Targeting PD-1 Research Articles

Abstract 6646: Inhibition of HuR/CD147/IL-6 axis enhances anti-PD1 immunotherapy response in cancer

Abstract Immune checkpoint blockade (ICB) targeting PD-1/PD-L1 has emerged as a revolutionizing treatment modality for multiple cancers. However, durable remission and objective response rates with ICB monotherapy remain low. Several immune resistance mechanisms have been revealed and used to develop combinational therapy. In consideration of the durable and impressive responses observed in ‘hot’ tumors, it will be valuable to make an endeavor to elucidate the tumor evasion mechanisms. Despite the well-established mechanisms in immunotherapy resistance, our study uncovered a new mechanism of tumor immune evasion through the RNA-binding protein HuR/CD147 axis. The RNA-binding protein Hu antigen R (HuR) is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs. The tumor-associated antigen CD147, a highly glycosylated transmembrane immunoglobulin, is widely and specifically expressed in multiple malignancies. We phenotypically observed the downregulation of CD147 in HuR-deficient cells and elucidated that HuR regulates CD147 mRNA via binding to the AU-rich elements of its 3' UTR. The direct binding of HuR to CD147 mRNA in multiple types of cells was validated by Ribonucleoprotein immunoprecipitation (RNP-IP) and RNA pulldown assay. These data together clarify HuR is engaged in CD147 post-transcription regulation. KH-39 is a HuR-specific small molecule inhibitor, which disrupts the HuR function by competing for the binding site with its target mRNAs. KH-39 treatment reduced CD147 level in a time-/dose-dependent manner. We also tested the anti-tumor action of HuR small molecular inhibitor KH-39 in tumor proliferation, clonogenicity, cell death, cytokine secretion, and in vivo tumor growth. The cytokine array assay indicated multiple immune-related cytokines changed after HuR inhibition and CD147 knockdown, including IL-6, which is proven to act as an immunomodulating agent in the tumor microenvironment. These data demonstrated the potential mechanism of overcoming immunoresistance by inhibiting the HuR/CD147/IL-6 axis. To test the combination strategy using HuR inhibitor and anti-PD-1 antibody in vivo, we established the EMT6 orthotopic mouse breast cancer model. A combination of HuR inhibitor KH-39 and anti-PD-1 antibody enhanced the anti-PD1 immunotherapy response and prolonged survival compared to the single agent group. Taken together, we preliminarily explored the HuR/CD147/IL-6 axis in tumor immune evasion, which might be a promising therapeutic target to overcome immune evasion and improve immunotherapy response in cancer. This finding may have reference significance for clinical cancer therapy. Citation Format: Qi Zhang, Lauren Dandreo, Xiaoqing Wu, Liang Xu. Inhibition of HuR/CD147/IL-6 axis enhances anti-PD1 immunotherapy response in cancer [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 6646.

Abstract 5226: Exome-wide association study identifies coding variant in IL17RA associated with survival in cancer patients treated with immunotherapy

Abstract Immune checkpoint inhibitors (ICI) targeting PD-1/PD-L1 and CTLA4 are effective treatments in multiple cancer types, but durable responses are seen only in a minority of patients. Resistance to ICI is likely to reflect a combination of tumor-intrinsic, such as tumor mutational burden, and tumor-extrinsic factors, such as systemic immune suppression and host genetics. With the advent of large-scale germline genotyping studies, there is emerging evidence that host-specific genetic factors can substantially modify both disease risk and responsiveness to therapy. There has been limited characterization to date of the interaction between host genetics and response to ICI. Here, we perform harmonized mutation calling in available normal exome sequencing from 668 metastatic cancer patients treated with ICI (derived from n=7 studies, spanning n=3 cancer types and PD-1/PD-L1/CTLA4-directed therapy) and performed exome-wide association studies in European ancestry patients. For each cancer type (melanoma, renal cell carcinoma, urothelial carcinoma), we conducted exome-wide Cox regression (with PC1-4 and sex as covariates) to identify coding single-nucleotide polymorphisms (SNPs) associated with overall survival (OS). On fixed-effect meta-analysis, we identified a single coding variant (rs140221307, C allele, MAF 0.5% in European populations) that met genome-wide significance (p=1.11 × 10−15, HR=4.86, 95% CI 3.30-7.15). This SNP causes a substitution of Trp to Arg at amino acid 320 at the interface between the transmembrane and extracellular domains of interleukin 17 receptor A (IL17RA). While the secretion mechanism is uncharacterized, soluble IL17RA can be readily detected in plasma and the survival-reducing variant is associated with substantially elevated plasma IL17RA (p=1.90 × 10−9, beta=1.01, Sun et al. 2018) as well as reduced monocyte counts in UK Biobank (p=2.20 × 10−308, beta=-0.60). We have established a prime editing strategy to induce this variant in cultured human monocytes to investigate its biochemical and functional consequences, and are performing validation studies in independent patient cohorts. Soluble IL17RA would be expected to function as a decoy receptor to antagonize IL17/IL17RA signaling, and so these data suggest that activation of IL-17 receptor signaling might improve responsiveness to ICI therapy. Citation Format: Sam O. Kleeman, Michael Chan, Matthew Chvasta, Tobias Janowitz. Exome-wide association study identifies coding variant in IL17RA associated with survival in cancer patients treated with immunotherapy. [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 5226.

Abstract 5106: The addition of radiation therapy to simultaneous targeting of PD-1 and IL2Rβγ enhances antigen-specific CD8 T cell anti-tumor immunity in pancreatic cancer models

Abstract Although IL-2 has been shown to critically mediate NK and T cell effector functions, when used in clinical trials this cytokine has failed due to toxicity and severe side effects of the systemic treatment. Our lab has investigated the use of a next-generation IL-2 compound, murine PD1-IL2v, which consists of an anti-PD-1 antibody fused to a IL2-variant with abolished binding to IL2Rα. Previous work shows this molecule results in tumor-antigen specific T cell expansion and differentiation towards immune effectors. Here, we report for the first time that radiation therapy amplifies the anti-tumor effect observed with PD1-IL2v treatment of murine orthotopic models of local tumor growth and metastasis. This anti-tumor effect is correlated with increases in proliferative and polyfunctional CD8 T cells across tumor, lymph node, and blood compartments. Furthermore, using an OVA-antigen specific orthotopic model, we found that radiation in combination with PD1-IL2v treatment enhances infiltration and activation of antigen-specific CD8 T cells, which have been shown to be critical for immunotherapy clinical success. A portion of tumor-bearing mice treated with radiation and PD1-IL2v fully eradicated their primary tumors and tumor rechallenges, which we show correlates with increased markers of memory in CD8 T cells of these mice. To demonstrate the clinical relevance of these preclinical findings, we performed RNA sequencing on paired pancreatic adenocarcinoma (PDAC) patient tumor tissue samples prior to and following radiation. We observed enhanced IL2Rβ and IL2Rγ expression as well as IL2R signaling in PDAC-patient responders to radiation therapy compared to non-responders. In conclusion, the antigen-specific, lasting memory response to preclinical PDAC models demonstrated here provides rationale for the treatment of PDAC with a radiation and PD1-IL2v combination. Citation Format: Maureen Hoen, Miles Piper, Laurel B. Darragh, Diemmy Nugyen, Jacob Gadwa, Maria Amann, Greta Durini, Michael Knitz, Benjamin Van Court, Tiffany Pham, Ross Kedl, Pablo Umana, Laura Codarri-Deak, Christian Klein, Angelo D’Alessandro, Sana D. Karam. The addition of radiation therapy to simultaneous targeting of PD-1 and IL2Rβγ enhances antigen-specific CD8 T cell anti-tumor immunity in pancreatic cancer models. [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 5106.

Abstract 6367: BSI-507, a first-in-class bispecific antibody targeting PD1 and PVRIG for cancer immunotherapy

Abstract Introduction: Despite the success of anti-PD1/PDL1 therapies, only a small fraction of patients benefit from these checkpoint inhibitors (CPIs). Novel approaches to improve outcomes for patients who are resistant to current CPIs are needed. PVRIG is expressed on CD4+ and CD8+ T cells, NKT and NK cells. PVRIG binds with high affinity with its ligand PVRL2, which is expressed on tumor cells and some myeloid cells. The PVRIG-PVRL2 axis exerts an inhibitory effect on the cytotoxic activity of lymphocytes. Bispecific antibodies that exhibit dual blockade of PD1 and PVRIG provide a promising strategy to enhance anti-tumor immune response. Methods: An anti-PD1 mAb was identified from PD1 KO mice immunized with PD1-ECD-Fc and screened by our proprietary H3 (High-throughput, High-content and High-efficiency) platform. An anti-PVRIG mAb was identified from rats immunized with recombinant PVRIG-ECD-Fc and screened by the H3 platform. Both anti-PD1 and anti-PVRIG antibodies were humanized, and the anti-PD1 scFv was fused to the N-terminus of the heavy chain of the anti-PD1 antibody via a flexible linker. The binding activities and affinities were evaluated by ELISA, FACS and SPR, and the ligand blocking activities were measured by ELISA and FACS. Cell-based reporter assays were used to evaluate the functions of the anti-PD1 and anti-PVRIG mAbs alone and the bispecific antibody. In addition, the activity of the bispecific antibody to reverse PD1 and PVRIG mediated suppression of CMV pp65495-503 antigen specific CD8+ T-cell cytotoxicity was evaluated. Results: BSI-507, an anti-PD1xanti-PVRIG bispecific antibody demonstrated comparable activity to the parental anti-PD1 antibody regarding PD1 binding and PD1/PDL1 blocking. It also exhibited comparable activity to the parental anti-PVRIG antibody in PVRIG binding and PVRIG/PVRL2 blocking. Based on cell-based reporter assays, BSI-507 was able to reverse either PD1 or PVRIG mediated T-cell suppression and exhibited comparable potency to the parental antibodies. BSI-507 was also able to show enhanced reversal of both PD1 and PVRIG mediated T-cell suppression, much better than anti-PD1 or anti-PVRIG alone. In addition, BSI-507 showed the ability to reverse PD1 and PVRIG mediated suppression of CMV pp65495-503 antigen specific CD8+ T-cell cytotoxicity, stronger than either the anti-PD1 or anti-PVRIG monoclonal antibody. Summary: BSI-507 is a first-in-class anti-PD1xanti-PVRIG bispecific antibody for dual blockade of PD-1 and PVRIG pathways to enhance reversal of T cell inhibition. BSI-507 demonstrates favorable biophysical and functional characteristics, supporting the initiation of development activities including manufacturing and IND-enabling studies. Citation Format: Zeyu Peng, Xiaodong F. Liu, Hongyan Li, Wenwen Dai, Jinyu Liu, Xiaoyao Hao, Shukai Xia, Qun Lv, Hugh M. Davis, Mingjiu Chen, Mark Z. Ma. BSI-507, a first-in-class bispecific antibody targeting PD1 and PVRIG for cancer immunotherapy. [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 6367.

Abstract 1846: Intratumoral mPH-762, a self-delivering RNAi therapeutic (INTASYL™) targeting mouse PD-1, generates systemic tumor-specific memory CD8 T cells, providing a mechanism for abscopal efficacy toward untreated tumors in a murine hepatocarcinoma model

Abstract Intratumoral (IT) administration of immune checkpoint inhibition (ICI) holds potential to enhance local activity while decreasing systemic toxicity, however, high molecular weight antibodies are not well-suited for this application. PH-762, a self-delivering RNAi compound targeting PD-1 built on proprietary INTASYL™ technology, rapidly enters tissues without need for further formulation or delivery vehicles. In preclinical studies, PH-762 and its murine-targeted analog (mPH-762) provide robust PD-1 silencing, anti-tumor efficacy and associated immunophenotypic changes in the tumor microenvironment (TME). PH-762 is currently under clinical investigation in a Phase 1b study as neoadjuvant therapy for advanced resectable melanoma. We have previously shown that IT mPH-762 provides abscopal efficacy toward untreated distal tumors in a bilateral Hepa1-6 model of murine hepatocellular carcinoma. Here, we identify potential mechanisms underlying this abscopal efficacy in follow-up studies in vivo and ex vivo. Equivalent inoculums of Hepa1-6 cells were subcutaneously implanted into bilateral flanks of C57BL/6J mice (N = 12/group). When tumors reached a mean volume of 150 mm3, vehicle (PBS) or mPH-762 (0.5 mg/dose or 2 mg/dose) were administered to one of the two tumors (the directly treated tumor), on Days 1, 4, 7, 10 and 13. Tumor volumes and body weights were recorded longitudinally. Directly treated (DT) and untreated distal tumors (UT), as well as mesenteric lymph nodes (mLNs) and spleens (SP) were isolated from a satellite group (N = 6) on Day 14. On-target immunomodulatory changes were assessed in DT and UT TME by immunostaining/flow cytometry. Additionally, systemic tumor specific T cells were expanded from bulk mLN and SP by 21-day culture with irradiated Hepa1-6 cells. Tumor specific memory reactivity was assessed by challenge with either intact Hepa1-6 cells; or intact CT26 BALB/c colon cancer cells, PMA/ionomycin, or PBS controls: and assessed by intracellular IFN-γ, TNF-α and surface CD107a staining. IT mPH-762 again provided an abscopal decrease in mean tumor volume of UT tumors compared to IT PBS. On-target immunomodulatory changes to TME appeared primarily confined to the DT only at this timepoint. However, IT mPH-762 increased both the frequency and reactivity of Hepa1-6-specific CD8 T cells expanded from mLN and SP compared to IT PBS. These data indicate that IT mPH-762 generates a systemic immune response of durable tumor-specific reactive memory CD8 T cells in systemic lymphoid organs, providing a mechanism for the abscopal efficacy of mPH-762. These data further support the clinical development of IT PH-762. Citation Format: Benjamin G. Cuiffo, Andrew Boone, Dingxue Yan, Melissa Maxwell, Brianna Rivest, James Cardia, Simon P. Fricker. Intratumoral mPH-762, a self-delivering RNAi therapeutic (INTASYL™) targeting mouse PD-1, generates systemic tumor-specific memory CD8 T cells, providing a mechanism for abscopal efficacy toward untreated tumors in a murine hepatocarcinoma model [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 1846.

Abstract 6043: Manipulation of mitochondrial metabolism, through SIRT3 agonists, in a novel immunoreactive melanoma organoids model

Abstract The interplay between tumor cell metabolism and immune cells in the tumor microenvironment has emerged as a determinant factor in cancer progression and treatment. Sirtuin 3 (SIRT3) is a mitochondrial deacetylase that modulates ROS levels and governs many enzymes that regulate mitochondrial metabolism and homeostasis. SIRT3 has a dual role in cancer; it is regarded as an oncogene and tumor suppressor gene simultaneously. It has tumor-promoting action via keeping ROS levels low and favoring cell proliferation; on the contrary, it can trigger cell death under stress conditions by modulating tumor cell glycolysis. Recent data suggest a role for SIRT3 in cancer immunology with PD-L1 levels are found to be inversely associated with SIRT3 level in cervical cancer, and CD8+ T cells from SIRT3 knockout donor mice exhibited low GVHD in recipients. In this study, we explored the potential role of SIRT3 agonists in sensitizing the immunoreactivity of melanoma cells in a novel immunoreactive melanoma organoids model. We used the B16-F10 melanoma cell line spheroids to determine the effects of SIRT3 agonists on cellular viability, using an ATP assay. Validation of SIRT3 expression was determined via Western blot. Non-toxic amounts of SIRT3 agonists were also applied to melanoma organoids prepared from patient-derived metastatic melanoma from a lymph node. Preliminary results revealed a significant decrease in the viability of the organoids treated with R-propranolol (20µM) and Imipramine blue (0.25µM) agonists. We further investigated the effect of SIRT3 agonists R-propranolol and Imipramine blue on immunotherapy targeting PD-1, Pembrolizumab (100 nM) and Nivolumab (100 nM), and CTLA-4 inhibitor Ipilimumab (100 nM). We found a limited effect of Pembrolizumab and Nivolumab. Anti-tumor cytotoxicity was markedly increased in combination of Imipramine blue with Pembrolizumab and R-propranolol with Nivolumab. We propose that these effects are mediated through the activation of the endogenous immune cells and/or via the regulation of the PD-L1 and NKD2 ligand expression levels in the melanoma cells to become more sensitized to immune cells. Further studies will aim to validate these results in immunoreactive organoids from both mice and human patients. Our studies will shed light on the immune and metabolic axes and the use of specific agonists to develop new immune therapy treatment strategies for melanoma. Citation Format: Azza M. El-derby, Nadeem Wajih, Jack Arbiser, Konstantinous Votanopoulos, Shay Soker. Manipulation of mitochondrial metabolism, through SIRT3 agonists, in a novel immunoreactive melanoma organoids model. [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 6043.

Abstract 5113: INTASYL self-delivering RNAi therapeutic dual targeting PD-1 and CTLA-4 provides synergistic antitumor efficacy in the treatment of murine colon cancer in vivo

Abstract Combination immune checkpoint inhibition (ICI) with antibodies targeting PD-1 and CTLA-4 provides superior outcomes compared to either monotherapy alone. This combination has been approved for advanced melanoma, metastatic colon cancer and others. However, combination therapy with anti-PD-1/CTLA-4 antibodies results in serious immune related adverse events (irAEs), presenting an obstacle for effective treatment with combination systemic anti-PD-1/CTLA-4. Intratumoral (IT) immunotherapy is a strategy to enhance local activity while decreasing systemic irAEs. While clinical testing of IT antibodies is underway, antibodies' high molecular weight may limit their local diffusion and retention time within tumors. RNAi is an emerging therapeutic modality well-suited for local clinical application of ICI. We have demonstrated that self-delivering RNAi therapeutics built on proprietary INTASYL™ technology specifically silence their targets in tissues without need for specialized formulations or delivery systems, and convey robust antitumor efficacy in vivo. Furthermore, multiple INTASYL compounds can be easily co-formulated into a multi-targeting therapeutic, providing specific silencing of multiple therapeutic targets in a single injection. Here, we present proof-of-concept (POC) in vivo data demonstrating synergistic efficacy of a novel INTASYL dual-targeting murine PD-1 (mPH-762) and CTLA-4 (27790) coformulation in PBS in a syngeneic CT26 model of murine colon cancer. Dual on-target silencing was first validated in murine T cells in vitro. In vivo, CT26 cells were implanted subcutaneously into female BALB/c mice. Vehicle (PBS) or INTASYL IT treatment commenced when tumors reached a mean threshold volume (150 mm3; Day 1) with doses given on Days 1, 4, 7, 10, and 13. Mice received a total 1 mg/dose with the dual-targeting INTASYL, comprised of 0.5 mg of each compound. Another group received the dual-targeting INTASYL at 2 mg/dose. Tumor volumes and body weights were recorded longitudinally and analyzed by area under the curve (AUC). Tumors were isolated on Day 15 and mechanistic immunomodulation of the tumor microenvironment (TME) was assessed by flow cytometry. IT INTASYL dual-targeting PD-1 and CTLA-4 elicited robust dose-associated antitumor efficacy that was superior to the identical total dose of either single-targeting INTASYL, when comparing mean tumor volume AUC, demonstrating antitumor synergy by the dual-targeting coformulation. On-target mechanistic immunomodulatory effects were observed in the TME. These data demonstrate POC synergistic efficacy of IT INTASYL dual-targeting CTLA-4/PD-1 in vivo, supporting further development to maximize efficacy and minimize irAEs. PH-762 is currently under clinical investigation in a Phase 1b study for advanced melanoma. Citation Format: Benjamin G. Cuiffo, Andrew Boone, Dingxue Yan, Melissa Maxwell, Brianna Rivest, James Cardia, Simon P. Fricker. INTASYL self-delivering RNAi therapeutic dual targeting PD-1 and CTLA-4 provides synergistic antitumor efficacy in the treatment of murine colon cancer in vivo. [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 5113.

Abstract 2963: CDX-585, a novel bispecific antibody targeting PD-1 and ILT4

Abstract Both PD-1 and ILT4 (LILRB2, CD85d) have well established immunosuppressive effects that enable tumors to evade anti-tumor immunity. Tumor expression of PD-L1 and ILT4 has been associated with poor clinical outcomes in several cancer types. Recently, ILT4 activation has been postulated as a resistance mechanism for PD-1/PD-L1 blockade prompting the investigation of dual blockade of these pathways. Siu, L.L. et al. (2022 Clin Cancer Res 28: 57-70) reported encouraging results of the initial clinical study combining the ILT4 antagonist mAb MK-4830 with pembrolizumab. The combination was generally well tolerated and led to several durable responses including in PD-1/PD-L1 refractory patients. We have combined novel PD-1 (mAb E1A9) and ILT4 (mAb 7B1) antagonist antibodies into a tetravalent IgG-scFv format bispecific antibody (bsAb, CDX-585). CDX-585 was engineered to avoid effector functionality by eliminating interaction with Fcγ receptors, and to increase its half-life by enhancing binding to FcRn. As expected, CDX-585 inhibits interaction of PD-1 and ILT4 with their ligands, results in potent inhibition of PD-1 signaling and enhances myeloid cell inflammatory responses to stimulation through toll like receptors (LPS) or CD40 (CD40 agonist mAb). In mixed lymphocyte reactions CDX-585 promoted greater T cell activation than the combination of the parental mAbs. Similarly, CDX-585 demonstrated superior anti-tumor activity over the combination of the parental mAbs in a humanized mouse model of melanoma. CDX-585 has completed GMP manufacturing and IND-enabling activities. Doses of CDX-585 up to 60 mg/kg were well tolerated in cynomolgus macaques and displayed a favorable pharmacokinetic profile. Together these data demonstrate that CDX-585 effectively combines PD-1 and ILT4 blockade into one molecule with favorable biophysical and functional characteristics supporting the initiation of a dose-escalation clinical trial in patients with advanced solid tumors. Citation Format: Lawrence J. Thomas, Laura A. Vitale, Michael Murphy, Collin Xia, Zeyu Peng, Asma Ejas, Montu Patel, James Boyer, April R. Baronas, Thomas O'Neill, Jenifer Widger, Laura Mills-Chen, Andrea Crocker, Mark Ma, Mingjiu Chen, Hugh M. Davis, Russ A. Hammond, Cherie Taglienti, Michael Yellin, Joel Goldstein, Diego Alvarado, Henry C. Marsh, Tibor Keler. CDX-585, a novel bispecific antibody targeting PD-1 and ILT4 [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 2963.

Abstract 5865: Combinatorial activity of amivantamab and pembrolizumab in head and neck squamous cell carcinoma and lung squamous cell carcinoma expressing wild-type EGFR and MET

Abstract Introduction: Unmet needs exist for immunotherapy targeting PD-1/PD-L1 in head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LUSC) due to its suboptimal response. Amivantamab, a bispecific antibody targeting epidermal growth factor receptor (EGFR) and c-Met, has been demonstrated to induce antibody-dependent cytotoxicity and trogocytosis in tumor cells. We hypothesized that combination of amivantamab with pembrolizumab may synergistically enhance antitumor immunity. In this study, we present comprehensive immunomodulatory and synergistic antitumor efficacy of amivantamab and pembrolizumab in humanized HNSCC and LUSC mice models. Methods: EGFR and MET-expressing tumors from a HNSCC and a LUSC patient were transplanted into Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) models. Tumor-bearing PDXs were treated with vehicle, pembrolizumab (10mpk, Q5D, n=10), amivantamab (10mpk, BIW, n=10), or a combination of pembrolizumab and amivantamab (n=10). Analysis of immune modulatory responses within the tumor microenvironment (TME) using multiplexed IHC, flow cytometry, and single cell RNA sequencing was performed. Results: Combination of amivantamab and pembrolizumab showed a significant reduction of tumor volume (p<0.001) compared to vehicle or single treatment in both models. Additionally, significantly longer survival was observed for combination treated compared to the vehicle treated groups (p<0.0001). Multispectral imaging of tumor indicated that granzyme B-producing CD8+ T cells were significantly increased within the tumor in the combination group (p<0.01). Further analysis of T cell subsets suggested that central memory type CD8+ T cells were increased upon combination treatment. This group also demonstrated significantly higher CEA-tetramer positive CD8+ T cells in the tumor (p<0.01), suggesting that cytotoxic T cells recognizing tumor specific antigens enhanced antitumor immune response. Single cell RNA sequencing analysis of HNSCC showed that an EGFRhighMEThigh cluster was enriched in the TME after pembrolizumab treatment. This subcluster had elevated glycolysis and lactic acid pathway-related genes compared to EGFRlowMETlow cluster. Lactate transporter, MCT4 (SLC16A3) and LDHA genes were dramatically increased in the EGFRhighMEThigh cluster. Elevated lactic acid pathway may lead to immune evasion in the tumor, dampening the activity of pembrolizumab. Interestingly, combination treatment with amivantamab could reduce EGFRhighMEThigh cluster, and could effectively control tumor via creating favorable immune TME. Conclusion: Our study demonstrated combinatorial benefits of amivantamab and pembrolizumab by effectively remodeling TME, providing a preclinical rationale to clinically combine amivantamab and PD-1 blockade treatments. Citation Format: Sun Min Lim, Chun-Bong Synn, Seong-san Kang, DongKwon Kim, Soo-Hwan Lee, Sujeong Baek, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Jii Bum Lee, Ji Yun Lee, Chang Gon Kim, Min Hee Hong, Kyoung-Ho Pyo, Joshua Curtin, Bharvin Patel, Isabelle Bergiers. Combinatorial activity of amivantamab and pembrolizumab in head and neck squamous cell carcinoma and lung squamous cell carcinoma expressing wild-type EGFR and MET [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 5865.

Abstract 2958: BSI-508, a novel bispecific fusion molecule targeting PD1 and CD47 for cancer immunotherapy

Abstract Introduction: While anti-PD1/PDL1 mAbs have shown success in the market only a small fraction of patients actually benefit from the therapies. Novel approaches to improve outcomes for patients who are resistant or refractory to current checkpoint inhibitors (CPIs) are needed. CD47 expressed on tumor cells can provide a “don’t eat me” signal to antigen-presenting cells (APCs) via the binding to SIRPα. Blocking this interaction, enhancing the phagocytosis of tumor cells, has emerged as an effective alternative to current CPIs. Here we report on the generation and characterization of a novel bispecific fusion molecule BSI-508, composed of an anti-PD1 antibody fused with a SIRPα extracellular domain (ECD). The SIRPα-ECD binds CD47 and blocks the CD47/SIRPα-mediated cell signaling pathway. BSI-508 is designed to target PD1 on T cells and CD47 on tumor cells, providing tumor killing by T cells and macrophages synergistically. Methods: An anti-PD1 mAb was identified from PD1 KO mice immunized with PD1-ECD-Fc and screened by our proprietary H3 (High-throughput, High-content and High-efficiency) platform. Human SIRPα-V2-ECD1 was fused to the N-terminus of the heavy chain of the anti-PD1 antibody via a flexible linker. The binding activities and affinities to PD1 and CD47 were evaluated by ELISA, FACS and SPR. Ligand blocking activity was measured by ELISA and FACS. A cell-based reporter assay was used to evaluate the function of the anti-PD1 portion and a macrophage mediated phagocytosis assay was used to evaluate the function of the SIRPα-V2-ECD1 portion of the bispecific. B-hPD-1/hPD-L1/hSIRPα/hCD47 mice bearing B-hPDL1/hCD47 MC38 tumors were used to evaluate the tumor inhibitory activity of the bispecific fusion molecule. Results: BSI-508 demonstrated comparable activity to the parental anti-PD1 monoclonal antibody regarding PD1 binding and PD1/PDL1 blocking. It also exhibited comparable activity to the SIRPα-V2-ECD1-Fc fusion protein in CD47 binding and CD47/SIRPα blocking. BSI-508 was able to reverse PD1 mediated T-cell suppression and exhibited comparable bioactivity to the parental anti-PD1 antibody. The bispecific molecule showed comparable bioactivity to the SIRPα-V2-ECD1-Fc fusion protein in inducing macrophage mediated phagocytosis of Jurkat cells. In addition, BSI-508 showed superior antitumor efficacy in vivo. Summary: BSI-508 is a novel anti-PD1xanti-CD47 bispecific fusion molecule combining both T-cell activation and macrophage mediated phagocytosis together for superior tumor killing. BSI-508 demonstrates favorable biophysical and functional characteristics, supporting the initiation of development activities including manufacturing and IND-enabling studies. Citation Format: Zeyu Peng, Xiaodong F. Liu, Hongyan Li, Wenwen Dai, Jinyu Liu, Xiaoyao Hao, Shukai Xia, Qun Lv, Hugh M. Davis, Mingjiu Chen, Mark Z. Ma. BSI-508, a novel bispecific fusion molecule targeting PD1 and CD47 for cancer immunotherapy [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 2958.

HX009, a novel BsAb dual targeting PD1 x CD47, demonstrates potent anti-lymphoma activity in preclinical models

Both PD1/PD-L1 and CD47 blockades have demonstrated limited activity in most subtypes of NHL save NK/T-cell lymphoma. The hemotoxicity with anti-CD47 agents in the clinic has been speculated to account for their limitations. Herein we describe a first-in-class and rationally designed bispecific antibody (BsAb), HX009, targeting PD1 and CD47 but with weakened CD47 binding, which selectively hones the BsAb for tumor microenvironment through PD1 interaction, potentially reducing toxicity. In vitro characterization confirmed: (1) Both receptor binding/ligand blockade, with lowered CD47 affinity; (2) functional PD1/CD47 blockades by reporter assays; (3) T-cell activation in Staphylococcal-enterotoxin-B-pretreated PBMC and mixed-lymphocyte-reaction. In vivo modeling demonstrated antitumor activity in Raji-B and Karpass-229-T xenograft lymphomas. In the humanized mouse syngeneic A20 B-lymphoma (huCD47-A20) HuGEMM model, which has quadruple knocked-in hPD1xhPD-L1xhCD47xhSIRPα genes and an intact autologous immune-system, a contribution of effect is demonstrated for each targeted biologic (HX008 targeting PD1 and SIRPα-Fc targeting CD47), which is clearly augmented by the dual targeting with HX009. Lastly, the expression of the immune-checkpoints PD-L1/L2 and CD47 seemed co-regulated among a panel of lymphoma-derived-xenografts, where HX009 maybe more effective in those with upregulated CD47. Our data warrants HX009’s further clinical development for treating NHLs.

Therapeutic efficacy of plant-produced Nivolumab in transgenic C57BL/6-hPD-1 mouse implanted with MC38 colon cancer

The therapeutic blockade of inhibitory PD-1 signaling has emerged as an effective approach for cancer immunotherapy. Nivolumab (Opdivo®), a monoclonal antibody (mAb) targeting the PD-1 immune checkpoint, is approved for treatment of several cancer indications. It functions by blocking the PD-1-mediated T-cell inhibition thus reinstating anticancer immune responses. Tremendous advances in plant biotechnology offer an alternative and economical strategy to produce therapeutic mAbs for immune-based therapies. In this study, recombinant anti-PD-1 Nivolumab was produced in Nicotiana benthamiana and the plant-produced anti-PD-1 mAb was exploited for cancer treatment in syngeneic mice model C57BL/6 mice that were used to test the antitumor efficacy of plant produced Nivolumab, along with commercial Opdivo®. C57BL/6 syngeneic mice treated with plant produced anti-PD-1 mAb exhibited reduction in the growth of established MC38 tumors. The plant produced Nivolumab treatment showed 82.9% antitumor effect in decreasing the tumor volume along with 50% tumor-free mice, whereas Opdivo® showed 90.26% reduction in volume without any tumor-free mice. Finally, plant-derived anti-PD-1 therapy was also well tolerated in tumor-bearing mice that correlated with no significant body weight changes. Overall, our plant-produced Nivolumab elicits significant inhibition of tumor growth in vivo and provides a proof-of-concept for the production of immunotherapy targeting PD-1.

Novel bispecific aptamer targeting PD-1 and nucleolin for cancer immunotherapy

BackgroundImmune checkpoint blockade (ICB) is a promising strategy for cancer treatment and has achieved remarkable clinical results. Further improvement of ICB efficacy may advance cancer immunotherapy and has evident medical importance. Here in this study, a PD-1 aptamer was functionalized with a tumor-homing nucleolin aptamer (AS1411) to build a novel bispecific agent (BiApt) for boosting the efficacy of ICB therapy.ResultsThe two aptamers were coupled together via sticky ends to form BiApt, which had an average size of 11.70 nm. Flow cytometry revealed that BiApt could bind with both the activated T cells and the nucleolin-expressing tumor cells. In addition, BiApt could recruit more T cells to the vicinity of nucleolin-positive tumor cells. Functionally, BiApt enhanced the PBMC-mediated anticancer cytotoxicity in vitro compared with free PD-1 aptamer. Moreover, in an animal model of CT26 colon cancer, BiApt significantly boosted the antitumor efficacy vs. free PD-1 aptamer.ConclusionThe results suggest that bispecific agent combining ICB and tumor-homing functions has potential to improve the efficacy of ICB immunotherapy.

Immunotherapy Targeting PD-1/PD-L1 in Early-Stage Triple-Negative Breast Cancer.

The advent of immunotherapy, especially immune checkpoint inhibitors (ICIs), has revolutionized antitumor therapy. Programmed cell death receptor 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are among the most promising targets for encouraging the immune system to eliminate cancer cells. PD-1/PD-L1 have made clinical remission for numerous solid tumors, including metastatic triple-negative breast cancer (TNBC). In recent years, integrating PD-1/PD-L1 inhibitors into existing treatments in early-stage TNBC has attracted wide attention. Herein, we summarize the clinical benefit of PD-1/PD-L1 inhibitors plus neoadjuvant chemotherapy, adjuvant chemotherapy, and targeted therapy in early-stage TNBC. Possible immunotherapy biomarkers, immune-related adverse events (irAEs), and the key challenges faced in TNBC anti-PD-1/PD-L1 therapy are also concluded. Numerous studies on immunotherapy are ongoing, and PD-1/PD-L1 inhibitors have demonstrated great clinical prospects in early-stage TNBC. To maximize the efficacy of anti-PD-1/PD-L1 therapy, further research into the challenges which still exist is necessary.

Tertiary Lymphoid Structure Raises Survival and Immunotherapy in HPV− HNSCC

Immune checkpoint blockade (ICB) targeting PD-1/PD-L1 has been used for the treatment of head and neck squamous cell carcinoma (HNSCC). However, the overall response rate to ICB therapy for HNSCC remains less than 20%. It has recently been reported that the appearance of tertiary lymphoid structures (TLSs) in tumor tissue is correlated with better prognosis and response to ICB treatment. Here, we demonstrated an immune classification for the tumor microenvironment (TME) of HNSCC by analyzing The Cancer Genome Atlas (TCGA)–HNSCC data set and found that immunotype D with TLS enrichment had a better prognosis and response to ICB treatment. Furthermore, we observed that TLSs were present in a part of tumor samples of human papillomavirus (HPV) infection negative HNSCC (HPV− HNSCC) and were associated with the densities of dendritic cell (DC)–LAMP+ DCs, CD4+ T cells, CD8+ T cells, and progenitor T cells in TME. We established an HPV− HNSCC mouse model with TLS-enriched TME by overexpressing LIGHT in a mouse HNSCC cell line. We found that the induction of TLS formation enhanced the response to PD-1 blockade treatment in the HPV− HNSCC mouse model, accompanied by increases in DCs and progenitor exhausted CD8+ T cells in the TME. Elimination of CD20+ B cells attenuated the therapeutic effect of PD-1 pathway blockade in TLS+ HPV− HNSCC mouse models. These results indicate that TLSs contribute to the favorable prognosis and antitumor immunity of HPV− HNSCC. Inducing TLS formation in HPV− HNSCC tumors is a potential therapeutic method for improving the ICB response rate in patients with HPV− HNSCC.

Retrospective Analysis of the Predictive Value of 18F-FDG PET/CT Metabolic Parameters for PD-L1 Expression in Cervical Cancer

Immunotherapy targeting PD-1/PD-L1 has been proven to be effective for cervical cancer treatment. To explore non-invasive examinations for assessing the PD-L1 status in cervical cancer, we performed a retrospective study to investigate the predictive value of 18F-FDG PET/CT. The correlations between PD-L1 expression, clinicopathological characteristics and 18F-FDG PET/CT metabolic parameters were evaluated in 74 cervical cancer patients. The clinicopathological characteristics included age, histologic type, tumor differentiation, FIGO stage and tumor size. The metabolic parameters included maximum standard uptake (SUVmax), mean standard uptake (SUVmean), total lesion glycolysis (TLG) and tumor metabolic volume (MTV). In univariate analysis, SUVmax, SUVmean, TLG, tumor size and tumor differentiation were obviously associated with PD-L1 status. SUVmax (rs = 0.42) and SUVmean (rs = 0.40) were moderately positively correlated with the combined positive score (CPS) for PD-L1 in Spearman correlation analysis. The results of multivariable analysis showed that the higher SUVmax (odds ratio = 2.849) and the lower degree of differentiation (Odds Ratio = 0.168), the greater probability of being PD-L1 positive. The ROC curve analysis demonstrated that when the cut-off values of SUVmax, SUVmean and TLG were 10.45, 6.75 and 143.4, respectively, the highest accuracy for predicting PD-L1 expression was 77.0%, 71.6% and 62.2%, respectively. The comprehensive predictive ability of PD-L1 expression, assessed by combining SUVmax with tumor differentiation, showed that the PD-L1-negative rate was 100% in the low probability group, whereas the PD-L1-positive rate was 84.6% in the high probability group. In addition, we also found that the H-score of HIF-1α was moderately positively correlated with PD-L1 CPS (rs = 0.51). The SUVmax and differentiation of the primary lesion were the optimum predictors for PD-L1 expression in cervical cancer. There was a great potential for 18F-FDG PET/CT in predicting PD-L1 status and selecting cervical cancer candidates for PD1/PD-L1 immune checkpoint therapy.

TROPHU-U-01 cohort 5: Evaluation of maintenance sacituzumab govitecan (SG) plus zimberelimab (ZIM), ZIM, or avelumab in cisplatin-eligible patients (pts) with unresectable or metastatic urothelial cancer (mUC).

TPS598 Background: SG is an antibody-drug conjugate composed of an anti-trophoblast cell-surface antigen-2 (Trop-2) antibody coupled to a topoisomerase-I inhibitor, SN-38, via a hydrolyzable linker. In Cohort 1 of the phase 2 TROPHY-U-01 trial, SG monotherapy resulted in a 27% objective response rate (ORR) and median overall survival (OS) of 10.9 months with an overall manageable toxicity profile in pts with locally advanced or mUC who previously received platinum-based therapy and a checkpoint inhibitor (CPI; Tagawa S, et al. J Clin Oncol. 2021). These results led to accelerated FDA approval of SG in this population. The CPI ZIM is a fully human IgG4 monoclonal antibody targeting PD-1. CPIs are active in mUC, including avelumab that is FDA-approved as switch maintenance therapy for locally advanced or mUC that has not progressed with first-line platinum-based chemotherapy. Cohort 5 of the TROPHY-U-01 trial will evaluate the safety, tolerability, and efficacy of SG + ZIM vs ZIM alone vs avelumab as switch maintenance in pts with mUC who have received gemcitabine (GEM)/cisplatin without progressive disease (PD). Methods: TROPHY-U-01 (NCT03547973) is a multicohort, open-label, global, phase 2 trial. Cohort 5 includes pts who have not progressed after completion of 4-6 cycles of GEM/cisplatin. Key eligibility requirements include ≥18 y; ECOG PS 0-1; available tissue for biomarker testing; adequate blood counts without transfusion or growth factor within 2 weeks of study drug initiation; creatinine clearance ≥30 mL/min. A safety lead-in of 6-8 pts will be conducted, where pts will be treated with SG 10 mg/kg IV on D1 and D8 of a 21-D cycle plus ZIM 360 mg IV q3wk on a 21-D cycle. On safety lead-in, if SG + ZIM is deemed safe, pts will be randomized 1:1:1. Those in Arm 1 will receive SG 10 mg/kg IV on D1 and D8 of a 21-D cycle followed by ZIM 360 mg IV, q3wk (D1 of a 21-D cycle). Pts in Arm 2 will receive avelumab 800 mg IV q2wk (D1 of a 14-D cycle). Pts in Arm 3 will receive ZIM 360 mg IV q3wk. All pts will continue treatment until PD, unacceptable toxicity, or loss of clinical benefit. The primary endpoint is progression-free survival based on central review by RECIST 1.1 criteria. Secondary endpoints include OS (all arms), safety/tolerability of SG in combination with ZIM (Arm 1). Cohort 5 aims to enroll an estimated 158 pts. A sample size of 50 pts per randomized arm is determined based on clinical consideration for exploring the activity of anticancer agents. Clinical trial information: NCT03547973 .

Expression of the immune targets in tumor-infiltrating immunocytes of gestational trophoblastic neoplasia.

Objectives: To evaluate the expression of emerging immune targets in the tumor-infiltrating immunocytes (TIIs) of human gestational trophoblastic neoplasia (GTN) specimens, and to analyze the correlation between the expression patterns and prognosis of GTN patients. Methods: Between January 2008 and December 2017, patients who were diagnosed histologically with GTN were included in this study. The expression densities of LAG-3, TIM-3, GAL-9, PD-1, CD68, CD8, and FOXP3 in the TIIs were assessed independently by two pathologists blinded to clinical outcomes. The expression patterns and correlation with patient outcomes were analyzed to identify prognostic factors. Results: We identified 108 patients with GTN, including 67 with choriocarcinoma, 32 with placental site trophoblastic tumor (PSTT), and 9 with epithelioid trophoblastic tumor (ETT). Almost all GTN patients showed expression of GAL-9, TIM-3, and PD-1 in TIIs (100%, 92.6%, and 90.7%, respectively); LAG-3 was expressed in 77.8% of the samples. The expression densities of CD68 and GAL-9 were significantly higher in choriocarcinoma than that in PSTT and ETT. The TIM-3 expression density in choriocarcinoma was higher than that in PSTT. In addition, the expression density of LAG-3 in the TIIs of choriocarcinoma and PSTT was higher than that in ETT. There was no statistical difference in the expression pattern of PD-1 among different pathological subtypes. The positive expression of LAG-3 in tumor TIIs was a prognostic factor for disease recurrence, and patients with positive expression of LAG-3 in the TIIs had poorer disease-free survival (p = 0.026). Conclusion: Our study evaluated the expression of immune targets PD-1, TIM-3, LAG-3, and GAL-9 in the TIIs of GTN patients and found that they were widely expressed but not associated with patients' prognoses, excepting the positive expression of LAG-3 was a prognostic factor for disease recurrence.

IFN-γ Signaling Sensitizes Melanoma Cells to BH3 Mimetics

Immunotherapy targeting PD-1 and/or CTLA4 leads to durable responses in a proportion of patients with melanoma. However, many patients will not respond to these immune checkpoint inhibitors, and up to 60% of responding patients will develop treatment resistance. We describe a vulnerability in melanoma driven by immune cell activity that provides a pathway towards additional treatment options. This study evaluated short-term melanoma cell lines (referred to as PD1 PROG cells) derived from melanoma metastases that progressed on PD-1 inhibitor-based therapy. We show that the cytokine IFN-γ primes melanoma cells for apoptosis by promoting changes in the accumulation and interactions of apoptotic regulators MCL-1, NOXA, and BAK. The addition of pro-apoptotic BH3 mimetic drugs sensitized PD1 PROG melanoma cells to apoptosis in response to IFN-γ or autologous immune cell activation. These findings provide translatable strategies for combination therapies in melanoma.

Enhancer RNA-based modeling of adverse events and objective responses of cancer immunotherapy reveals associated key enhancers and target genes.

Immune checkpoint inhibitors (ICI) targeting PD-1/PD-L1 or CTLA-4 are emerging and effective immunotherapy strategies. However, ICI-treated patients present heterogeneous responses and adverse events, thus demanding effective ways to assess benefit over risk before treatment. Here, by integrating pan-cancer clinical and molecular data, we tried to predict immune-related adverse events (irAEs, risk) and objective response rates (ORRs, benefit) based on enhancer RNAs (eRNAs) expression among patients receiving anti-PD-1/PD-L1 therapies. We built two tri-variate (eRNAs) regression models, one (with ENSR00000326714, ENSR00000148786, and ENSR00000005553) explaining 71% variance (R=0.84) of irAEs and the other (with ENSR00000164478, ENSR00000035913, and ENSR00000167231) explaining 79% (R=0.89) of ORRs. Interestingly, target genes of irAE-related enhancers, including upstream regulators of MYC, were involved in metabolism, inflammation, and immune activation, while ORR-related enhancers target PAK2 and DLG1 which participate in T cell activation. More importantly, we found that ENSR00000148786 probably enhanced TMEM43/LUMA expression mainly in B cells to induce irAEs in ICI-treated patients. Our study provides references for the identification of immunotherapy-related biomarkers and potential therapeutic targets during immunotherapy.