PD-1 Interaction Research Articles

Illuminating the impact of CD38-induced adenosine formation in B-cell lymphoma

The expression of CD38 by cancer cells may mediate an immune-suppressive effect by producing Extracellular Adenosine (ADO) acting through G-protein-coupled cell surface receptors on cellular components and tumor cells. This can increase PD-1 expression and interaction with PD-L1, suppressing CD8 + cytotoxic T cells. This study examines the impact of heightened CD38 expression and extracellular ADO on various hematological and clinical parameters in patients with mature B-cell lymphoma, alongside their correlation with the soluble counterparts of the PD-1/PD-L1 axis. Our study was conducted on 90 patients, CD38-positive and CD38-negative (measured by flow cytometry), with mature B-cell lymphoma divided into CLL and B-NHL subtypes. Their serum ADO, soluble PD-1, and PD-L1 levels were measured using a sandwich ELISA. Our study revealed a positive correlation between CD38 expression, sADO, sPD-1, and sPD-L1 in mature B-cell lymphoma patients. CD38-positive patients had higher sADO, sPD-1, and sPD-L1 levels. Higher CD38 expression and extracellular ADO negatively affected HB level and PLT count and positively correlated with the higher risk stratification in mature B-cell lymphoma patients. This study explored the potential impact of CD38 expression and elevated extracellular ADO on B-cell lymphoma alongside their link with the PD-1/PD-L1 axis. Our findings underscore the influence of extracellular ADO on the neoplastic process of mature B-cell lymphoma. We also propose targeting the CD38-induced-ADO formation pathway, which could serve as a promising therapeutic immune target with multifaceted effects within mature B-cell neoplasms.

VSTM2A reverses immunosuppression in colorectal cancer by antagonizing the PD-L1/PD-1 interaction

Immunoglobulin (Ig) VSTM2A (V-set and transmembrane domain containing 2A) is a top-ranked secretory protein frequently silenced during colorectal carcinogenesis; however, its role in immune modulation remains largely unknown. Bioinformatic and immunohistochemistry analysis of human colorectal specimens and Vstm2a+/- knockout mice indicated that VSTM2A positively correlated with CD8a and immune infiltration in both physiological and pathological conditions. We then utilized liquid chromatography-mass spectrometry to pinpoint programmed death ligand 1 (PD-L1) as a membrane receptor of VSTM2A. A series of invitro biochemistry assays further revealed the binding pattern and kinetics between VSTM2A and PD-L1 proteins through their IgV domains ata dissociation constant of 0.7-2.5nM. Recombinant VSTM2A protein inhibited the PD-1/PD-L1 interaction and induced NFAT response element (RE) luciferase activity dose dependently. Furthermore, interleukin (IL)-2 production from DO11.10 Tcells upon co-culture with mouse non-T splenocytes was upregulated in the presence of VSTM2A conditioned medium. Finally, tumor killing assay and exvivo data from human peripheral blood mononuclear cells and autologous dendritic cell-T cell co-culture demonstrated that VSTM2A significantly enhanced immune activation via the release of granzyme B and interferon (IFN)-γ cytokines. In conclusion, our study demonstrates the tumor-extrinsic role of VSTM2A in sterically blocking the PD-L1/PD-1 interaction at a picomole to nanomole affinity, which leads to the enhanced anti-tumor effect of cytotoxic Tcells.

Design, synthesis, and antitumor activity evaluation of 1,2,3-triazole derivatives as potent PD-1/PD-L1 inhibitors

A series of 1,2,3-triazole derivatives targeting the PD-1/PD-L1 pathway were designed, synthesized, and evaluated both in vitro and in vivo. Among them, compound III-4 demonstrated exceptional inhibitory activity against the interaction of PD-1/PD-L1 and showed great binding affinity with hPD-L1, with an IC50 value of 2.9 nM and a KD value of 3.33 nM. In the co-culture of Hep3B/OS-8/hPD-L1 cells and CD3+ T cells assay, III-4 relieved the inhibition of PD-L1 on PD-1 and promoted the expression of IFN-γ, which shared a comparable effect to that of the PD-1 monoclonal antibody Pembrolizumab (5 μg/mL). Moreover, compound III-5, an ester prodrug derived from III-4, demonstrated significant antitumor effects in the hPD-L1-MC38 C57BL/6 mouse model (TGI: 49.6 %) by oral administration. These findings suggest that compound III-5 holds promise as an inhibitor of the PD-1/PD-L1 interaction for cancer immunotherapy.

Nonsymmetrically Substituted 1,1'-Biphenyl-Based Small Molecule Inhibitors of the PD-1/PD-L1 Interaction.

Therapeutic antibodies directed against either programmed cell death-1 protein (PD-1) or its ligand PD-L1 have demonstrated efficacy in the treatment of various cancers. In contrast with antibodies, small molecules have the potential for increased tissue penetration; better pharmacology; and therefore, improved antitumor activity. A series of nonsymmetric C2 inhibitors were synthesized and evaluated for PD-1/PD-L1 interaction inhibition. These compounds induced PD-L1 dimerization and effectively blocked PD-L1/PD-1 interaction in a homogeneous time-resolved fluorescence (HTRF) assay with most inhibitors exhibiting IC50 values in the single-digit nM range and below. Their high inhibitory potency was also demonstrated in a cell-based coculture PD-1 signaling assay where 2 exhibited an EC50 inhibitory activity of 21.8 nM, which approached that of the PD-L1 antibody durvalumab (EC50 = 0.3-1.8 nM). Structural insight into how these inhibitors interact with PD-L1 was gained by using NMR and X-ray cocrystal structure studies. These data support further preclinical evaluation of these compounds as antibody alternatives.

PD-L2 drives resistance to EGFR-TKIs: dynamic changes of the tumor immune environment and targeted therapy.

There is a lack of effective treatments to overcome resistance to EGFR-TKIs in EGFR mutant tumors. A deeper understanding of resistance mechanisms can provide insights into reducing or eliminating resistance, and can potentially deliver targeted treatment measures to overcome resistance. Here, we identified that the dynamic changes of the tumor immune environment were important extrinsic factors driving tumor resistance to EGFR-TKIs in EGFR mutant cell lines and syngeneic tumor-bearing mice. Our results demonstrate that the acquired resistance to EGFR-TKIs is accompanied by aberrant expression of PD-L2, leading a dynamic shift from an initially favorable tumor immune environment to an immunosuppressive phenotype. PD-L2 expression significantly affected EGFR mutant cell apoptosis that depended on the proportion and function of CD8+ T cells in the tumor immune environment. Combined with single-cell sequencing and experimental results, we demonstrated that PD-L2 specifically inhibited the proliferation of CD8+ T cells and the secretion of granzyme B and perforin, leading to reduced apoptosis mediated by CD8+ T cells and enhanced immune escape of tumor cells, which drives EGFR-TKIs resistance. Importantly, we have identified a potent natural small-molecule inhibitor of PD-L2, zinc undecylenate. In vitro, it selectively and potently blocks the PD-L2/PD-1 interaction. In vivo, it abolishes the suppressive effect of the PD-L2-overexpressing tumor immune microenvironment by blocking PD-L2/PD-1 signaling. Moreover, the combination of zinc undecylenate and EGFR-TKIs can synergistically reverse tumor resistance, which is dependent on CD8+ T cells mediating apoptosis. Our study uncovers the PD-L2/PD-1 signaling pathway as a driving factor to mediate EGFR-TKIs resistance, and identifies a new naturally-derived agent to reverse EGFR-TKIs resistance.

A Synergistic Chemoimmunotherapy System Leveraging PD-L1 Blocking and Bioorthogonal Prodrug Activation.

Novel strategies to facilitate tumor-specific drug delivery and restore immune attacks remain challenging in overcoming the current limitations of chemoimmunotherapy. An antitumor chemoimmunotherapy system comprising bioorthogonal reaction-ready group tetrazine (TZ) modified with an anti-PD-L1 antibody (αPD-L1TZ) and TZ-activatable prodrug vinyl ether-doxorubicin (DOX-VE) for self-reinforced anti-tumor chemoimmunotherapy is proposed. The αPD-L1TZ effectively disrupts the PD-L1/PD-1 interaction and activates the DOX prodrug in situ through the bioorthogonal click reaction of TZ and VE. Conversely, the activated DOX upregulates PD-L1 on the surface of tumor cells, facilitating tumor accumulation of αPD-L1TZ and enhancing DOX-VE activation. Furthermore, the activated DOX-induced immunogenic cell death of tumor cells, substantially improving the response efficiency of αPD-L1 in an immune-suppressive tumor microenvironment. Thus, PD-L1 blocking and bioorthogonal in situ prodrug activation synergistically enhance the antitumor efficacy of the chemoimmunotherapy system. Therefore, the system significantly enhances αPD-L1 tumor accumulation and prodrug activation and induces a robust immunological memory effect to prevent tumor recurrence and metastasis. Thus, a feasible chemoimmunotherapy combination regimen is presented.

GNUV201, a novel human/mouse cross-reactive and low pH-selective anti-PD-1 monoclonal antibody for cancer immunotherapy

BackgroundSeveral PD-1 antibodies approved as anti-cancer therapies work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. These PD-1 antibodies lack inter-species cross-reactivity, necessitating surrogate antibodies for preclinical studies, which may limit the predictability and translatability of the studies.ResultsTo overcome this limitation, we have developed an inter-species cross-reactive PD-1 antibody, GNUV201, by utilizing an enhanced diversity mouse platform (SHINE MOUSE™). GNUV201 equally binds to human PD-1 and mouse PD-1, equally inhibits the binding of human PD-1/PD-L1 and mouse PD-1/PD-L1, and effectively suppresses tumor growth in syngeneic mouse models. The epitope of GNUV201 mapped to the “FG loop” of hPD-1, distinct from those of Keytruda® (“C’D loop”) and Opdivo® (N-term). Notably, the structural feature where the protruding epitope loop fits into GNUV201’s binding pocket supports the enhanced binding affinity due to slower dissociation (8.7 times slower than Keytruda®). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (5.6 times strong than at pH 7.4), which mimics the hypoxic and acidic tumor microenvironment (TME). This phenomenon is not observed with marketed antibodies (Keytruda®, Opdivo®), implying that GNUV201 achieves more selective binding to and better occupancy on PD-1 in the TME.ConclusionsIn summary, GNUV201 exhibited enhanced affinity for PD-1 with slow dissociation and preferential binding in TME-mimicking low pH. Human/monkey/mouse inter-species cross-reactivity of GNUV201 could enable more predictable and translatable efficacy and toxicity preclinical studies. These results suggest that GNUV201 could be an ideal antibody candidate for anti-cancer drug development.

PD-1 knockout on cytotoxic primary murine CD8+ T cells improves their motility in retrovirus infected mice.

Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies.

Characterization of a Trispecific PD-L1 Blocking Antibody That Exhibits EGFR-Conditional 4-1BB Agonist Activity.

Immune checkpoint blockade has changed the treatment paradigm for advanced solid tumors, but the overall response rates are still limited. The combination of checkpoint blockade with anti-4-1BB antibodies to stimulate tumor-infiltrating T cells has shown anti-tumor activity in human trials. However, the further clinical development of these antibodies has been hampered by significant off-tumor toxicities. Here, we generated an anti-4-1BB/EGFR/PD-L1 trispecific antibody consisting of a triple-targeting tandem trimerbody (TT) fused to an engineered silent Fc region. This antibody (IgTT-4E1-S) was designed to combine the blockade of the PD-L1/PD-1 axis with conditional 4-1BB costimulation specifically confined to the tumor microenvironment (TME). The antibody demonstrated simultaneous binding to purified EGFR, PD-L1, and 4-1BB in solution, effective blockade of the PD-L1/PD1 interaction, and potent 4-1BB-mediated costimulation, but only in the presence of EGFR-expressing cells. These results demonstrate the feasibility of IgTT-4E1-S specifically blocking the PD-L1/PD-1 axis and inducing EGFR-conditional 4-1BB agonist activity.

The C1q and gC1qR axis as a novel checkpoint inhibitor in cancer.

Understanding at the molecular level of the cell biology of tumors has led to significant treatment advances in the past. Despite such advances however, development of therapy resistance and tumor recurrence are still unresolved major challenges. This therefore underscores the need to identify novel tumor targets and develop corresponding therapies to supplement existing biologic and cytotoxic approaches so that a deeper and more sustained treatment responses could be achieved. The complement system is emerging as a potential novel target for cancer therapy. Data accumulated to date show that complement proteins, and in particular C1q and its receptors cC1qR/CR and gC1qR/p33/HABP1, are overexpressed in most cancer cells and together are involved not only in shaping the inflammatory tumor microenvironment, but also in the regulation of angiogenesis, metastasis, and cell proliferation. In addition to the soluble form of C1q that is found in plasma, the C1q molecule is also found anchored on the cell membrane of monocytes, macrophages, dendritic cells, and cancer cells, via a 22aa long leader peptide found only in the A-chain. This orientation leaves its 6 globular heads exposed outwardly and thus available for high affinity binding to a wide range of molecular ligands that enhance tumor cell survival, migration, and proliferation. Similarly, the gC1qR molecule is not only overexpressed in most cancer types but is also released into the microenvironment where it has been shown to be associated with cancer cell proliferation and metastasis by activation of the complement and kinin systems. Co-culture of either T cells or cancer cells with purified C1q or anti-gC1qR has been shown to induce an anti-proliferative response. It is therefore postulated that in the tumor microenvironment, the interaction between C1q expressing cancer cells and gC1qR bearing cytotoxic T cells results in T cell suppression in a manner akin to the PD-L1 and PD-1 interaction.

Abstract 2039: Cellular characterization of small molecule PD-L1 inhibitors reveal their novel mechanisms of action

Abstract Introductions: Immune checkpoint inhibitors, including PD-1/L1, are key regulators of immune response and promising targets in cancer immunotherapy. Like anti-PD-1/L1 antibodies, small molecule PD-L1 inhibitors that have been discovered by us and others could also efficiently block PD-1 and PD-L1 interaction and exhibit anti-tumor efficacy in preclinical and clinically settings. In this study, we explore the cellular mechanism of small molecule PD-L1 inhibitors, unveiling their novel mechanisms of action in the regulation of PD-L1 and its functions. Materials and Methods: The cellular effects of the small molecule PD-L1 inhibitor on PD-L1 protein and RNA levels were assessed through Western blot and real-time qPCR experiments. HTRF assay and cellular luciferase reporter assay were used to evaluate the blockade of PD-1 and PD-L1 interaction in vitro and in cells. Flow cytometry and confocal microscopy were used to investigate the internalization of PD-L1 upon treatment with inhibitors. MSD assay was used to evaluate the alteration of soluble PD-L1 proteins. Results: Small molecule PD-L1 inhibitors discovered by Abbisko Therapeutics potently blocked PD-1/PD-L1 interaction in vitro and in cellular system. It could also rapidly and strongly induce PD-L1 internalization in cells. Further profiling suggested that small molecule PD-L1 inhibitors specifically disrupt PD-L1 N-glycosylation, resulting in aberrant PD-L1 glycosylation. This, in turn, may hinder the proper translocation of newly synthesized PD-L1 to the cell surface, thereby abolishing its interaction with PD-1 and functions in immune regulation. Conclusion: Taken together, these results for the first time revealed the distinctive mechanisms of our small molecule PD-L1 inhibitors. With their multi-layer inhibitory effects stemming from various mechanisms, small molecule PD-L1 inhibitors may offer potentially improved activities and an alternative therapeutic treatment for the cancer patients. Citation Format: Weiling Pan, Yongxian Zhang, Fangfei Qi, Wenqun Xin, Zhigang Feng, Zhui Chen, Haiyan Ying. Cellular characterization of small molecule PD-L1 inhibitors reveal their novel mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2039.

Abstract 2719: Papiliximab, a bispecific nanobody targeting CD47 and PDL1 retards tumor growth without hemolysis

Abstract Cancer cells are known to express immune checkpoint proteins, enabling them to evade immune surveillance. In response, anti-cancer therapies targeting these proteins have been developed. While immune checkpoint inhibitors targeting PD-L1 have been clinically approved, their effectiveness as standalone therapies is limited, with only a 20% response rate. To improve responsiveness, bispecific monoclonal antibodies (MoAbs) targeting both CD47 and PD-L1 have been developed. These bispecific antibodies activate both the innate (by blocking CD47) and adaptive (by blocking PD-L1) immune systems simultaneously. However, conventional antibodies targeting CD47 have been dropped during development due to the risk of hemolysis. In this study, we developed a bispecific single-domain antibody (nanobody, Nb) that targets both CD47 and PD-L1 with a minimal hemagglutination risk. Using phage display libraries from alpacas immunized with recombinant antigens, we screened for an anti-CD47 Nb and an anti-PD-L1 Nb. The affinities (Kd) of these Nbs for their antigens were 7.0 × 10−9 and 9.7 × 10−9, respectively. To maximize affinity for both antigens, a bispecific antibody (Papiliximab) was designed to tandemly array anti-CD47 and PD-L1 Nbs with the Fc region of IgG4. Papiliximab has a lower affinity for RBC (up to 3 μM) than conventional anti-CD47 MoAbs reported earlier. Papiliximab successfully inhibited interactions between CD47/SIRP-α and PD-L1/PD-1, with IC50 values of 7.09 nM and 2.67 nM, respectively. Papiliximab induced the expression of IFN-γ by inhibiting the PD-L1/PD-1 interaction in the PBMC-based MLR (Mixed Lymphocyte reaction) assay. In addition, Papiliximab demonstrated the induction of a phagocytosis effect through CD47 blocking and its IgG4 Fc domain. Therefore, Papiliximab is a multifunctional hybrid bispecific antibody that modulates both innate and acquired immunity. Papiliximab exhibited no cross-reactivity with mouse antigens, but did with cynomolgus antigens. We tested its efficacy on the growth of human B cell lymphoma (Raji cells) and human breast cancer (MDA-MB-231 cells) in humanized NSG mice. Papiliximab was more effective in inhibiting tumor growth than mono-specific Abs and Nbs for PD-L1 or CD47, and their combination. These observations suggest that Papiliximab might be able to improve clinical remission more effectively than the combination of monotherapies without the risk of anemia associated with anti-CD47 MoAbs. We are now working to elucidate the mechanisms behind Papiliximab's superior efficacy and safety compared to the combination of monotherapies. Citation Format: Bum Seo Baek, Min Geun Kim, Jong Hyun Kim, In Young Jang, Sung Min Kim, Jeong Hwan Kim, Sang Beum Lee, Hyoung Tae Kim, Seung-Yong Seong. Papiliximab, a bispecific nanobody targeting CD47 and PDL1 retards tumor growth without hemolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2719.

Abstract 5188: Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 7th most common cancer globally with a poor 5-year survival rate of ~50%. With the emerging successes with immune checkpoint blockade (ICB) in the metastatic and recurrent setting, there is a need for predictive biomarkers to identify patients likely to achieve clinical benefit. Whilst PD-L1 expression on tumour cells and PD-1 on immune cells have been investigated, these remain inconclusive biomarkers. To better understand and map the PD-L1/PD-1 receptor-ligand interactions, we evaluated a novel in situ proximity ligation assay (is PLA) across whole tissue sections of pre-treatment HNSCC patients to received ICB therapy. Pre-treatment FFPE whole tissue samples were collected from n=25 advanced stage HNSCC patients across two major Queensland Hospitals (Royal Brisbane and Women’s Hospital and the Princess Alexandra Hospital). Tissues were stained with multiplex immunofluorescence and the Navinci Diagnostics fluorescent is PLA assay for PD-1/PD-L1. Tissues were incubated with the PD1/PD-L1 antibodies and subsequent incubation with oligonucleotide-tagged secondary probes enabled PLA, which highlights PD1 and PD-L1 interactions. Following the application of secondary probes, the sections underwent a ligation step, then a post-blocking, and an amplification process to enhance the detection of protein interactions. Next, the sections were treated with a detection solution to visualize the amplification product. The PLA assay findings were measured against clinical endpoints (PFS/OS criteria). In n=5/25 HNSCC tissues, positive PD-1/PD-L1 interactions were measured across the tumour microenvironment (TME), specifically in the immune rivers on the immediate tumour periphery. These cells showed highly specific binding of the PLA product. These areas positive by the PLA assay were in stark contrast to the tumour bulk/nests which were absent in PD-1/PD-L1 interactions. Notably, the PLA PD-1/PD-L1 do not correlate with tumour PD-L1 expression or tumour proportion scores (TPS). Rather, our study highlighted the difference in spatially mapping PD-1/PD-L1 interactions which may better recapitulate the tumour-immune recognition and activity in the TME. Taken together, these data provide a novel approach for determining clinically relevant receptor/ligand interactions in the HNSCC TME. Citation Format: Habib Sadeghirad, Vahid Yaghoubi Naei, James Monkman, Subham Basu, Agata Wicher, Rahul Ladwa, Brett GM Hughes, Arutha Kulasinghe. Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5188.

Abstract 5354: High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach

Abstract The immune checkpoint involving PD-1 and PD-L1 generates suppressive signals in T cells, which help to prevent autoimmunity by inducing a state of immune exhaustion. In the context of the tumor microenvironment (TME), however, cancer cells can manipulate these pathways to camouflage themselves from an immune attack. Blocking these checkpoints has thus emerged as a key immunotherapeutic tactic. Nonetheless, the success rate of immune checkpoint inhibitors (ICIs) has been mixed, even for patients who test positive for relevant diagnostic biomarkers. Patient stratification currently depends on immunohistochemical staining for checkpoint proteins like PD-L1, but these tests do not provide adequate stratification. A more comprehensive stratification, one that includes immune profiling of the TME plus an evaluation of immune checkpoint interactions, might provide better patient stratification and ICI responsiveness. Here we describe mIF that is augmented with a PD-1 and PD-L1 protein-protein interaction assay. Human formalin-fixed, paraffin-embedded (FFPE) tissue sections were subjected to standard histological processing and then incubated with primary antibodies against PD1 and PD-L1. Sections were then treated with oligonucleotide-modified secondary probes suitable for in situ Proximity Ligation Assay (isPLA), enabling the detection of PD1 and PD-L1 interactions. The PLA protocol utilized a fluorescent probe complementary to amplified nucleic acid to enhance the visibility of the protein interactions. The sample was then mounted in a closed microfluidic chamber and imaged on the CellScape™ platform for Precise Spatial Multiplexing. After imaging of the isPLA signal, the CellScape cyclic multiplex mIF staining approach was used to iteratively stain immune and structural markers on the tissue sample utilizing the VistaPlex™ Spatial Immune Profiling Assay Kit. This proof-of-concept study demonstrates that high-plex mIF can be augmented with an isPLA on human FFPE samples. The combined application of both methods allows visualization of PD1/PD-L1 protein-protein interactions and integrates this interaction within the spatial context of the surrounding cell populations. This approach allows for a more comprehensive insight into the interplay of different immune cell and non-immune cell populations during checkpoint activation processes in normal and neoplastic tissues. Citation Format: Arne Christians, Jannik Boog, Charles E. Jackson, Matthew H. Ingalls, J Spencer Schwarz, Sara Bodbin, Agata Zieba Wicher, Subham Basu, Oliver Braubach. High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5354.

Abstract 6361: KY-HC-mouse: A low-cost and rapid nanobody discovery platform

Abstract Nanobodies, primarily derived from camelid animals, have gained prominence in antibody-drug development due to their small molecular weight and ease of modification. However, the high cost associated with camelid animals for nanobody discovery led us to develop a strain of heavy-chain mice known as KY-HC-mouse. This genetically modified mouse expresses IgG without the CH1 domain, resulting in the production of nanobodies in IgG format. Utilizing hybridoma techniques, we developed a variety of nanobodies against multiple targets, such as PSMA, CD38, PD-L1, CD28, and 4-1BB, from KY-HC-mouse. These nanobodies all demonstrate high affinity, with KD ranging from 0.01 nM to 10 nM. We also selected antagonist nanobodies that block the interaction of PD1 and PDL1, as well as agonist nanobodies that stimulate 4-1BB signaling in T cells. After humanization, most nanobodies have a half-life of more than five days in the blood of both mice and monkeys. Subjected to various treatments, such as high concentration, low pH, repeated freezing and thawing, and high temperature, most humanized nanobodies show neither aggregation nor degradation, suggesting good developability of these nanobodies. Finally, we have developed multiple bispecific antibodies (BsAbs) targeting both PDL1 and 4-1BB, PSMA and 4-1BB, or B7H3 and 4-1BB. These BsAbs exhibit good developability and efficacy. In conclusion, our KY-HC-mouse is a powerful nanobody discovery platform. Citation Format: Guojin Wu, Feng Hao, Feng He, Hao Peng, Yi Gu, Jinying Ning. KY-HC-mouse: A low-cost and rapid nanobody discovery platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6361.

Abstract 2380: The bispecific antibody KA-3004, targeting PD-L1 and 4-1BB, is designed to enhance anti-tumor activity

Abstract Checkpoint inhibitors (CPI) have transformed the treatment landscape for advanced solid tumors; however, a majority of cancers still do not respond to immune checkpoint therapy. In this study, we developed a bispecific antibody (BsAb) named KA-3004, targeting PD-L1 and human 4-1BB, with the aim of enhancing the functionality and survival of stimulated CD8+ T cells in the tumor microenvironment. This antibody was designed using our nanobody antibody discovery platform. KA-3004, in IgG1 format, incorporates mutations in the Fc domain to eliminate ADCC, ADCP, and CDC activity. It features two identical nanobodies at the C-terminal against 4-1BB to stimulate 4-1BB signaling in T cells and binds to PD-L1 with two Fabs at the N-terminal. KA-3004 binds to PD-L1 with high affinity, blocking the interaction of PD-1 and PD-L1. It also binds to 4-1BB expressed on 293T cells and activated human T cells. In the presence of a low concentration of CD3 antibody to stimulate T cell activation, co-culture of tumor cells expressing PD-L1 with primary T cells isolated from human PBMC enhances T cell activation and proliferation. In PBMC humanized immunodeficient mouse tumor models, KA-3004 effectively suppresses the growth of NCI-H292 tumors expressing PD-L1. Additionally, KA-3004 has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this antibody with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, and KA-3004 shows neither aggregation nor degradation. In conclusion, the BsAb KA-3004, developed with our nanobody antibody discovery platform, stands out as a promising pre-clinical candidate drug for the treatment of solid cancers. Citation Format: Jiabei Liang, Zhengcheng Guo, Hao Peng, Feng Hao, Tongtong Liu, Jinying Ning, Guojin Wu. The bispecific antibody KA-3004, targeting PD-L1 and 4-1BB, is designed to enhance anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2380.

Abstract 7517: Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging

Abstract Background: Programmed cell death protein 1 (PD1) and its ligand PD-L1 are pivotal components of the immune checkpoint machinery, playing a crucial role in the regulation of immune responses in cancer. The interaction between PD1 and PD-L1 is a significant therapeutic target in oncology, as its inhibitors have shown improved outcomes across a variety of malignancies. However, the treatment response to immunotherapies such as pembrolizumab has shown a modest response (~30%) in previous studies. This could be explained by the lack of molecular insight pertaining to the interaction of PD1 on T-cells and PD-L1 in the tumor cells in malignant tissues at the time of clinical diagnosis. The recent development of in situ Proximity Ligation Assay (isPLA), marks a significant advancement in the field. This method allows for the visualization of direct protein interactions within tissues, for example between PD1 and PD-L1. This adds an important layer of information compared to multiplexed immunofluorescence, which is useful for deep phenotyping of cell types in a tissue but lacks the ability to detect direct interactions between specific cells. Methods: PD-L1-PD1 interactions in human FFPE tonsil and bladder cancer tissue were identified using the NaveniFlex Tissue Atto647N kit, following the guidelines provided by the manufacturer. This method is capable of detecting proteins within a proximity of less than 40 nm, utilizing oligonucleotide-antibody conjugates that generate an enhanced fluorescent signal. After completing the Naveni® detection process, we performed multiplexed immunofluorescence (IF) using the Phenocycler platform and a panel of 14 barcoded antibodies, to generate isPLA and multiplexed IF data on the same tissue section. Results & Discussion: We have successfully established an automated workflow for the isPLA protocol. In this work, we aim to continue this effort and combine the isPLA, with downstream highly multiplexed immunofluorescence using the Phenocycler platform. We have generated proof of concept data for the successful integration of the two methods, using tonsil and bladder cancer tissue to detect protein interactions between PD1 and PD-L1, as well as phenotypic profiling of the tumor microenvironment with a 14 plex panel. This panel allows us to phenotype the relevant cell types (B cells, dendritic cells, T cells, macrophages, tumor cells and their proliferation status) and subsets of T-cells like cytotoxic CD8+ cells. The integration of the isPLA and multiplexed IF is poised to significantly advance our understanding of the TME and hopefully aid in patient stratification for immune checkpoint inhibition therapy. The clinical significance of this method integration will later be explored in a retrospective cohort of muscle invasive bladder cancer, receiving immune checkpoint inhibition therapy with pembrolizumab as a second line treatment. Citation Format: Tony Ullman, David Krantz, Hampus Elofsson, Carolina Oses Sepulveda, Agata Zieba-Wicher, Päivi Östling, Anders Ullén, Subham Basu, Charlotte Stadler. Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7517.

Anti-Tumor Potential of Post-Translational Modifications of PD-1.

Programmed cell death protein-1 (PD-1) is a vital immune checkpoint molecule. The location, stability, and protein-protein interaction of PD-1 are significantly influenced by post-translational modification (PTM) of proteins. The biological information of PD-1, including its gene and protein structures and the PD-1/PD-L1 signaling pathway, was briefly reviewed in this review. Additionally, recent research on PD-1 post-translational modification, including the study of ubiquitination, glycosylation, phosphorylation, and palmitoylation, was summarized, and research strategies for PD-1 PTM drugs were concluded. At present, only a part of PD-1/PD-L1 treated patients (35-45%) are benefited from immunotherapies, and novel strategies targeting PTM of PD-1/PD-L1 may be important for anti-PD-1/PD-L1 non-responders (poor responders).

Assessment of PD-1 and PD-L1 tissue expression levels in lichen planus patients: a case–control study

Programmed cell death protein-1 (PD-1) is an immune checkpoint protein, PD-1 interaction with PD ligand-1 (PD-L1) is essential for maintaining immunological tolerance. The study aimed to study and compare the levels of PD-1 and PD-L1 in lesional and nonlesional skin of lichen planus (LP) patients and compare these levels to normal healthy controls to assess their role in the pathogenesis of LP. This case–control study involved 30 patients with LP and 30 healthy age-and sex-matched controls. After clinical assessment of the severity by LP severity index score (LPSI), skin biopsies were taken from lesional and nonlesional skin of LP patients and from normal skin in healthy controls for assessment of the tissue levels of PD-1 and PD-L1 by ELISA. The tissue levels of both PD-1 and PD-L1 were significantly higher in healthy controls than in both lesional and nonlesional skin of LP patients (P < 0.001). Also, significantly higher PD-l and PD-L1 levels in nonlesional skin than in lesional skin of LP patients were reported (P < 0.001). No significant correlations were found between lesional and nonlesional PD-1, PD-L1 levels, or LPSI score. Based on the fact that PD-1/PD-L1 interaction is important to maintain tolerance and protection against autoimmune diseases, in addition to our study results that revealed lower levels of PD-1/PD-L1 in LP skin than in healthy skin, we can conclude that PD-1/PDL-1 may be incriminated in the pathogenesis of LP. ClinicalTrials.govID: NCT04892381.

Association between spatial distribution of leukocyte subsets and clinical presentation of head and neck squamous cell carcinoma.

Interactions between tumor cells and cells in the microenvironment contribute to tumor development and metastasis. The spatial arrangement of individual cells in relation to each other influences the likelihood of whether and how these cells interact with each other. This study investigated the effect of spatial distribution on the function of leukocyte subsets in the microenvironment of human head and neck squamous cell carcinoma (HNSCC) using multiplex immunohistochemistry (IHC). Leukocyte subsets were further classified based on analysis of two previously published HNSCC single-cell RNA datasets and flow cytometry (FC). IHC revealed distinct distribution patterns of leukocytes differentiated by CD68 and CD163. While CD68hiCD163lo and CD68hiCD163hi cells accumulated near tumor sites, CD68loCD163hi cells were more evenly distributed in the tumor stroma. PD-L1hi and PD-1hi cells accumulated predominantly around tumor sites. High cell density of PD-L1hi CD68hiCD163hi cells or PD-1hi T cells near the tumor site correlated with improved survival. FC and single cell RNA revealed high variability within the CD68/CD163 subsets. CD68hiCD163lo and CD68hiCD163hi cells were predominantly macrophages (MΦ), whereas CD68loCD163hi cells appeared to be predominantly dendritic cells (DCs). Differentiation based on CD64, CD80, CD163, and CD206 revealed that TAM in HNSCC occupy a broad spectrum within the classical M1/M2 polarization. Notably, the MΦ subsets expressed predominantly CD206 and little CD80. The opposite was observed in the DC subsets. The distribution patterns and their distinct interactions via the PD-L1/PD-1 pathway suggest divergent roles of CD68/CD163 subsets in the HNSCC microenvironment. PD-L1/PD-1 interactions appear to occur primarily between specific cell types close to the tumor site. Whether PD-L1/PD-1 interactions have a positive or negative impact on patient survival appears to depend on both the spatial localization and the entity of the interacting cells. Co-expression of other markers, particularly CD80 and CD206, supports the hypothesis that CD68/CD163 IHC subsets have distinct functions. These results highlight the association between spatial leukocyte distribution patterns and the clinical presentation of HNSCC.