Human PD-1 Research Articles

Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery

ABSTRACT The potential of antibodies, especially for the bispecific antibodies, are limited by high cost and complex technical process of development and manufacturing. A cost-effective and rapid platform for the endogenous antibodies expression via using the in vitro transcription (IVT) technique to produce nucleoside-modified mRNA and then encapsulated into lipid nanoparticle (LNP) may turn the body to a manufactory. Coinhibitory pathway of programmed death ligand 1 (PD-L1) and programmed cell death protein 1 receptor (PD-1) could suppress the T-cell mediated immunity. We hypothesized that the coblocking of PD-L1 and PD-1 via bispecific antibodies may achieve more potential antitumor efficacies compare with the monospecific ones. Here, we described the application of mRNA to encode a bispecific antibody with ablated Fc immune effector functions that targets both human PD-L1 and PD-1, termed XA-1, which was further assessed the in vitro functional activities and in vivo antitumor efficacies. The in vitro mRNA-encoded XA-1 held comparable abilities to fully block the PD-1/PD-L1 pathway as well as to enhance functional T cell activation compared to XA-1 protein from CHO cell source. Pharmacokinetic tests showed enhanced area under curve (AUC) of mRNA-encoded XA-1 compared with XA-1 at same dose. Chronic treatment of LNP-encapsulated XA-1 mRNA in the mouse tumor models which were reconstituted with human immune cells effectively induced promising antitumor efficacies compared to XA-1 protein. Current results collectively demonstrated that LNP-encapsulated mRNA represents the viable delivery platform for treating cancer and hold potential to be applied in the treatment of many diseases. Abbreviations: IVT: in vitro transcription; LNP: lipid nanoparticle; hPD-1: human PD-1; hPD-L1: human PD-L1; ITS-G: Insulin-Transferrin-Selenium; Pen/Strep: penicillin-streptomycin; FBS: fetal bovine serum; TGI: tumor growth inhibition; IE1: cytomegalovirus immediate early 1; SP: signal peptide; hIgLC: human immunoglobulin kappa light chain; hIgHC: human IgG1 heavy chain; AUC: area under the curve; Cl: serum clearance; Vss: steady-state distributed volume; MLR: mixed lymphocyte reaction.

Ruxolitinib Plus Nivolumab in Patients with R/R Hodgkin Lymphoma after Failure of Check-Point Inhibitors: Preliminary Report on Safety and Efficacy

Background: Recent studies demonstrated that classical Hodgkin lymphoma (cHL) is characterized by chromosome 9p24.1 amplification with associated overexpression of PD-1 ligands as well as JAK2 tyrosine kinase activation. Immune evasion mediated by the PD-1/PD-1 ligand axis can be inhibited with IgG4 agonist antibody specific for human PD-1 nivolumab which has shown excellent overall response rate (ORR) of over 60% in relapsed cHL. JAK2 induces PD-1 ligand expression and augments tumor cell proliferation. JAK2 signaling can be inhibited with clinical grade small molecules (i.e. ruxolitinib). Given this biology, we designed a Phase I/II study to target both pathways in cHL patients who had failed PD-1 blocking agents.Methods: This is a Phase I/II, multicenter, open-label, dose escalation/dose-expansion study to evaluate the safety and tolerability of ruxolitinib when combined with nivolumab (fixed dose 3mg/kg IV every 2 weeks) in patients with relapsed or refractory (R/R) cHL (NCT03681561). Eligible patients had to fail prior check-point inhibitor (CPI; nivolumab or pembrolizumab). Three dose levels of ruxolitinib were tested: 10 mg orally twice a day (bid), 15 mg bid, and 20 mg bid. Planned duration of therapy was 2 years. The primary objective was to find the maximum tolerated dose (MTD) of ruxolitinib when given with nivolumab and characterize the safety and tolerability of this combination. The phase 2 objective was to evaluate the overall response rate (ORR).Results: We enrolled 19 patients. Median age was 38 years (range 22-76); 68% were males. Patients were a median of 3.4 years from the initial diagnosis (range 0.9-16.7 years), 89% had stage III-IV disease and all had experienced progressive disease following CPI. 17 (89%) had prior autologous HCT and 1 had prior allogeneic HCT. All patients received nivolumab combined with ruxolitinib, and the highest ruxolitinib dose 20mg BID (MTD) was reached without DLTs. The combination was well tolerated; most AEs were grade 1 and 2. Only 3 patients required hospitalization (2 had pneumonia, one for disease progression). Three patients experienced immune mediated adverse events (LFT elevation: 1 Gr 1 and 1 Gr 2; 1 Gr 3 pneumonitis) and all were reversible. Three subjects were not evaluable for response (2 patients who received < 1 month of therapy due to rapid disease progression and Gr 2 immune hepatitis and 1 patient with short follow-up is not yet evaluable for response). Median follow-up was 13 months (range 3-27 months).In 16 patients evaluable for response, best ORR was 75% (12 of 16); including 3 CRs (19%), 2 PRs (13%), 6 patients had stable disease (SD, 44%) with tumor bulk reduction ranging from 10-45%. One patient had indeterminate response (negative biopsy of new PET-avid site). Duration of responses were 12.5 months (ranged from 3.7 to 20.4 months); 5 responders continue study therapy and 2 completed 2 years; 1 went on to autologous HCT in PR, and 3 progressed while on therapy after 6, 6 and 23 months. One patient died after later experimental therapy. Progression-free survival at 1 year was 64% (95%CI 34-84%).Conclusions: Inhibition of JAK2 combined blockade of the PD-1 pathway represent complementary rational therapeutic targets in cHL. Therapy combining ruxolitinib with nivolumab is well tolerated and yield encouragingly high remission rates and durable responses in patients who had all failed previous CPI. Pharmacodynamic and correlative analyses on the mechanism of synergism are underway. DisclosuresBachanova: KaryoPharma: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; FATE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding. Janakiram: ADC Therapeutics: Research Funding; FATE Therapeutics: Research Funding; Takeda Pharmaceuticals: Research Funding; Kyowa Kirin Therapeutics: Honoraria. Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding. Farooq: Kite, a Gilead Company: Honoraria. OffLabel Disclosure:ruxolitinib for Hodgkin lymphoma

Molecular features underlying differential SHP1/SHP2 binding of immune checkpoint receptors.

A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors.

A HER2 target antibody drug conjugate combined with anti-PD-(L)1 treatment eliminates hHER2+ tumors in hPD-1 transgenic mouse model and contributes immune memory formation.

Disitamab vedotin (RC48) is an HER2-directed antibody-drug conjugate, emerging as an effective strategy for cancer therapy, which not only enhances antitumor immunity in previous animal models but also improves clinical outcomes for patients such as with gastric cancer, urothelium carcinoma, and HER2 low-expressing breast cancer. Here, we explore the combination therapeutic efficacy of this novel HER2-targeting ADC with immune checkpoint inhibitors in a human HER2-expressing syngeneic breast cancer model. The human HER2+ cancer cell line is constructed by stable transfection and individual clones were isolated by single-cell sorting. Flow cytometry was performed to determine its binding activity. Cytotoxic effect was determined using an MTT assay with the supplement of RC48. Human PD-1 transgenic mice were used to analyze the in vivo antitumor effects of the ADC and its combination therapy with PD-1/PD-L1 antibody. The combination of RC48 and PD-1/PD-L1 immune checkpoint inhibition significantly enhanced tumor suppression and antitumor immunity. Tumor rejection in the synergistic groups was accompanied by massive T cell infiltration and immune marker activation. Furthermore, the combination therapy promoted immunological memory formation in the tumor eradication animals, protecting them from tumor rechallenge. A novel HER2-targeting ADC combined with immune checkpoint inhibitors can achieve remarkable effects in mice and elicit long-lasting immune protection in a hHER2+ murine breast cancer model. This study provides insights into the efficacy of RC48 therapeutic activity and a rationale for potential therapeutic combination strategies with immunotherapy.

Monitoring PD-1 Phosphorylation to Evaluate PD-1 Signaling during Antitumor Immune Responses.

PD-1 expression marks activated T cells susceptible to PD-1-mediated inhibition but not whether a PD-1-mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho-PD-1). We showed PD-1+ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1+TIM-3+ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8+ TILs. Phospho-PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.

Prec-O2-04 - Functional remodeling of macrophages with anti-PD-L1 and lenalidomide in cutaneous T cell lymphoma

<h3>Background:</h3> M2-like tumor-associated macrophages (TAMs) are the most abundant phenotype that promote the growth of cutaneous T cell lymphoma (CTCL) by inducing immunosuppression. Although PD1/PD-L1blockade has demonstrated efficacy in CTCL, little is known about the effects on tumor-associated macrophages. Here, we report that anti–PD-L1 treatment favorably impacts the phenotype and function of tumor macrophages by polarizing the macrophage compartment toward a more pro-inflammatory phenotype. Understanding the mechanisms of macrophage reprogramming will identify novel therapeutic targets to reverse impaired immunity. <h3>Methods and results:</h3> We used multiplex immunofluorescence staining of lesional skin samples of CTCL patients demonstrating co-localization of PD1 on CD163+ M2 macrophages. Moreover, RNA-seq analysis performed on tissue sections from same CTCL specimens revealed an up-regulation of the TLR, NF-κB and JAK/STAT signaling pathways. To further confirm PD1 expression on M2 TAMs in CTCL, we cultured CD14+ cells from healthy donor-derived peripheral blood in conditioned media (CM) from MyLa cells. We observed macrophage differentiation towards an PD1+ M2 phenotype, with significant CD163, CD206 and PD1 upregulation but not CD80 expression. The TRIF-stimulated NF-κB and JAK/STAT signaling pathways, downstream of both TLR3 and TLR4 were activated in PD1+ M2-like TAMs. Furthermore, our RT-PCR results show significantly elevated IL-10 levels, but decreased IL-1β, CXCL-10 and CXCL-11 compared to control. To determine whether PD1+ M2-like TAMs could be reprogrammed, anti-PD-L1 (durvalumab) and lenalidomide were used for treatment of MyLa-conditioned media induced human peripheral blood monocyte-derived PD1+ M2-like TAMs. The results show that anti-PD-L1 and lenalidomide synergistically increased IL-1β, CXCL-10, and CXCL-11 expression, but significantly decreased IL-10 level compared with the untreated control in vitro through ablation of TLR3, TLR4 and the downstream signaling pathways, which was linked with functional changes in phagocytic activity and cell migration.

The PD1 inhibitory pathway and mature dendritic cells contribute to abacavir hypersensitivity in human leukocyte antigen transgenic PD1 knockout mice

Based on recent genome-wide association studies, abacavir-induced hypersensitivity is highly associated with human leukocyte antigen (HLA)-B*57:01 allele. However, the underlying mechanism of this occurrence is unclear. To investigate the underlying mechanism, we developed HLA-B*57:01 transgenic mice and found that application of abacavir could cause CD8 T cell activation with elevation in PD1 expression; however, severe skin hypersensitivity was not observed. To eliminate the immunosuppressive effect of PD1, HLA-B*57:01 transgenic/PD1 knockout (01Tg/PD1) mice were generated by mating HLA-B*57:01 transgenic mice and PD1 knockout mice. Thereafter, 01Tg/PD1 mice were treated with abacavir. Similar to the above results, severe skin hypersensitivity was not observed. Therefore, we treated 01Tg/PD1 mice with an anti-CD4 antibody to deplete CD4 T cells, followed by abacavir topically and orally. Severe abacavir-induced skin hypersensitivity was observed in 01Tg/PD1 mice after depletion of CD4 T cells, in addition to significant CD8 T cell activation and dendritic cell maturation. Taken together, we succeeded in reproducing severe skin hypersensitivity in a mouse model. And we found that through the combined depletion of PD1 and CD4 T cells, CD8 T cells could be activated and could proceed to clonal proliferation, which is promoted by mature dendritic cells, thereby eventually inducing severe skin hypersensitivity.

Enhanced anti-tumor response elicited by a novel oncolytic HSV-1 engineered with an anti-PD-1 antibody.

Oncolytic viruses as cancer vaccines modulate the tumor microenvironment and act synergistically with immune checkpoint inhibitors to overcome resistance. Taking advantage of the loading capacity for exogenous genes, we generated a recombinant herpes simplex virus type 1 (HSV-1), HSV-aPD-1, carrying a full-length humanized anti-PD-1 monoclonal antibody (anti-PD-1 mAb) that replicates and expresses anti-PD-1 mAbs in tumor cells in vitro and in vivo. Its anti-tumor effect was assessed in human PD-1 knock-in mice by analyzing tumor inhibition, cell populations and RNA expression in tumors, and serum cytokine levels. Enhanced anti-tumor immune responses and T-cell infiltration were induced by HSV-aPD-1 compared with unloaded virus or anti-PD-1 therapy in both MC38 and B16-F10 models, resulting in improved treatment efficacy in the latter. Moreover, compared with unloaded HSV-1 or HSV-1 loaded with GM-CSF/IL-2 combined with anti-PD-1 mAbs, HSV-aPD-1 displayed similar therapeutic control of tumor growth. Finally, tumor RNAseq analysis in the B16-F10 model showed upregulated IFN pathway and antigen processing and presentation genes, and downregulated angiogenesis and cell adhesion genes, which all contribute to tumor inhibition. These findings indicate the clinical potential of HSV-aPD-1 as monotherapy or combination therapy, especially in tumors resistant to immune checkpoint inhibitors.

1017P Preclinical evaluation of KD033, a human anti-PD-L1/IL-15 bispecific protein, in human PD-1/PD-L1 transgenic C57/Bl6 mice with PD-L1 positive and negative tumors

KD033 is a clinical-stage bispecific fusion molecule consisting of a high-affinity anti-human-PD-L1 antibody and human IL-15. Previous preclinical studies with mouse anti-PD-L1/IL-15 (KD033 surrogate) have demonstrated that targeting IL-15 with anti-PD-L1 antibody resulted in increased efficacy, safety and maximal tolerated dose of the fusion protein compared to administrations of free IL-15. Reduction of tumor growth in both PD-L1 positive and negative tumor models was also observed. (Mol Cancer Ther, February 1 2021 (20) (2) 347-356) The goal of the current study is to evaluate the efficacy of KD033, the human anti-PD-L1/IL-15, in a human PD-1/PD-L1 transgenic mice, with human-PD-L1 positive and negative tumor cells.

973P A phase II study of PDR001 among patients with recurrent and/or metastatic esophageal squamous cell carcinoma: An interim analysis of KM-12

Programmed cell death protein 1 (PD-1) inhibitors emerged as a promising treatment option for esophageal cancer refractory to standard treatment. PDR001 (spartalizumab) is a humanized IgG4 monoclonal antibody targeting PD-1. We report the results of a single-arm phase II study of PDR001 in the treatment of esophageal squamous cell carcinoma (ESCC) in Korea.

949P Thermal ablation plus toripalimab in patients with advanced hepatocellular carcinoma: Phase I results from a multicenter, open-label, controlled phase I/II trial (IR11330)

Thermal ablation has been showed to induce immunogenic cell death and enhance the effect of anti-programmed death-1 (PD-1) in preclinical study. The open label phase I/II trial evaluated the safety and efficacy of ablation plus toripalimab (a humanized PD-1 antibody) in patients with advanced hepatocellular carcinoma (HCC). Phase I results on time scheduling, safety and preliminary antitumor activity are reported.

782P A retrospective study of toripalimab combined with concurrent chemoradiotherapy in patients with recurrent/advanced cervical cancer

Cervical cancer constitutes a leading cause of morbidity and cancer deaths in women throughout the world. The prognosis of patients with metastatic cervical cancer is poor with a median survival of 8-13 months. Toripalimab, a humanized IgG4 antibody specific for human PD-1 receptor, was first approved to treat second-line metastatic melanoma in China in 2018. Favorable anti-tumor activity and favorable toxicities in advanced cervical cancer patients were also observed. We hence conducted this retrospective study to further assess the efficacy and safety of toripalimab combined with chemoradiotherapy in recurrent or advanced cervical cancer patients.

Engineered extracellular vesicles for concurrent Anti-PDL1 immunotherapy and chemotherapy

Immune checkpoint inhibitors (ICI) targeting PD-1/PD-L1 have been approved for the treatment of a variety of cancers. However, the efficacy of antibody-based ICIs could be further improved by mitigating anti-drug antibodies, proteolytic cleavage, and on-target off-tumor toxicity. One strategy for accomplishing this is through the use of extracellular vesicles (EVs), cell derived submicron vesicles with many unique properties. We constructed an engineered MDA-MB-231 cell line for harvesting EVs. This was accomplished by overexpressing a high-affinity variant human PD-1 protein (havPD-1), while simultaneously knocking out intrinsic PD-L1 and beta-2 microglobulin. The engineered havPD-1 EVs reduced PD-L1 overexpressing cancer cell proliferation and induced cellular apoptosis. Moreover, the EVs were shown to efficiently block PD-L1 mediated T cell suppression. Meanwhile antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity were not observed. The havPD-1 EVs treatment resulted in robust anti-tumor activity in both preventative co-implantation and therapeutic xenograft tumor models reconstituted with human T cells. The efficacy of the havPD-1 EVs was shown to be comparable to clinical anti-PD1 monoclonal antibodies. Additionally, loading the havPD-1 EVs with a potent PARP inhibitor was shown to further augment treatment efficacy. In brief, the engineered universal EVs harboring havPD-1 proteins can be used for cancer concurrent immunotherapy and chemotherapy.

Computational design of a synthetic PD-1 agonist

Programmed cell death protein-1 (PD-1) expressed on activated T cells inhibits T cell function and proliferation to prevent an excessive immune response, and disease can result if this delicate balance is shifted in either direction. Tumor cells often take advantage of this pathway by overexpressing the PD-1 ligand PD-L1 to evade destruction by the immune system. Alternatively, if there is a decrease in function of the PD-1 pathway, unchecked activation of the immune system and autoimmunity can result. Using a combination of computation and experiment, we designed a hyperstable 40-residue miniprotein, PD-MP1, that specifically binds murine and human PD-1 at the PD-L1 interface with a Kd of ∼100 nM. The apo crystal structure shows that the binder folds as designed with a backbone RMSD of 1.3 Å to the design model. Trimerization of PD-MP1 resulted in a PD-1 agonist that strongly inhibits murine T cell activation. This small, hyperstable PD-1 binding protein was computationally designed with an all-beta interface, and the trimeric agonist could contribute to treatments for autoimmune and inflammatory diseases.

Abstract 493: A novel humanized B-hPD-1/hPD-L1/hTIGIT mouse model reveals enhanced efficacy of combined TIGIT and PD-L1 blockade against cancer

Abstract Immune checkpoint blockade targeting PD-1/PD-L1 has enjoyed tremendous success in the clinic against various advanced stage cancers, unleashing potent and durable immune-mediated control of tumors while vastly improving patient survival. However, only a portion of patients truly benefit, while other cancers, such as breast, prostate, pancreatic, and colon cancers, are largely resistant to PD-1/PD-L1 therapy alone. Targeting additional immunosuppressive proteins could boost the efficacy of anti-PD-1/PD-L1 therapy and bring the benefits of immunotherapy to more patients. TIGIT is a recently identified immune checkpoint protein mainly expressed by T and NK cells. CD155 (PVR) is a high-affinity receptor of TIGIT, while CD112 and CD113 bind it with weaker affinity. CD155 and CD112 can engage CD226, an NK activating receptor also expressed on some CD8+ T cells, and trigger killing of tumors. Competitive binding of TIGIT to CD155 and CD112 would prevent signaling through CD226. Therefore, anti-TIGIT blocking antibodies may enhance the function of NK cells and T cells. Preclinical studies have shown that inhibition of TIGIT promotes proliferation and function of T cells. Combined blockade of both TIGIT and PD1/PD-L1 has also shown superior efficacy over monotherapy against tumors in early phase clinical trials. Given the intensive interest in combined TIGIT and PD-1/PD-L1 therapies for cancer, Biocytogen has established a novel human PD-1/PD-L1/TIGIT triple knock-in (TKI) mouse as a vital tool to pre-clinically evaluate the in vivo efficacy of combined human TIGIT and PD1/PD-L1 blocking agents against tumors compared to either alone. Exon 2 of the mouse PD-1 gene, exon 3 of mouse PD-L1, and exon 2 of mouse TIGIT were replaced by their human homologs in B-hPD-1/hPD-L1/hTIGIT mice. Human PD-1, PD-L1, and TIGIT proteins were expressed in homozygous B-hPD-1/hPD-L1/hTIGIT but not wildtype mice. Immune profiles of B-hPD-1/hPD-L1/hTIGIT mice were similar to those in wild-type C57BL/6 mice, and abnormalities were not observed. Importantly, we showed that combining anti-human PD-L1 and anti-human TIGIT antibodies significantly inhibited the growth of MC38-hPD-L1 tumor cells implanted into B-hPD-1/hPD-L1/hTIGIT mice. Thus, our novel hPD-1/hPD-L1/hTIGIT TKI mice will be very useful for investigating the in vivo efficacy of TIGIT antibodies currently in company pipelines both alone and combined with PD1/PD-L1 antibody drugs. Citation Format: Chonghui Liu, Xiaofei Zhou, Yanan Guo, Eugene Lin, Luke (Zhaoxue) Yu, Yuelei Shen. A novel humanized B-hPD-1/hPD-L1/hTIGIT mouse model reveals enhanced efficacy of combined TIGIT and PD-L1 blockade against cancer [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 493.

Abstract 1791: LBL-015, a novel anti-PD-1 fused with TGF-βRII, shows a great anti-tumor activity in a mouse MC38 model

Abstract Objective: Blocking PD-1 with monoclonal antibodies is a highly promising onco-immune therapy and has elicited durable antitumor responses and long-term remissions with a broad spectrum of cancers, However, the response to anti-PD-1 therapy is restricted to only a subset of patients with around 25% response rate in cancer patients. TGF-β, a growth factor, plays a pivotal role in tumor development and progression, by inducing tumor immune evasion, activating cancer associated fibroblasts with induction of migration and invasiveness of tumor cells, and promoting angiogenesis in tumor microenvironment. Therefore, a tetravalent bispecific fusion protein (LBL-015), targeting both PD-1/PD-L1 axis and TGF-β signaling pathway was developed to achieve maximal anti-tumor immunity, while blocking the factors implicated in the tumor microenvironment and overcoming the resistance of tumor cells to anti-PD-1 therapy. Methods: The bispecific fusion protein was designed as 2:2 antibody-cytokine fusion format, using our unique Mab fusion platform. The binding affinity of LBL-015 to human PD-1 and TGF-β1 was determined by Fortebio. The activity of LBL-015 was determined using several in vitro assays, including PD-1/NFAT, TGFβ1/SBE-Luc reporter gene and PBMC activation assays. The in vivo anti-tumor activity of LBL-015 was investigated in the human PD-1 knock-in mouse model (Shanghai Model Organisms) implanted with MC38-OVA cells. Results: The binding affinity of LBL-015 was measured as 5.24 nM to human PD-1 and 0.14 nM to human TGF-β1, respectively. In vitro LBL-015 could block PD-1-PD-L1 interaction, recovering NFAT reporter gene signal, with EC50 of 0.1565 nM, which was much more potent than M7824 (EC50 of 0.5412 nM). In TGF-β1/SBE-Luc reporter gene assay, LBL-015 inhibited SMAD signaling induced by TGF-β1, with IC50 of 0.00576 nM, which was comparable to M7824. In pre-activated human PBMCs, the release of IFN-γ by PBMCs was completely suppressed with TGF-β1 addition. LBL-015 could relieve the suppression of IFN-γ release induced by PD-1 or TGF-β1, in a dose dependent manner. Surprisingly, M7824 didn't show any effect in this assay system. In a model of huPD-1 knock-in mice (Shanghai Model Organisms) implanted with MC38-OVA cells. LBL-015 was shown significant tumor growth inhibition (TGI) of 84.36% and 55.27% at the dose of 20 mg/kg and 10 mg/kg respectively (p&amp;lt;0.001 compared with vehicle treated mice), indicating its potent anti-tumor activity of this bi-functional fusion protein. Conclusions: LBL-015, an anti-PD-1 and TGF-β bispecific fusion protein was shown a great synergetic anti-tumor efficacy in vitro to antagonize both tumor immune evasion and aberrant microenvironment induced by PD-1 and TGF-β1, also in a mouse tumor model, LBL-015 strongly inhibited tumor growth in a dose dependent manner, hence a promising bispecific fusion protein for further clinical development. Citation Format: Xiao Huang, Jianming Sun, Yurong Qin, Huan Lin, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-015, a novel anti-PD-1 fused with TGF-βRII, shows a great anti-tumor activity in a mouse MC38 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 1791.

Abstract 693: ASKG843, a bifunctional fusion protein of PD1/TGF-βRII, demonstrates potent in vitro/in vivo activities, and excellent pharmacokinetic properties in non-human primates (NHPs) with good safety profiles

Abstract Introduction: Antibodies targeting the immune checkpoint PD1/PD-L1 are emerging as effective cancer immunotherapies, but most cancers fail to respond to these as single agents. Tumor cells and the tumor microenvironment (TME) frequently express transforming growth factor-β (TGF-β) to either drive immune cell dysfunction in the microenvironment or to exclude immune cell infiltration into tumors. Concurrently targeting the immunosuppressive/immune-excluding TGF-β pathway represents a rational and promising strategy to enhance PD1/PD-L1-based immunotherapies. ASKG843 is a bifunctional molecule designed to simultaneously block PD1/PD-L1 pathway and disable all three TGF-β isoforms in the TME. Here we report the functional evaluation of ASKG843 in vitro, anti-tumor activity in vivo, pharmacokinetic (PK) and pharmacodynamic (PD) properties in cynomolgus monkeys as well as its safety profiles following 5-repeat weekly dosing in NHPs. Methods: ASKG843 was constructed comprising αhPD1 Ab with the C-terminus of the heavy chain fused to the ECD of human TGF-βRII via flexible linkers. Bispecific binding properties were evaluated by Fortebio and bispecific ELISA. Respective signaling blockade for PD1 and TGF-β were assessed using either PD1/NFAT-luciferase reporter system or TGF-β/SMAD-luciferase reporter cell line. The anti-tumor activity of ASKG843 was determined in human PBMC-engrafted NSG mice-bearing A375 xenografts. The PK/PD and safety profiles of ASKG843 were assessed in NHPs. Results: Fortebio and bispecific ELISA analyses demonstrated the ability of ASKG843 to simultaneously bind both human PD1 and TGF-β1-3. ASKG843 not only dose-dependently blocked PD1/PD-L1 inhibitory axis with similar potency to parental αPD1 Ab in PD1 bioassay, but also thwarted TGF-β canonical signaling with IC50 =14.7 ng/mL in TGF-β bioassay. In human PBMC-reconstituted A375 xenograft model ASKG843 promoted strong anti-tumor response with efficacy comparable to the reference molecule (PD-L1/TGF-βRII) at the same dose/dosing frequency. In cynomolgus monkeys ASKG843 demonstrated superior PK to PD-L1/TGF-βRII reference molecule resulting in sustained depletion of TGF-β in peripheral blood for at least 10 days following single injection at 5 mg/kg dose, and no toxicity-related sign observed which contrasted with the reference molecule. Most encouragingly ASKG843 maintained very good safety profiles in cynomolgus monkeys following multiple injections at dose up to 100 mg/kg. Conclusions: ASKG843 demonstrated potent bi-functional activities in vitro and in vivo endowed by its design. Superior PK to the reference molecule yet maintaining better safety profiles in NHPs support further development of ASKG843 as an effective immunotherapeutic candidate. Citation Format: Liqin Liu, Shiwen Zhang, Kurt Shanebeck, Mouzhong Xu, Yunxia Yuan, Ming Li, Lu Li, Ray Chuang, Yong Wen, Yuefeng Lu, Jeff Lu. ASKG843, a bifunctional fusion protein of PD1/TGF-βRII, demonstrates potent in vitro/in vivo activities, and excellent pharmacokinetic properties in non-human primates (NHPs) with good safety profiles [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 693.

Abstract 2759: Reprogramming of PD1+ M2-like tumor-associated macrophages with anti-PD-L1 and Lenalidomide in cutaneous T cell lymphoma

Abstract M2-like tumor-associated macrophages (TAMs) are abundant and influence cutaneous T cell lymphoma (CTCL) development by inducing immunosuppression. Although it is well established that depletion of M2-like TAMs delays CTCL development, little is known about the underlying mechanisms that shape programmed cell death 1+ (PD1+) M2-like TAMs phenotype in CTCL. Here we identified PD1+ M2-like TAMs accumulated in CTCL lesional skin. Moreover, RNA-seq analysis of human CTCL tissues revealed an up-regulation of the TLR/NF-κB and JAK/STAT signaling pathways. In vitro, PD1+ TAMs exhibited an M2-like profile, with a significant increase in the expression of CD163, CD206, and IL-10, and a clear decrease in the expression of CD80, IL-1β, CXCL-10, and CXCL-11, and the activation of TLR/NF-κB and JAK/STAT signaling pathways. To determine whether PD1+ M2-like TAMs could be reprogrammed, anti-PD-L1 (durvalumab) and lenalidomide were used for treatment of MyLa-conditioned media induced human peripheral blood monocyte-derived PD1+ M2-like TAMs. The results show that anti-PD-L1 and lenalidomide synergistically reshaped M2-like TAMs to M1-like TAMs in vitro through ablation of the TLR/NF-κB and JAK/STAT signaling pathways, which was linked with functional changes in phagocytic activity and cell migration. In conclusion, TLR/NF-κB and JAK/STAT signaling pathways may drive PD1+ M2-like TAMs programming in the CTCL tumor microenvironment, anti-PD-L1 and lenalidomide may reshape PD1+ M2-like TAMs and induce functional changes through ablation of these specific pathways in CTCL. Corresponding author: Christiane Querfeld, MD, PhD. Div. of Dermatology and Beckman Research Institute, City of Hope. 1500 E. Duarte Road, Duarte, CA 91010. Email: cquerfeld@coh.org Phone: 626-634-4436 Fax: 626-218-6190 Citation Format: Zhen Han1,5, Chingyu Su1,5, Xiwei Wu2,5, Hanjun Qin2,5, Steven T. Rosen3,5, Christiane Querfeld1,3,4,5. Reprogramming of PD1+ M2-like tumor-associated macrophages with anti-PD-L1 and Lenalidomide in cutaneous T cell lymphoma [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 2759.

Abstract 494: Novel human B7-H3 knock-in mice demonstrate efficacy of anti-B7-H3 immunotherapy in vivo

Abstract In recent years, immune checkpoint inhibitors (ICIs), particularly against PD-1/PD-L1, have revolutionized cancer treatment. However, ICI monotherapy only benefits a portion of cancer patients, while co-blockade of PD-1/PD-L1 and CTLA-4 is associated with increased IrAEs. Combined targeting of other immune checkpoints with PD-1/PD-L1 rather than CTLA-4 may be safer. B7-H3, also known as CD276, is a newer member of the same B7/CD28 superfamily as PD-1 and PD-L1. Its transcripts are ubiquitously expressed in normal tissues and solid tumors, but the protein is expressed in tumor tissues and by antigen-presenting cells, such as DC or macrophages, where it inhibits T cells and contributes to tumor immune evasion. Importantly, B7-H3 is overexpressed on a wide range of human solid cancers and correlates with negative prognosis and poor clinical outcome in patients. Recent studies have shown that B7-H3 also drives tumor growth, invasion, migration, angiogenesis, and metastasis beyond its immune regulatory roles. Thus, B7-H3 is an interesting new target for immunotherapy. To investigate B7-H3 function in vitro and evaluate in vivo efficacy of B7-H3 antibodies, Biocytogen generated a B7-H3 humanized mouse (B-hB7-H3) and B7-H3-expressing MC38 cell line (MC38-hB7-H3). In the mice, exons 3-4 of the mouse B7-H3 gene, which encode the extracellular domain, are replaced by human B7-H3 exons 3-6. Human B7-H3 mRNA was detectable in spleen and bone marrow of only B-hB7-H3 mice, and B7-H3 antibodies bound well to hB7-H3+ MC38 cells. Furthermore, using the MC38-hB7-H3 and EL4 tumor models, we showed robust efficacy of anti-human B7-H3 antibodies in inhibiting tumor growth in vivo, which was amplified in mice receiving combined human B7-H3 and mouse PD-1 blockade. Taken together, B7-H3 humanized mice are a useful tool for in vivo efficacy evaluation and selection of candidate human B7-H3 antibodies for advancement to clinical trials. In addition, Biocytogen has generated B7-H3/PD-1 and B7-H3/PD-1/PD-L1 multi-gene humanized mice to evaluate B7-H3/PD-1/PD-L1 combination therapies in vivo. Citation Format: Lei Zhao, Huilin Li, Yujie Liu, Zhaoxue Yu, Eugene Lin. Novel human B7-H3 knock-in mice demonstrate efficacy of anti-B7-H3 immunotherapy in vivo [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 494.

Abstract 2711: Modeling pancreatic and non-small cell lung cancer using a humanized mouse platform

Abstract The development of immuno-oncology agents against human targets is actively pursued in the preclinical space using immune deficient mice with reconstituted human immune systems. We utilized a human peripheral blood mononuclear cell (hPBMC) based humanized mouse model to investigate treatment response in human pancreatic and non-small cell lung cancers. Three human PBMC donors were identified that exhibited consistent T cell engraftment, allowing for a four-week treatment window, and robust growth of two human xenograft models in NSG mice, MiaPaCa-2 (pancreatic) and A549 (non-small cell lung). Anti-tumor efficacy of pembrolizumab (Keytruda®) targeting human PD-1 was also assessed in these humanized models. All animal work was performed in an AAALAC accredited facility, in alignment with applicable animal welfare regulations and with predetermined humane euthanasia criteria on all studies. Control growth of MiaPaCa-2 and A549 tumors were similar with and without hPBMC engraftment. Both tumor models progressed uniformly to a mean tumor volume of 1000 mm3 without apparent hPBMC impact. Engraftment was assessed at 28 and 38 days (MiaPaCa-2) or 42 days (A549) after hPBMC administration by immunophenotypic analysis using flow cytometry in peripheral blood. At all timepoints, hCD45+ cells were detected. While intragroup variability occurred, engraftment remained constant or increased over time. hPBMC engraftment was reproducibly higher for animals harboring A549 compared to MiaPaCa-2 tumors, with hCD45+ cells in blood ranging from 31-52% and 19-33% of total live cells, respectively. It is unknown if this was a result of normal variability or whether this finding is model dependent. Pembrolizumab treatment in the hPBMC-engrafted mice did not inhibit A549 or MiaPaCa-2 tumor growth under the conditions tested (median ΔT/ΔC of 76-118% (Day 41) and 60-106% (Day 22), respectively). However, in vitro, CD8+ T cells from all donors responded to CD3/CD28 stimulation, demonstrating at least 36-fold upregulation of the Ki67 proliferation marker and at least 9-fold upregulation of interferon gamma release, indicative of cytotoxic activity. This result indicated that the hPBMCs were healthy and responsive, but the extent of activation varied by donor. Investigation of human T cell infiltration into human tumors following treatment with pembrolizumab are currently being performed. Despite the lack of response of pembrolizumab against A549 or MiaPaCa-2, these models are promising translational tools for assessing efficacy of novel therapeutics using rational combination strategies with pembrolizumab. This humanized mouse model represents a powerful preclinical platform to examine the effects of novel agents that harness human T cells to direct anti-tumor activity both as monotherapy and in combination with FDA approved or investigational agents. Citation Format: Sheri Barnes, David Draper, Stacey Roys, Philip Lapinski, Maryland Rosenfeld Franklin. Modeling pancreatic and non-small cell lung cancer using a humanized mouse platform [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 2711.