Anti-tumor Immunological Memory Research Articles

Abstract 5293: Preclinical development of NI-3201, a PD-L1xCD28 bispecific antibody mediating CD28 costimulation upon PD-L1 blockade

Abstract NI-3201 is a PD-L1xCD28 bispecific antibody generated on the κλ body platform that blocks the PD-L1/PD-1 immune checkpoint pathway and conditionally provides T cell activation “signal 2” when engaging PD-L1+ tumor or immune cells (e.g., antigen-presenting cells). By combining two distinct mechanisms of action into a single molecule, NI-3201 overcomes two major immune suppression mechanisms commonly observed in a variety of solid tumors, namely: [1] suppression of T cell response (induced by PD-L1+ cells engaging PD-1 on T cells) and [2] restricted/limited T cell activation (due to the lack of costimulatory signal associated to the scarcity of CD28 ligands in the TME). Importantly, NI-3201 was designed to require primary T cell stimulation (“signal 1”) to afford its activity: in the absence of TCR engagement, NI-3201 is unable to activate T cells, thus preventing unintended systemic inflammation. The single agent pharmacological activity of NI-3201 was assessed in vitro through a Cytomegalovirus (CMV) recall assay in which CMV+ PBMCs were cocultured with PD-L1+ cancer cell lines ectopically loaded with CMV-specific MHC-I peptides, thus providing “signal 1”. NI-3201 induced the activation and proliferation of CMV-specific T cells which resulted in killing of the target tumor cells. In vivo, the single agent activity of NI-3201 was shown in immunocompetent huCD28-transgenic mice engrafted with huPD-L1-expressing murine colon adenocarcinoma MC38 cells, a model in which NI-3201 induced durable anti-tumor immunological memory response. In humanized mouse models where T cell engagers (TCEs) were ineffective, NI-3201 was able to synergize with TCEs to control tumor growth, even inducing tumor regression in one model. The pharmacokinetics (PK) and tolerability of NI-3201 were investigated in non-human primates (NHP) after single and repeated-dosing. NI-3201 demonstrated favorable PK with close to dose-proportional concentrations being achieved. Upon repeated NI-3201 administration, no hypercytokinemia was observed; only a mild and transient IL-6 release and associated increase in C-reactive protein (CRP) levels were seen, with no clinical signs reported. The favorable profile seen in NHP was supported by data generated in vitro further de-risking the cytokine release syndrome (CRS) potential. Quantitative systems pharmacology (QSP) modelling integrating both in vitro and NHP data predicted a drug concentration required for optimal NI-3201 activity in the TME, and preliminary data suggests optimal patient activity at Q2W-Q3W dosing schedule. The anti-tumor activity as a single agent or as universal combination partner for TCEs, shown in vitro and in vivo, together with the favorable safety profile of NI-3201, support its clinical development planned to start in Q1 2025. Citation Format: Sara Majocchi, Nadia Anceriz, Alizée Viandier, Adeline Lesnier, Laura Cons, Lise Nouveau, Emily A. Pace, Bjorn L. Millard, Bruno Daubeuf, Valéry Moine, Susana Salgado-Pires, José Saro, Limin Shang, Krzysztof Masternak, Walter G. Ferlin. Preclinical development of NI-3201, a PD-L1xCD28 bispecific antibody mediating CD28 costimulation upon PD-L1 blockade [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 5293.

Bacteria colonization in tumor microenvironment creates a favorable niche for immunogenic chemotherapy

The tumor microenvironment (TME) presents differential selective pressure (DSP) that favors the growth of cancer cells, and monovalent therapy is often inadequate in reversing the cancer cell dominance in the TME. In this work, we introduce bacteria as a foreign species to the TME and explore combinatorial treatment strategies to alter DSP for tumor eradication. We show that cancer-selective chemotherapeutic agents and fasting can provide a strong selection pressure against tumor growth in the presence of bacteria. Moreover, we show that an immunogenic drug (oxaliplatin), but not a non-immunogenic one (5-FU), synergizes with the bacteria to activate both the innate and adaptive immunity in the TME, resulting in complete tumor remission and a sustained anti-tumor immunological memory in mice. The combination of oxaliplatin and bacteria greatly enhances the co-stimulatory and antigen-presenting molecules on antigen-presenting cells, which in turn bridge the cytotoxic T cells for cancer-cell killing. Our findings indicate that rational combination of bacterial therapy and immunogenic chemotherapy can promote anticancer immunity against the immunosuppressive TME.

Novel vaccination strategies based on optimal stimulation of CD4+ T helper cells for the treatment of oral squamous cell carcinoma.

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant tumor of the oral cavity. Despite recent advances in the field of oral cancer therapy, including the introduction of immunotherapeutic approaches, the 5-year survival rate remains steadily assessed around 50%. Thus, there is an urgent need for new therapeutic strategies. After the characterization of the immune phenotype of three human OSCC cell lines (CAL-27, SCC-25, and SCC-4) and one mouse OSCC cell line (MOC2) showing their similarities to resected patient tumors, we explored for the first time an experimental preclinical model of therapeutic vaccination with mouse OSCC MOC2 cell line stably expressing MHC class II antigens after CIITA gene transfection (MOC2-CIITA). Mice injected with MOC2-CIITA reject or strongly retard tumor growth; more importantly, vaccinated animals that fully reject MOC2-CIITA tumors display anti-tumor immunological memory protective against challenge with parental MOC2 tumor cells. Further experiments of adoptive cell transfer or in vivo cell depletion show that both CD4+ and CD8+ T lymphocytes prove fundamental in tumor rejection. This unprecedented approach for oral cancer opens the way for possible future translation of novel immunotherapeutic strategies to the human setting for the treatment of this tumor.

Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory.

Current immunotherapies have proven effective in strengthening antitumor immune responses, but constant opposing signals from tumor cells and the surrounding microenvironment eventually lead to immune escape. We hypothesized that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system would provide a robust and long-term antitumor effect by creating immunologic memory against tumors. To achieve this, we developed CARG-2020, a genetically modified virus-like vesicle (VLV) that is a self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three immune modulators: (i) the pleiotropic antitumor cytokine IL12, in which the subunits (p35 and p40) are tethered together; (ii) the extracellular domain (ECD) of the protumor IL17RA, which serves as a dominant-negative antagonist; and (iii) a shRNA targeting PD-L1. Using a mouse model of ovarian cancer, we demonstrated the oncolytic effect and immune-modulatory capacities of CARG-2020. By enhancing IL12 and blocking IL17 and PD-L1, CARG-2020 successfully reactivated immune surveillance by promoting M1, instead of M2, macrophage differentiation, inhibiting MDSC expansion and establishing a potent CD8+ T cell-mediated antitumoral response. Furthermore, we demonstrated that this therapeutic approach provided tumor-specific and long-term protection against the establishment of new tumors. Our results provide a rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance antitumor responses and prevent a recurrence.

Abstract B141: Evaluation of antigen-agnostic anti-tumor activity and immunological memory induced by CBX-15 (alphalexTM-MMAE) in the rat syngeneic breast cancer model

Abstract CBX-15 is a peptide-drug conjugate consisting of alphalexTM-monomethyl auristatin E (MMAE). The alphalexTM is a unique variant of pH-Low Insertion Peptide1 (pHLIP®) designed to target the low pH microenvironment of cancer cells, a universal feature of all rapidly growing tumors. The peptide of CBX-15 forms an alpha helix only in low pH conditions, resulting in unidirectional insertion of the peptide and delivery of MMAE across the cancer cell membrane, and avoidance of delivery to healthy tissues, including immune cells. Efficacy and anti-tumor immunological memory induced by CBX-15 was evaluated in Fischer 344 rats bearing syngeneic 13762 mammary adenocarcinoma tumors. The development of anti-tumor immunological memory was examined in CBX-15-cured animals by in vivo/ex vivo rechallenge with live tumor cells and subsequent assessment of tumor rejection, cytokine release by T cells, tumor immune cell infiltration, and memory T cell composition of the bone marrow. CBX-15 rapidly regressed rat tumors, resulting in complete responses while sparing healthy tissues such as bone marrow. Cured rats rejected live tumor rechallenge and exhibited a doubling of bone marrow-resident CD4 memory T cells 50 days post-dose. Splenocytes and lymph node suspensions derived from cured rats demonstrated formation of a Th1-mediated IFNƳ response when exposed ex vivo to tumor cells. The ability of CBX-15 to induce immunogenic cell death was established by vaccinating syngeneic animals with CBX-15 treated tumor cells and subsequent tumor rechallenge, which demonstrated anti-tumor immunity induced by CBX-15. These preclinical data demonstrate the anti-tumor efficacy of CBX-15 in the rat as well as the ability of CBX-15 to enhance tumor immunogenicity through immunogenic cell death by utilizing a universal pH-based tumor targeting mechanism.1 Wyatt LC et al. 2017. Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotech. Citation Format: Sophia Gayle, Timothy Paradis, Qing Zhang, Laurie Tylaska, Connor Hagen, Theresa Pasqualini, Vishwas Paralkar. Evaluation of antigen-agnostic anti-tumor activity and immunological memory induced by CBX-15 (alphalexTM-MMAE) in the rat syngeneic breast cancer model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B141.

Dosimetric and biologic intercomparison between electron and proton FLASH beams

Background and purposeThe FLASH effect has been validated in different preclinical experiments with electrons (eFLASH) and protons (pFLASH) operating at an average dose rate above 40 Gy/s. However, no systematic intercomparison of the FLASH effect produced by eFLASHvs. pFLASH has yet been performed and constitutes the aim of the present study. Materials and methodsThe electron eRT6/Oriatron/CHUV/5.5 MeV and proton Gantry1/PSI/170 MeV were used to deliver conventional (0.1 Gy/s eCONV and pCONV) and FLASH (≥110 Gy/s eFLASH and pFLASH) dose rates. Protons were delivered in transmission. Dosimetric and biologic intercomparisons were performed using previously validated dosimetric approaches and experimental murine models. ResultsThe difference between the average absorbed dose measured at Gantry 1 with PSI reference dosimeters and with CHUV/IRA dosimeters was −1.9 % (0.1 Gy/s) and + 2.5 % (110 Gy/s). The neurocognitive capacity of eFLASH and pFLASH irradiated mice was indistinguishable from the control, while both eCONV and pCONV irradiated cohorts showed cognitive decrements. Complete tumor response was obtained after an ablative dose of 20 Gy delivered with the two beams at CONV and FLASH dose rates. Tumor rejection upon rechallenge indicates that anti-tumor immunity was activated independently of the beam-type and the dose-rate. ConclusionDespite major differences in the temporal microstructure of proton and electron beams, this study shows that dosimetric standards can be established. Normal brain protection and tumor control were produced by the two beams. More specifically, normal brain protection was achieved when a single dose of 10 Gy was delivered in 90 ms or less, suggesting that the most important physical parameter driving the FLASH sparing effect might be the mean dose rate. In addition, a systemic anti-tumor immunological memory response was observed in mice exposed to high ablative dose of electron and proton delivered at CONV and FLASH dose rate.

Integration of Protein Nanocage with CpG Motifs: A Virus-Mimicked Core-Shell Nanostructure to Ignite Antitumor Immunity.

The tumor microenvironment typically possesses immunosuppressive properties that hinder the effectiveness of antitumor immune responses, even in the context of immunotherapies. However, it is observed that pathogenic microorganisms can trigger strong immune responses during infection, offering a potential means to counteract the immunosuppressive environment of tumors. In this study, a protein nanocage called CpG@HBc nanocages (NCs) is developed, which mimics the structure of the hepatitis B virus and combines with an immunostimulatory component known as cytosine phosphoguanosine oligonucleotide (CpG). By delivering these immunostimulatory agents, CpG@HBc NCs are able to effectively reverse the suppressive tumor microenvironment, resulting in the inhibition of poorly immunogenic tumors in mice. Through high-dimensional mass cytometry (CyTOF) analysis, remarkable alterations in immune responses is observed induced by CpG@HBc. Treatment with immunogenic CpG@HBc NCs, along with co-injection of an OX40 agonist, sensitized colorectal cancer tumors to T cell immune responses, resulting in significant impairment of tumor growth and robust immune activation. Furthermore, CpG@HBc NCs induced long-term antitumor immunological memory, protecting tumor-cured mice from tumor rechallenge. Overall, these findings highlight the potential of a virus-inspired protein nanocage to mimic anti-viral immunity and offer a unique therapeutic approach for cancer immunotherapy.

1676P Active immunization with a multi-peptide B cell vaccine, targeting trastuzumab and pertuzumab binding sites, prevents the formation of HER-2/neu expressing lung metastases

The passive application of Trastuzumab and Pertuzumab has demonstrated remarkable success in the clinical outcome of patients with Her-2/neu positive metastatic breast cancer. However, active immunization with mimotope/B cell epitope-based vaccines can induce the production of the corresponding mAbs by the patient’s immune system. We have developed a B cell peptide-based Her-2/neu vaccine (HerVaxx) comprising Trastuzumab’s binding site. In clinical settings, the vaccine has been shown to reduce primary tumor growth by inducing polyclonal anti-tumor immune responses and immunological memory. The vaccine was also shown to result in loss of Her-2/neu expression, a phenomenon similarly observed in the clinic after treatment with Trastuzumab. Applying a Her-2/neu lung metastasis mouse model, here we tested a multi-peptide B cell vaccine containing HerVaxx and a mimotope of Pertuzumab for the prevention of lung metastases formation and investigated the loss of Her-2/neu expression. Mice were vaccinated with the multi-peptide vaccine, tail-vein injected with mammary carcinoma cells expressing human Her-2/neu, and histological evaluations of the mice lungs were carried out. A significantly reduced lung metastasis formation by active immunization with either the mimotope of Pertuzumab, HerVaxx, or the combination of both, was observed and associated with decreased lung weights and increased CD4+ and CD8+ T cells infiltration. Markedly, along with the overall reduction of lung weights and Her-2 positive metastases, the formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our data might result in a multi-peptide B cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Furthermore, the loss of Her-2/neu expression suggests a combination therapy involving an anti-PD-L1 immune checkpoint inhibitor or an active immunization with a mimotope from PD-L1. Such combination therapy to alternately target Her-2/neu and PD-L1 might be adapted to the stage and progression phase of the disease and result in the remission of the metastases.

An armed oncolytic virus enhances the efficacy of tumor-infiltrating lymphocyte therapy by converting tumors to artificial antigen-presenting cells in situ

The full potential of tumor-infiltrating lymphocyte (TIL) therapy has been hampered by the inadequate activation and low persistence of TILs, as well as inefficient neoantigen presentation by tumors. We transformed tumor cells into artificial antigen-presenting cells (aAPCs) by infecting them with a herpes simplex virus 1 (HSV-1)-based oncolytic virus encoding OX40L and IL12 (OV-OX40L/IL12) to provide local signals for optimum Tcell activation. The infected tumor cells displayed increased expression of antigen-presenting cell-related markers and induced enhanced Tcell activation and killing in coculture with TILs. Combining OV-OX40L/IL12 and TIL therapy induced complete tumor regression in patient-derived xenograft and syngeneic mouse tumor models and elicited an antitumor immunological memory. In addition, the combination therapy produced aAPC properties in tumor cells, activated Tcells, and reprogrammed macrophages to a more M1-like phenotype in the tumor microenvironment. This combination strategy unleashes the full potential of TIL therapy and warrants further evaluation in clinical studies.

Abstract 3432: A novel anti-CLDN6-CD137 bispecific antibody (NBL-028) for treating Claudin 6 positive solid tumors

Abstract Claudin-6 is a tetraspan membrane protein associated with tight junction formation. Its expression in normal tissues is restricted to the fetal organs but is not detected in the adult tissues. Increased expression of CLDN6 has been shown in several human malignancies such as testicular, ovarian, uterine, liver and lung adenocarcinoma, and is associated with poor prognosis in these cancer patients. Therefore, CLDN6 is a promising tumor-associated antigen (TAA) for tumor-targeting therapeutics such as CART and T cell engaging bispecific antibodies. CD137 co-stimulation has been reported to lead to extended T-cell proliferation, reactivating anergic T cells, promoting memory T cell formation and maintenance. Activating CD137 with agonistic antibodies presents a great opportunity to improve the therapeutic efficacy of immune checkpoint inhibitors (ICIs) or overcome resistance to ICIs. Additionally, a bispecific antibody that activates CD137 signaling only in the presence of TAA would help reduce the dose-dependent hepatotoxicity that was observed in clinical trials with monoclonal anti-CD137 agonistic antibody due to the activation of CD137 signaling in liver resident Kupffer cells. NBL-028 is a novel tetravalent bispecific antibody uniquely designed to activate the CD137 co-stimulatory pathway in the tumor microenvironment through the CLDN6-mediated clustering of CD137. NBL-028 binds to Claudin 6 with high affinity and specificity, activates CD137 in a CLDN6-dependent manner with fast-on and fast-off CD137 binding properties. NBL-028 demonstrated strong CLDN6-dependent T-cell activation and T-cell mediated cytotoxicity in vitro. NBL-028 elicits potent anti-tumor effects and immunological memory in mouse models without detectable liver damages or systemic toxicity. The tumor infiltrated Lymphocyte (TIL) analysis also demonstrated that NBL-028 treatment induced CD45+ immune cells infiltration, decreased Treg/CD8 ratio and increased M1/M2 like macrophage ratio. Furthermore, NBL-028 is designed upon a proprietary molecular scaffold with superior developability features, demonstrated by stability, yield and ease to purify. NBL-028 is currently at the stage of IND-enabling activities with the aim to enter clinical studies for treating Claudin 6 positive solid tumors in 1Q 2023. Citation Format: Raymond Yu, Yong Tong, Huarui Lu, Makenzie Danton, Samantha Dillman, Joshua Dewe, Joshua Karchin, Samruddhi Patil, Danielle Lavery, Haichun Huang, James Pei, Zhong Liu, Han Li, Ming Lei. A novel anti-CLDN6-CD137 bispecific antibody (NBL-028) for treating Claudin 6 positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3432.

Intravesical VAX014 Synergizes with PD-L1 Blockade to Enhance Local and Systemic Control of Bladder Cancer.

Emerging clinical evidence indicates that the combination of local administration of immunotherapy with systemic immune-checkpoint blockade targeting the PD-1/PD-L1 pathway improves response rates in select solid tumor indications; however, limited clinical experience with this approach exists in advanced bladder cancer patients. VAX014 is a novel bacterial minicell-based, integrin-targeted oncolytic agent undergoing clinical investigation for intravesical (IVE) treatment of nonmuscle-invasive bladder cancer. Here, we demonstrated that the antitumor activity of VAX014 following IVE administration was dependent upon CD4+ and CD8+ T cells in two syngeneic orthotopic bladder tumor models (MB49 and MBT-2). PD-L1 upregulation was found to be an acquired immune-resistance mechanism in the MB49 model, and the combination of VAX014 with systemic PD-L1 blockade resulted in a significant improvement in bladder tumor clearance rates and development of protective antitumor immunologic memory. Combination treatment also led to enhanced systemic antitumor immune responses capable of clearing distal intradermal tumors and controlling pulmonary metastasis. Distal tumors actively responding to combination therapy demonstrated a phenotypic shift from regulatory T cell to Th1 in intratumoral CD4+ T cells, which was accompanied by a higher percentage of activated CD8+ T cells and higher IFNγ. Finally, VAX014's target integrins α3β1 and α5β1 were overexpressed in tumor biopsies from advanced-stage bladder cancer patients, as well as in both the MB49 and MBT-2 orthotopic mouse models of bladder cancer. These collective findings provide a rationale for the clinical investigation of VAX014 and systemic PD-1/PD-L1 blockade in advanced-stage bladder cancer.

Platelet Pharmacytes for the Hierarchical Amplification of Antitumor Immunity in Response to Self-Generated Immune Signals.

Systemic immunosuppression mediated by tumor-derived exosomes is an important cause for the resistance of immune checkpoint blockade (ICB) therapy. Herein, self-adaptive platelet (PLT) pharmacytes are engineered to mediate cascaded delivery of exosome-inhibiting siRNA and anti-PD-L1 (aPDL1) toward synergized antitumor immunity. In the pharmacytes, polycationic nanocomplexes (NCs) assembled from Rab27 siRNA (siRab) and a membrane-penetrating polypeptide are encapsulated inside the open canalicular system of PLTs, and cytotoxic T lymphocytes (CTLs)-responsive aPDL1 nanogels (NGs) are covalently backpacked on the PLT surface. Upon systemic administration, the pharmacytes enable prolonged blood circulation and active accumulation to tumors, wherein PLTs are activated to liberate siRab NCs, which efficiently transfect tumor cells, silence Rab27a, and inhibit exosome secretion. The immunosuppression is thus relieved, leading to the activation, proliferation, and tumoral infiltration of cytotoxic T cells, which trigger latent aPDL1 release. As such, the competitive aPDL1 exhaustion by PD-L1-expressing exosomes is minimized to sensitize ICB. Synergistically, siRab and aPDL1 induce strong antitumor immunological response and memory against syngeneic murine melanoma. This study reports a bioinspired mechanism to resolve the blood circulation/cell internalization contradiction of polycationic siRNA delivery systems, and renders an enlightened approach for the spatiotemporal enhancement of antitumor immunity.

Active immunization with a Her-2/neu-targeting Multi-peptide B cell vaccine prevents lung metastases formation from Her-2/neu breast cancer in a mouse model

In pre-clinical and clinical settings, active immunization with a Her-2/neu vaccine (HerVaxx), comprising B-cell peptide from Trastuzumab binding site, has been shown to reduce primary tumor growth via induction of polyclonal anti-tumor immune responses and immunological memory. Here, we tested the combination of HerVaxx and the recently identified B-cell epitope/mimotope of Pertuzumab, i.e. a multi-peptide B-cell vaccine, for preventing Her-2/neu lung metastases formation in a mouse model. Active immunization with the multi-peptide vaccine was associated with decreased lung weights, and histological evaluation of the lungs showed that the significant reduction of lung metastases was associated with increased CD4+ and CD8+ T cell infiltration. Notably, along with the overall reduction of lungs weights and Her-2 positive metastases, a formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our results might pave the way to a multi-peptide B-cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Moreover, combination therapy targeting PD-L1 may result in total remission of metastases. Such a therapy may be used clinically to alternately target Her-2/neu and PD-L1 in metastatic breast cancer.

22P Active immunization with a multi-peptide B cell vaccine, targeting trastuzumab and pertuzumab binding sites, prevents the formation of Her-2/neu-expressing lung metastases

We have developed a B cell peptide-based Her-2/neu vaccine (HerVaxx) comprising trastuzumab’s binding site. Previous work in pre-clinical and clinical settings has shown that the vaccine reduces primary tumor growth by inducing polyclonal anti-tumor immune responses and immunological memory. Treatment with the monoclonal antibodies (mAbs) pertuzumab and trastuzumab have demonstrated remarkable success in the clinical outcome of patients with Her-2/neu-positive metastatic breast cancer. However, active immunization with B cell peptide-based vaccines can induce the production of the corresponding mAbs by the patient’s immune system.

VAX014 for instillation in subjects with nonmuscle-invasive bladder cancer.

488 Background: VAX014 is a novel tumor targeted oncolytic agent for intravesical administration. It is comprised of recombinant bacterial minicells, that actively target a3b1 and a5b1 integrins expressed on NMIBC and deliver an active cytotoxic/oncolytic protein payload, perfringolysin O. VAX014 is being investigated for the intravesical treatment of NMIBC in an ongoing multi-center Phase 1 marker lesion trial in subjects with low grade Ta disease (NCT 03854721). Methods: Subjects with up to 5 pathologically confirmed TaG1 lesions ≥ 5mm but ≤ 15mm are eligible to receive six (6) weekly intravesical instillations of VAX014. Subjects with presence or history of high-grade bladder cancer are ineligible. Phase 1a will establish the MTD using a 3+3 design. After the DSMB establishes the RP2D, a Phase 1b expansion will use this dose to treat an additional 10 patients. Results: No serious adverse events or dose limiting toxicities have been recorded through two dose cohorts (3 + 3 design) and evidence of clinical response has been observed. Treatment related adverse events have been limited to Grade 1/2 hematuria, dysuria, and urgency. Preclinical studies indicate the pharmacodynamic activity of VAX014 following weekly intravesical administration to immune competent mice bearing bladder tumors is immune dependent and synergizes with systemic PD-L1 blockade to eliminate organ confined tumors and extravesical tumors. Surviving mice are capable of rejecting tumor rechallenge due to development of protective antitumor immunologic memory in response to therapy. Exploratory immunohistochemical target validation studies in bladder tumor biopsy specimens from subjects with more advanced stage tumors (T1-T3), indicate overexpression of VAX014’s target integrins, a3b1 and a5b1, in most samples tested. Conclusions: These clinical and preclinical data support the further development of VAX014 for the intravesical treatment of NMIBC and set a foundation for combination therapy with systemically administered immune checkpoint blockade in subjects with advanced disease. Clinical trial information: NCT03854721.

In situ vaccination using unique TLR9 ligand K3-SPG induces long-lasting systemic immune response and synergizes with systemic and local immunotherapy

Although checkpoint inhibitors (CPIs) have changed the paradigm of cancer therapy, low response rates and serious systemic adverse events remain challenging. In situ vaccine (ISV), intratumoral injection of immunomodulators that stimulate innate immunity at the tumor site, allows for the development of vaccines in patients themselves. K3-SPG, a second-generation nanoparticulate Toll-like receptor 9 (TLR9) ligand consisting of K-type CpG oligodeoxynucleotide (ODN) wrapped with SPG (schizophyllan), integrates the best of conventional CpG ODNs, making it an ideal cancer immunotherapy adjuvant. Focusing on clinical feasibility for pancreaticobiliary and gastrointestinal cancers, we investigated the antitumor activity of K3-SPG-ISV in preclinical models of pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). K3-SPG-ISV suppressed tumor growth more potently than K3-ISV or K3-SPG intravenous injections, prolonged survival, and enhanced the antitumor effect of CPIs. Notably, in PDAC model, K3-SPG-ISV alone induced systemic antitumor effect and immunological memory. ISV combination of K3-SPG and agonistic CD40 antibody further enhanced the antitumor effect. Our results imply that K3-SPG-based ISV can be applied as monotherapy or combined with CPIs to improve their response rate or, conversely, with CPI-free local immunotherapy to avoid CPI-related adverse events. In either strategy, the potency of K3-SPG-based ISV would provide the rationale for its clinical application to puncturable pancreaticobiliary and gastrointestinal malignancies.

Intratumoral expression of IL-12 from lentiviral or RNA vectors acts synergistically with TLR4 agonist (GLA) to generate anti-tumor immunological memory.

Systemic interleukin-12 (IL12) anti-tumor therapy is highly potent but has had limited utility in the clinic due to severe toxicity. Here, we present two IL12-expressing vector platforms, both of which can overcome the deficiencies of previous systemic IL12 therapies: 1) an integrating lentiviral vector, and 2) a self-replicating messenger RNA formulated with polyethyleneimine. Intratumoral administration of either IL12 vector platform resulted in recruitment of immune cells, including effector T cells and dendritic cells, and the complete remission of established tumors in multiple murine models. Furthermore, concurrent intratumoral administration of the synthetic TLR4 agonist glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE) induced systemic memory T cell responses that mediated complete protection against tumor rechallenge in all survivor mice (8/8 rechallenged mice), whereas only 2/6 total rechallenged mice treated with intratrumoral IL12 monotherapy rejected the rechallenge. Taken together, expression of vectorized IL12 in combination with a TLR4 agonist represents a varied approach to broaden the applicability of intratumoral immune therapies of solid tumors.

Abstract PR18: A novel immunomodulatory strategy of targeting glyco-immune checkpoints using EAGLE technology to treat cancer

Abstract Cancer therapy has been revolutionized by inhibiting immune checkpoints to harness the power of the immune system in fighting cancer. However, the majority of patients are resistant to the current immuno-oncology drugs. There is a strong need to identify novel mechanisms of immune evasion in cancer and explore novel therapeutic modalities. Recently, glyco-immune checkpoints have been identified as an important modulator in cancer immune escape. Upon ligation of cancer-specific sialylated glycans, glyco-immune checkpoints suppress multiple facets of cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation anticancer T-cell immunity. However, due to the complexity and heterogeneity of cancer sialylated glycans and the profound species differences of the glyco-immune checkpoints between human and mouse, this immunomodulatory pathway remains unexplored for therapeutic interventions. Here we described a novel multifunctional antibody-like therapeutic modality, named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), to target this glyco-immune checkpoint axis. EAGLE overcomes the complexity of cancer glycans and specifically cleaves the immunosuppression-mediating key sugar moieties, the terminal sialic acids of various sialylated glycans on tumor cells. We hypothesized that EAGLE could block the immunosuppressive glyco-immune checkpoints in the tumor microenvironment and potentiate innate and adaptive immune responses to treat cancer. Indeed, systematic delivery of an EAGLE molecule decreased the levels of immunosuppressive sialic acids on tumor cells, and increased T-cell infiltration and activation in syngeneic tumor models. EAGLE treatment led to 50% complete regressions of established tumors as a monotherapy, and 100% cures in combination with anti-PD1 mAb. Furthermore, cured mice from EAGLE treatment completely rejected the rechallenge of tumor cells, suggesting that EAGLE induced antitumor immunologic memory. In summary, we demonstrated that this novel therapeutic modality, EAGLE, can release glyco-immune checkpoint-mediated immunosuppression in the tumor microenvironment and potentiate innate and adaptive antitumor immunity, which offers a novel and promising immunomodulatory strategy to treat cancer. This abstract is also being presented as Poster A91. Citation Format: Li Peng, Adam Petrone, Lizhi Cao, James Broderick. A novel immunomodulatory strategy of targeting glyco-immune checkpoints using EAGLE technology to treat cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR18.

Intratumoral administration of DNA-damaging chemotherapy-treated tumor cells to enhance therapeutic benefit of systemic immune checkpoint blockade in mouse cancer models.

77 Background: Immune checkpoint inhibition or ICI (antibodies to PD-1 and CTLA4), has shown promise in the treatment of some tumor types, especially in inducing durable remissions in advanced stage cancer patients. However, the majority of patients do not respond to ICI. Identifying combinations to enhance response to ICI is an urgent medical need. Methods: Murine tumor cell lines B16-Ova and MC-38-Ova were treated with DNA-damaging chemotherapeutic drugs, co-cultured with primary murine bone marrow derived dendritic cells (BMDC) followed by addition of OT-1 CD8+ T-cells and flow cytometric analysis of IFN-γ+ CD8+ T-cells. Mice bearing B16-Ova or MC-38 flank tumors were injected intra-tumorally with ex vivo chemotherapy-treated B16-Ova cells with or without systemic ICI. Tumor cross-sectional area was measured using calipers. Intra-tumoral DC and circulating H2-Kb/SIINFEKL-specific CD8+ T-cells were analyzed by flow cytometry. Results: Etoposide and mitoxantrone-treated B16-Ova and MC-38-Ova tumor cells, when co-cultured with BMDC, efficiently promote IFN- γ induction in OT-1 CD8+ T-cells. This was abrogated by co-treatment of tumor cells with Necrostatin-1 but not ZVAD-FMK. Intra-tumoral injection of ex vivo etoposide-treated tumor cells, with systemic ICI, increases the numbers of intra-tumoral CD103+ DC, the frequency of circulating H2-Kb/SIINFEKL-specific CD8+ T-cells and significantly improves survival. The tumor cell vaccine/systemic ICI combination, but not ICI alone, induced complete tumor regressions in a subset of mice. This is abrogated in BATF3-deficient mice. Conclusions: Etoposide and mitoxantrone-treated tumor cells efficiently promote BMDC-mediated CD8+ T-cell priming, in a tumor cell RIPK1 activity-dependent but caspase-independent manner. Intra-tumoral administration of the DNA-damage induced tumor cell vaccine in vivo, in combination with systemic ICI, enhances anti-tumor CD8+ T-cell responses, tumor-free and overall survival, and anti-tumor immunological memory. This enhancement in therapeutic efficacy is dependent on BATF3+ DC in vivo.

Abstract 3197: BI-907828, a novel and potent MDM2-p53 antagonist, acts synergistically in a triple combination with anti-PD-1 and anti-LAG-3 antibodies in syngeneic mouse models of cancer

Abstract MDM2-p53 antagonists block the interaction between the Tumor Protein p53 and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2-p53 antagonists are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2-p53 antagonists are currently being evaluated in early clinical development. BI-907828 is a novel and potent MDM2-p53 antagonist with optimized drug-like properties that has shown efficacy in human tumor xenograft models at daily low oral dose as well as intermittent high dose schedules. Recent preclinical studies in syngeneic mouse models of cancer have demonstrated that BI-907828, apart from its direct tumor-targeting activity, also has immunomodulatory activity shown to contribute to efficacy. Single-agent BI-907828 induced anti-tumor immunological memory that controlled tumor growth in a re-challenge study. Moreover, a dual combination with an anti-mouse PD-1 checkpoint inhibitor resulted in synergistic efficacy in a syngeneic mouse model of cancer (AACR 2018, abstract 4866). Here we present data for the triple combination of BI-907828 with two checkpoint inhibitors. In syngeneic mouse tumor models (Colon-26 and B16-F10), the combination of BI-907828 with tool antibodies against mouse PD-1 and mouse LAG-3 shows high response rates of 50-90% with tumor regressions observed for even very large tumors. Moreover, efficacy of the triple combination is superior to each single agent and all dual combinations. An antibody-mediated depletion study suggests a contribution of CD8+ T cells but not of CD4+ T cells to full efficacy of the triple combination. FACS analysis of tumors isolated from Colon-26 tumor-bearing mice indicates that treatment with the triple combination leads to expansion of tumor-infiltrating CD8+ T cells. In summary, BI-907828 is a novel, potent, orally bioavailable MDM2-p53 antagonist that shows synergistic efficacy in a triple combination with antibodies targeting the immune checkpoints PD-1 and LAG-3 in syngeneic mouse models of cancer. BI-907828 is currently under evaluation in a Phase I clinical study (NCT03449381). Citation Format: Dorothea Rudolph, Ulrike Weyer-Czernilofsky, Markus Reschke, Martina Sykora, Jörg Rinnenthal, Sophia Blake, Gabriela Gremel, Andreas Wernitznig, Andreas Gollner, Norbert Kraut, Jürgen Moll. BI-907828, a novel and potent MDM2-p53 antagonist, acts synergistically in a triple combination with anti-PD-1 and anti-LAG-3 antibodies in syngeneic mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3197.