSCLC. However, patients treated with ICI monotherapy have low response rates; hence, there is an urgent need to expand the population responding to immunotherapy treatment. This study aimed to explore the efficacy and safety of the combination of ICI with chemotherapy or anti-angiogenic therapy compared to ICI monotherapy in patients previously treated for advanced NSCLC. Methods: Data was collected from previously treated patients with NSCLC who further received ICI monotherapy or combination therapy. The distribution of clinical variables was assessed using the chi-square test or Fisher's exact test. The relationship between descriptive variables and survival was described using Kaplan–Meier curves and compared by log-rank test. Cox proportional-hazards models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI) to confirm survival differences. Results: A total of 145 patients were included in this study, of which 63 were in the ICI monotherapy group, 57 were in the ICI with chemotherapy group, and 25 were in the ICI with anti-angiogenic therapy group. Compared with the ICI monotherapy group, the ICI with chemotherapy group had significantly higher ORR (31.6% vs. 11.1%, P = 0.006), DCR (84.2% vs. 61.9%, P= 0.006), PFS (mPFS: 6.37 vs. 3.47 months, P< 0.0001; HR = 0.42, 95% CI: 0.29–0.63, P< 0.0001), and OS (mOS: 18.60 vs. 8.47 months, P< 0.0001; HR = 0.40, 95% CI: 0.25–0.64, P= 0.0001). In addition, the ICI with anti-angiogenic therapy group also had significantly elevated DCR (88% vs. 61.9%, P= 0.02), PFS (mPFS: 8.17 vs. 3.47 months, P< 0.0001; HR = 0.30, 95% CI: 0.17–0.53, P< 0.0001), and OS (mOS: 19.20 vs. 8.47 months, P= 0.006; HR = 0.44, 95% CI: 0.24–0.80, P= 0.007) compared with the ICI monotherapy group. There was no significant difference between the ICI with chemotherapy group and the ICI with anti-angiogenic therapy group. Meanwhile, the addition of chemotherapy or anti-angiogenics did not increase immune-related adverse events and had a good safety profile. Conclusions: For previously treated patients with advanced NSCLC, treatment with ICI in combination with chemotherapy led to enhanced ORR, DCR, PFS, and OS compared to treatment with ICI monotherapy. Moreover, treatment with ICI combined with anti-angiogenic therapy led to enhanced DCR, PFS, and OS compared to treatment with ICI monotherapy. The combination of immunotherapies is a promising second-line treatment option for some of the patients in this study, and the findings of this study necessitate the need for further exploration.

Emerging tumor immunotherapy methods encompass bispecific antibodies (BSABs), immune checkpoint inhibitors (ICIs), and adoptive cell immunotherapy. BSABs belong to the antibody family that can specifically recognize two different antigens or epitopes on the same antigen. These antibodies demonstrate superior clinical efficacy than monoclonal antibodies, indicating their role as a promising tumor immunotherapy option. Immune checkpoints are also important in tumor immunotherapy. Programmed cell death protein-1 (PD-1) is a widely acknowledged immune checkpoint target with effective anti-tumor activity. PD-1 inhibitors have demonstrated notable therapeutic efficacy in treating hematological and solid tumors; however, more than 50% of patients undergoing this treatment exhibit a poor response. However, ICI-based combination therapies (ICI combination therapies) have been demonstrated to synergistically increase anti-tumor effects and immune response rates. In this review, we compare the clinical efficacy and side effects of BSABs and ICI combination therapies in real-world tumor immunotherapy, aiming to provide evidence-based approaches for clinical research and personalized tumor diagnosis and treatment.

85 Background: Prior clinical trials in melanoma have demonstrated higher rates of irAEs from combination ICI therapy compared to monotherapy. However, this has not been well studied in the real-world where patients often have greater co-morbidities and less organ reserve. We aim to compare irAEs hospitalizations for melanoma patients on combination vs monotherapy ICIs. Methods: We performed a single centre retrospective chart review (Princess Margaret Cancer Centre, Toronto, ON) for all melanoma patients receiving ICI as standard of care (2012-2017) admitted with irAEs. Data collected include demographics, investigations, management and outcomes of hospitalizations. Descriptive analyses were performed to characterize hospitalizations and compare between ICI combination vs monotherapy groups. Results: Among 381 melanoma patients identified on standard of care ICI, 41 (11%) were admitted for irAE. Among those admitted, 10% received monotherapy with nivolumab, 22% pembrolizumab, 39% ipilimumab and 29% combination ICI. Admission rates were higher among patients receiving combination ICI compared to monotherapy (20% vs 8% p = 0.003). Prevalence of the most common irAEs were similar between combination and monotherapy groups: colitis (58% vs 59%), pneumonitis (8% vs 14%) and hepatitis (8% vs 10%). Less than half received invasive diagnostic tests (i.e, endoscopy) (42% combination vs 35% monotherapy, p = 0.50) with 3 (60%) and 5 (50%) confirming irAEs, respectively. Rates of infliximab use were similar between the combination and monotherapy group (25% vs 21%, p = 0.70). Average length of stay was shorter for patients on combination ICI compared to monotherapy (5 days vs 15 days, p = 0.08). irAE readmission rates were similar between patients receiving combination ICI compared to monotherapy (20% vs 17%, p = 0.65). Conclusions: Despite higher admission rates among patients receiving combination ICI, there was a trend towards shorter hospitalizations. Other outcomes including diagnoses, investigations and management were not significantly different between patients receiving combination vs ICI monotherapy.

CD47 is a ubiquitous cell surface glycoprotein that serves as a negative regulator of numerous innate immune functions such as phagocytosis, neutrophil inflammatory responses or dendritic cell maturation and activation. In particular, the interaction of CD47 with Signal Regulatory Protein Alpha (SIRP alpha) on myeloid cells is a mechanism of self-recognition, a ‘don't eat me’ signal used by healthy cells to impede their elimination by phagocytes. Cancer cells often up-regulate CD47 expression, which helps them to evade immune surveillance and killing. What is more, increased CD47 levels are generally predictive of poor clinical outcome. Inhibiting CD47-SIRP alpha interaction represents therefore an attractive, generally applicable therapeutic strategy. Yet, achieving that goal with an anti-CD47 monoclonal antibody (Mab) may be challenging in practice, given the ubiquitous expression of the target. The “drug sink” represented by erythrocytes, platelets and other CD47-expressing cells may lead to a rapid elimination of an anti-CD47 Mab through target-mediated drug disposition; another likely consequence of ubiquitous CD47 expression are on-target toxicities like, e.g., anemia. To overcome these limitations, we have developed dual-targeting bispecific antibodies to CD47 and different Tumor-Associated Antigens (TAAs). Dual targeting CD47/TAA bispecific antibodies bind preferentially to TAA-expressing cancer cells and enable selective CD47 neutralization at the same time sparing healthy TAA-negative cells. Various TAAs are currently being pursued at Novimmune (e.g., CD19, mesothelin, or glypican 3). Such dual targeting CD47/TAA bispecific antibodies have the kappa/lambda body format i.e., they are fully human bispecific IgGs composed of a high-affinity anti-TAA arm, a CD47-neutralizing arm, and an unmodified IgG1 Fc. So far, only CD47/CD19 kappa/lambda bodies have been thoroughly characterized in vitro and in vivo. Various human B cell leukemia and lymphoma lines were used to demonstrate selective, CD19-dependent binding of these CD47/CD19 kappa/lambda bodies. As anticipated, the neutralization of the CD47-SIRP alpha interaction was also CD19-dependent, and led to the enhancement of Fc-mediated cancer cell killing through ADCP (antibody mediated cellular phagocytosis) or ADCC (antibody mediated cellular cytotoxicity). Efficient killing of CD19-positive cancer cells was also observed in vivo using a localized tumor xenograft model with NOD/SCID mice implanted with Raji Burkitt lymphoma cells. A dose dependent inhibition of tumor growth or tumor regression was observed, depending on the potency of the CD47-neutralizing arm used to generate the kappa/lambda body. In parallel, a single dose PK study in cynomolgus demonstrated favorable elimination kinetics, comparable to human IgG Mab. Moreover, no effects on hematological parameters were apparent at either of the doses tested (0.5 and 10 mg/kg). The example of CD47/CD19 kappa/lambda bodies illustrates the power of the dual-targeting approach: A favorable safety profile and pharmacokinetics could be achieved in parallel to potent inhibition of the CD47-SIRP alpha interaction and effective cancer cell killing. Antagonizing CD47-SIRP alpha interaction with bispecific antibodies is therefore an attractive immune-potentiating strategy. Because of its distinct mechanism of action and the ability to that harnesses the innate immunity to fight cancer, CD47/TAA bispecific antibodies would be good candidates for combination therapies with immune checkpoint inhibitors or other immunotherapeutics. Citation Format: Krzysztof Masternak, Zoe Johnson, Vanessa Buatois, Francois Rousseau, Giovanni Magistrelli, Valery Moine, Ulla Ravn, Franck Gueneau, Lucile Broyer, Susana Salgado Pires, Maureen Deehan, Nicolas Fischer, Walter Ferlin, Marie Kosco-Vilbois. Antagonizing CD47-SIRP alpha interaction with a bispecific antibody: A novel cancer immunotherapy approach. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B54.

9552 Background: Immune checkpoint inhibitors (ICI) and combination chemotherapy (chemo) plus ICI (Chemo-ICI) have been shown in RCTs to have improved OS compared to chemo in the 1L treatment of advanced NSCLC. However, the benefit of chemo-ICI compared with ICI alone is unknown. Methods: Systematic review using MEDLINE, Embase and Cochrane CENTRAL was done to identify relevant studies up to December 2019. Phase 3 RCTs that evaluated the efficacy of 1L ICI or chemo-ICI and reported outcomes stratified by PD-L1 status (<1%, 1-49%, ≥50%) were included. ICI was defined as single-agent PD-1 axis inhibitor or dual checkpoint blockade with PD-1 axis inhibitor plus CTLA-4 inhibitor. Comparison for PD-L1<1% included chemo-ICI vs ipi/nivo and for PD-L1 1-49% and PD-L1>50% included chemo-ICI vs ipi/nivo or single-agent ICI. OS, PFS, and ORR were extracted. Network meta-analysis (NMA) was done in Bayesian random-effects regression models. Results: Ten phase 3 RCTs (7971 screened) involving 7,218 patients assigned to ICI (pembro or atezo or ipi/nivo) or chemo-ICI (platinum-doublet + atezo, pembro, or nivo) were included. In PD-L1 <1% patients, NMA comparing chemo-ICI with ipi/nivo failed to show a statistically significant difference in OS, PFS or ORR. In PD-L1 1-49% patients, there was no significant difference between chemo-ICI vs ICI in OS or ORR; PFS could not be analyzed due to lack of available data. In PD-L1 >50% patients, chemo-ICI was associated with improved PFS and ORR compared to ICI alone, but without any OS difference (Table). Conclusions: Although the addition of chemo to ICI appears to improve ORR and PFS in PD-L1 ≥50% patients when compared to ICI alone, chemo-ICI does not confer an OS benefit in the 1L treatment of NSCLC patients regardless of PD-L1 status. Prospective trials comparing chemo-ICI, ICI monotherapy, and combination ICI are needed to confirm these findings. OS, PFS and ORR with chemo-ICI vs ICI. [Table: see text]

Immune checkpoints are differentially expressed on various immune cells to regulate immune responses in tumor microenvironment. Tumor cells can activate the immune checkpoint pathway to establish an immunosuppressive tumor microenvironment and inhibit the anti-tumor immune response, which may lead to tumor progression by evading immune surveillance. Interrupting co-inhibitory signaling pathways with immune checkpoint inhibitors (ICIs) could reinvigorate the anti-tumor immune response and promote immune-mediated eradication of tumor cells. As a milestone in tumor treatment, ICIs have been firstly used in solid tumors and subsequently expanded to hematological malignancies, which are in their infancy. Currently, immune checkpoints have been investigated as promising biomarkers and therapeutic targets in hematological malignancies, and novel immune checkpoints, such as signal regulatory protein α (SIRPα) and tumor necrosis factor-alpha-inducible protein 8-like 2 (TIPE2), are constantly being discovered. Numerous ICIs have received clinical approval for clinical application in the treatment of hematological malignancies, especially when used in combination with other strategies, including oncolytic viruses (OVs), neoantigen vaccines, bispecific antibodies (bsAb), bio-nanomaterials, tumor vaccines, and cytokine-induced killer (CIK) cells. Moreover, the proportion of individuals with hematological malignancies benefiting from ICIs remains lower than expected due to multiple mechanisms of drug resistance and immune-related adverse events (irAEs). Close monitoring and appropriate intervention are needed to mitigate irAEs while using ICIs. This review provided a comprehensive overview of immune checkpoints on different immune cells, the latest advances of ICIs and highlighted the clinical applications of immune checkpoints in hematological malignancies, including biomarkers, targets, combination of ICIs with other therapies, mechanisms of resistance to ICIs, and irAEs, which can provide novel insight into the future exploration of ICIs in tumor treatment.

Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

The use of immune checkpoint inhibitors (ICIs) in combination therapy is an emerging trend in tumor immunology. However, the value of combination immunotherapy remains controversial, because of the toxic effects induced by combination. The added benefit of each additional drug has not been assessed against the added toxicity. We searched for clinical trials that evaluated ICI monotherapies and combination therapies in lung cancer and melanoma patients. The overall response rate (ORR), grade 3/4 treatment-related adverse event rate, overall survival (OS), and progression-free survival (PFS) were extracted from the most recently published studies to determine the relative risk (RR), hazard ratios (HRs), and 95% confidence intervals (CIs). Seven randomized controlled trials and one open-label study were identified (n = 3,097). Treatments included combinations of several ICIs, a combination of an ICI and dacarbazine, two combinations of an ICI, paclitaxel and carboplatin, and a combination of an ICI and gp100 vaccine. Higher ORR (RR: 1.51, 95% CI: 1.03-2.20, p = 0.034), OS (HR: 0.86, 95% CI: 0.78-0.95, p = 0.000), and PFS (HR: 0.93, 95% CI: 0.72-1.14, p = 0.000) values were observed in combination therapy than in monotherapy. In addition, the toxicity of combination ICI immunotherapy was higher (RR: 1.50, 95% CI: 1.03-2.19, p = 0.036) than that of monotherapy. This meta-analysis showed that the addition of nivolumab to ipilimumab better benefits PFS and ORR. Adding sargramostim was associated with better OS and safety. The efficacy and safety of a nivolumab-ipilimumab-sargramostim combination should be investigated further.

Introduction: Immune checkpoint inhibitors (ICIs) are an effective strategy to engage the adaptive arm of the immune system in several solid cancers. While approved for metastatic triple negative breast cancer and breast cancer with microsatellite instability or mismatch repair deficiencies, ICIs are still under investigation in HER2+ breast cancer. In preclinical studies, the effects of ICIs are dampened by immunosuppressive cells, such as myeloid derived suppressor cells (MDSC), in the breast tumor microenvironment (TME). Previous studies in mice showed that addition of entinostat, a histone deacetylase inhibitor, to ICIs improved survival and reduced immunosuppression in an early HER2+ breast tumor model. Here, we evaluated the addition of entinostat to ICIs in a mouse model of metastatic HER2+ breast cancer. We hypothesize that the different immune cell composition within the TMEs of breast and lung metastases will affect mechanisms of response to this treatment combination. Methods: mmTV-NeuN transgenic mice (NeuN) were challenged with syngeneic NT2.5-LM cells that spontaneously metastasize to the lungs. Mice were treated with different combinations of entinostat, anti-CTLA-4, anti-PD-1, and anti-HER2 for 3 weeks for analysis of survival and metastatic tumor burden. For analyses of tumor-infiltrating immune cells in pulmonary metastases, flow cytometry of dissociated lungs was done 6 weeks after tumor injection. Results: Unlike the previously published model of early-stage HER2+ breast cancer, combinations of entinostat and ICIs did not improve survival in NT2.5-LM bearing mice. Anti-HER2 therapy was the only agent to improve survival, and its effect was hindered by the addition of entinostat and ICIs. The numbers of pulmonary metatastases among treatment groups were not significantly different. Although the combination of entinostat and ICIs increased the percentage of cytotoxic CD8 T cells in the lungs, it also increased the percentage of the more suppressive monocytic-MDSCs and decreased the percentage of less suppressive granulocytic-MDSCs. Whereas entinostat decreased suppressive activity of MDSCs from primary tumors, entinostat did not significantly change functional markers of MDSCs in the lung. Conclusion: Entinostat and ICIs did not reduce breast metastases in the lung, nor did their combination improve survival. Previous studies showed that entinostat and ICIs reduced MDSC suppressive function within the TME. Reduced suppression was not observed in lung metastases. Investigations are underway to define mechanisms responsible for the differential effect of entinostat on MDSC phenotype in primary tumors but not in the metastatic niche. Citation Format: Julie K. Jang, Christine Rafie, Sofi Castanon, Brian J. Christmas, Kayla A. Cruz, Skylar Woolman, Evanthia T. Roussos Torres. Breast pulmonary metastases and associated myeloid derived suppressor cells are resistant to the effects of entinostat with checkpoint inhibitor in a murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2730.