Agonistic anti-CD40 Research Articles

BT7480, a novel fully synthetic Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA™) induces tumor localized CD137 agonism

BackgroundCD137 (4-1BB) is an immune costimulatory receptor with high therapeutic potential in cancer. We are creating tumor target-dependent CD137 agonists using a novel chemical approach based on fully synthetic constrained...

Boosting the Abscopal Effect Using Immunogenic Biomaterials With Varying Radiation Therapy Field Sizes

Persistent immunosuppression in the tumor microenvironment is a major limitation to boosting the abscopal effect, whereby radiation therapy at 1 site can lead to regression of tumors at distant sites. Here, we investigate the use of radiation and immunogenic biomaterials (IBM) targeting only the gross tumor volume/subvolume for boosting the abscopal effect in immunologically cold tumors. To evaluate the abscopal effect, 2 syngeneic contralateral tumors were implanted in each mouse, where only 1 tumor was treated. IBM was administered to the treated tumor with 1 fraction of radiation and results were compared, including as a function of different radiation therapy field sizes. The IBM was designed similar to fiducial markers using immunogenic polymer components loaded with anti-CD40 agonist. Tumor volumes of both treated and untreated tumors were measured over time, along with survival and corresponding immune cell responses. Results showed that radiation with IBM administered to the gross tumor subvolume can effectively boost abscopal responses in both pancreatic and prostate cancers, significantly increasing survival (P < .0001 and P < .001, respectively). Results also showed equal or superior abscopal responses when using field sizes smaller than the gross tumor volume compared with irradiating the whole tumor volume. These results were buttressed by observation of higher infiltration of cytotoxic CD8+ T-lymphocytes in the treated tumors (P < .0001) and untreated tumors (P < .0001) for prostate cancer. Significantly higher infiltration was also observed in treated tumors (P < .0001) and untreated tumors P < .01) for pancreatic cancer. Moreover, the immune responses were accompanied by a positive shift of proinflammatory cytokines in both prostate and pancreatic tumors. The approach targeting gross tumor subvolumes with radiation and IBM offers opportunity for boosting the abscopal effect while significantly minimizing healthy tissue toxicity. This approach proffers a radioimmunotherapy dose-painting strategy that can be developed for overcoming current barriers of immunosuppression especially for immunologically cold tumors.

1011P FORTITUDE phase I study of NG-350A, a novel tumour-selective adenoviral vector expressing an anti-CD40 agonist antibody: Monotherapy dose escalation results

Multiple anti-CD40 antibodies (Abs) have been evaluated clinically, however they have shown limited efficacy as monotherapy. NG-350A is a novel transgene ‘armed’ tumour-selective adenoviral vector designed to re-engineer immunosuppressive tumours by expressing a fully human agonistic anti-CD40 Ab. As NG-350A selectively replicates in tumour cells, the anti-CD40 Ab is only expressed in the tumour microenvironment (TME), thereby limiting systemic exposure. Unlike many viral vectors, NG-350A is stable in blood allowing intravenous (IV) delivery to multiple tumor sites. In this phase I trial (NCT03852511) NG-350A was dose-escalated in patients (pts) with metastatic or advanced epithelial tumours in a 3+3 design. Here we present initial safety and pharmacodynamic data after completion of monotherapy dose-escalation. A total of 16 pts were treated at 3 IV dose levels, from 1x1011 virus particles (vp) on Days (D) 1, 3 and 5 to 1x1012vp on D1, followed by 6x1012vp on D3 and 5. Overall, NG-350A was well-tolerated, with no cases of Grade≥3 CRS nor evidence of raised liver enzymes. Dose-dependent specific increases in IL-12 and IFNγ were detected in serum of evaluable pts treated at higher dose levels, beginning at ∼Week2. In the majority of pts, IL-12 and IFNγ levels remained at ≥40pg/ml and 5x baseline levels at 7 weeks after dosing. NG-350A also led to the expansion of T-cell clones in blood with the majority of these being newly detected clones. These data suggest that NG-350A replicates in tumour cells and produces functional anti-CD40 within the TME, leading to higher and more sustained elevations in IL-12 and IFNγ than are typically observed with systemic anti-CD40 Abs. The elevations observed suggest innate immune cell stimulation and Th1-type T-cell activation, consistent with the mechanism of action of CD40 agonists. Together, these initial safety and biomarker data indicate that NG-350A may contribute to immunological re-engineering of the TME through localized production of anti-CD40, while avoiding the associated toxicity of systemic dosing, and support further clinical assessment of NG-350A in combination with anti-PD-1 Abs in FORTITUDE.

Abstract 1567: IL-15 and anti-PD-1 augment the abscopal efficacy of agonistic intratumoral anti-CD40 in the TRAMP-C2 murine tumor model

Abstract IL-15 promotes activation and maintenance of natural killer (NK) and CD8+ T-effector memory (TEM) cells making it a potential immunotherapeutic agent for the treatment of cancer. However, high doses of γc cytokines lead to the paralysis/depression of naïve CD4 but not CD8 T-cells that is mediated through transient expression of suppressor of cytokine signaling 3 (SOCS3). Thus, the out-of-normal order addition of γc cytokines is associated with generation of “helpless” tumor antigen nonspecific CD8 T cells. The role of CD4 helper T cells can be alternatively mediated by CD40 signaling through the addition of agonistic anti-CD40 antibodies. However, parenteral administration of an agonistic anti-CD40 antibody is associated with unacceptable toxicity that can be avoided by intratumoral administration. Intratumoral immunotherapy that aims at generating a potent low toxic priming of antitumor immunity uses the tumor as its own vaccine. We investigated the combination of IL-15 with an intratumoral anti-CD40 monoclonal antibody using a dosing schedule based on our previous study in the TRAMP-C2 murine prostate cancer model. Here we demonstrated that given intratumorally anti-CD40 had abscopal efficacy, and that the combination of IL-15 with the intratumoral anti-CD40 showed an augmented immune response with an increase in the number of tumor specific tetramer positive CD8+ T-cells. Furthermore, anti-PD-1 antibody was additive to the anti-CD40 IL-15 combination. These studies support the initiation of a clinical trial in patients with cancer involving IL-15 in association with intratumoral optimized anti-CD40. Citation Format: Wei Chen. IL-15 and anti-PD-1 augment the abscopal efficacy of agonistic intratumoral anti-CD40 in the TRAMP-C2 murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1567.

Abstract 1724: Microinjection of Nectin-4/CD137 tumor-targeted immune cell agonist (TICA™) activates the local tumor microenvironment

Abstract A new generation of both systemic and targeted CD137 agonists are entering clinical development following agonistic anti-CD137 (4-1BB) antibody development nearly a decade ago. However, each of these molecules rely on biologic agents with potentially suboptimal properties for CD137 agonism due to their relatively large size and long circulating half-lives which may limit their tissue penetration, cause sustained agonism resulting in overstimulation and activation-induced lymphocyte cell death. Fully synthetic constrained bicyclic peptides (Bicycles™) with antibody-like affinities and target selectivity are uniquely suited to circumvent limitations of other targeted CD137 agonistic therapeutics. BT7480 and BCY11864 are tumor targeted immune cell agonists (TICA) designed to deliver a highly potent CD137 agonist to Nectin-4 overexpressing tumors with a flexible dosing schedule maximizing anti-tumor activity while circumventing the need for continuous systemic exposure. The Comparative In Vivo Oncology (CIVO) platform has been developed to enable in situ investigation of multiple microdosed drugs simultaneously in human tumors with safety and feasibility of this platform recently demonstrated in patients with soft tissue sarcomas. CIVO percutaneously injects drug microdoses directly into tumor tissue as trackable columns where tissue can be analyzed after resection for the effect of the drug treatment in the tumor. Here we report on an evaluation of the feasibility of using the CIVO -platform to demonstrate the mechanism of action of our tumor target-dependent CD137 agonist TICAs. Both BT7480 and BCY11864 demonstrate extremely potent Nectin-4-dependent CD137 agonism in primary human PBMC/tumor cell co-culture assays. Systemic administration of BT7480 in Nectin-4 expressing tumors in immunocompetent mice leads to profound reprogramming of the tumor immune microenvironment including a rapid increase in T cell chemotactic cytokine transcripts and macrophage gene signatures followed by an increase in cytotoxic cell gene signature and increase in intratumoral CD8+ T cells. CIVO was used to demonstrate the in vivo mechanism of action of a Nectin-4/CD137 TICA. Micro-injection of BCY11864 into Nectin-4 expressing tumors in immunocompetent mice led to dose dependent induction of immune activation markers in the tumor microenvironment and activation of the tumor resident cytotoxic T cells was evident 24 hours after BCY11864 micro-injection. Akin to our findings with transcriptional profiling of the tumor response to BT7480, BCY11864 activity was shown not to be limited to T cells but also included other CD137 positive cell populations such as myeloid and NK cells. We hypothesize that tumor targeted CD137 agonism may lead to initial myeloid cell response that enhances the cytotoxic T cell recruitment and activation in the tumor tissue. Citation Format: Johanna Lahdenranta, Stephen J. Blakemore, Kristen Hurov, Marc O. Grenley, Punit Upadhyaya, Carly Campbell, Elizabeth Repash, Eric Haines, Philip E. Brandish, Richard A. Klinghoffer, Nicholas Keen. Microinjection of Nectin-4/CD137 tumor-targeted immune cell agonist (TICA™) activates the local tumor microenvironment [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 1724.

Abstract 1851: Responses of a novel tumor selective anti-CD137 agonist antibody activated by elevated extracellular ATP in tumor microenvironment

Abstract Background: CD137 (4-1BB) is a co-stimulatory molecule which exerts activation signals in multiple immune cell subsets. However, agonist antibodies targeting CD137 have not been clinically successful due to severe systemic toxicity. Since conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigen, we instead exploited extracellular adenosine triphosphate (exATP), which is known to be elevated in tumor microenvironment (TME) while remaining tightly regulated in normal tissues, as a broadly tumor selective switch. Here we report a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. Methods &amp; Results: STA551 bound to human CD137 in an ATP dependent manner. Consistent with the binding profile, STA551 induced IFN-γ only in the presence of ATP, whereas urelumab induced IFN-γ regardless of ATP concentration in a human T cell assay. We examined the anti-tumor efficacy of Sta-MB, which has the same variable region as STA551 using a surrogate mouse Fc, in human CD137 knock-in (hCD137-KI) mice. STA551 demonstrated potent and broad anti-tumor efficacy against all the tumors we tested both as a monotherapy and in combination with anti-PD-L1 antibody. To confirm the immune responses in tumors, we evaluated gene expression and analyzed tumor infiltrating lymphocytes by flow cytometry. Immune-related gene expression changed after treatment with Sta-MB. We also confirmed that Sta-MB treatment increased the number of activated CD8+ T cells in tumors. For all tumor responses, Sta-MB tended to be more potent than Ure-MB. Whereas other anti-CD137 conventional antibodies showed significant CD137 mediated clearance and systemic T cell activation in draining lymph node (DLN), spleen, and liver, STA551 demonstrated significant intratumor T cell activation, but not in normal tissues and without CD137 mediated systemic clearance in mice. Furthermore, STA551 was well-tolerated in cynomolgus monkeys in a repeated-dose toxicity study*. Conclusion: These results suggest that STA551, due to its tumor selective agonistic activity, has potent anti-tumor efficacy and a wide therapeutic window, providing a strong rationale for its clinical testing against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. STA551 is currently being tested in a phase 1 clinical study. We will here disclose the clinical study design for the first time. Citation Format: Mika Kamata-Sakurai, Yoshinori Narita, Ryo Uchikawa, Kenji Taniguchi, Sayuri Horikawa, Koki Hamada, Yuji Hori, Takayuki Nemoto, Shoichi Metsugi, Michiyasu Inatani, Tomoyuki Igawa. Responses of a novel tumor selective anti-CD137 agonist antibody activated by elevated extracellular ATP in tumor microenvironment [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 1851.

Antitumor efficacy and reduced toxicity using an anti-CD137 Probody therapeutic

Costimulation via CD137 (4-1BB) enhances antitumor immunity mediated by cytotoxic T lymphocytes. Anti-CD137 agonist antibodies elicit mild liver inflammation in mice, and the maximum tolerated dose of Urelumab, an anti-human CD137 agonist monoclonal antibody, in the clinic was defined by liver inflammation-related side effects. A protease-activated prodrug form of the anti-mouse CD137 agonist antibody 1D8 (1D8 Probody therapeutic, Pb-Tx) was constructed and found to be selectively activated in the tumor microenvironment. This construct, which encompasses a protease-cleavable linker holding in place a peptide that masks the antigen binding site, exerted antitumor effects comparable to the unmodified antibody but did not result in liver inflammation. Moreover, it efficaciously synergized with both PD-1 blockade and adoptive T-cell therapy. Surprisingly, minimal active Pb-Tx reached tumor-draining lymph nodes, and regional lymphadenectomy did not abrogate antitumor efficacy. By contrast, S1P receptor-dependent recirculation of T cells was absolutely required for efficacy. The preferential cleavage of the anti-CD137 Pb-Tx by tumor proteases offers multiple therapeutic opportunities, including neoadjuvant therapy, as shown by experiments in which the Pb-Tx is given prior to surgery to avoid spontaneous metastases.

Memory T Cells from Minor Histocompatibility Antigen-Vaccinated Donors Improve Engraftment with Low Intensity Conditioning

While allogeneic hematopoietic stem cell transplantation (alloSCT) is largely employed to treat malignant disorders, it can also cure inherited and acquired nonmalignant disorders. For example, inherited diseases such as sickle cell anemia, thalassemias, and varied immunodeficiencies can be cured with alloSCT. Autoimmune diseases can also be cured via the abilities of both the conditioning regimen and alloimmune T cells to eradicate autoimmune T and B cells. And importantly, tolerance to solid organ grafts can be created via transplantation of hematopoietic cells from the organ donor. However, the utility of alloSCT in these settings is limited by the morbidity and mortality of the procedure, including graft-vs-host disease (GVHD). Some progress has been made in lowering the toxicities in these settings. However, these approaches frequently result in mixed chimerism, which may not be sufficient to treat autoimmune diseases, and may lead to unreliable engraftment overall. Previously we reported that in mouse models of alloSCT memory CD8+ T cells from mice vaccinated against the single minor histocompatibility antigen (miHA) H60, which is mostly expressed on hematopoietic cells, dramatically improves graft-vs-leukemia (GVL) against H60-expressing leukemias, without causing GVHD. In the present work we have tested the utility of memory CD8 cells reactive to H60 (TMH60) in promoting engraftment. Following 6 Gy or 8Gy of total body irradiation (TBI), B6.H60 (H-2b, Ly9.1-, H60+) mice were transplanted with C3H.SW (H-2b, Ly9.1+, H60-) bone marrow (BM), with memory T cells from H60 vaccinated mice (TMH60) containing 3500 tetramer+ cells (TetH60) or with TM from unvaccinated mice as negative control. At TBI doses of 8 Gy and 6 Gy, the addition of TM from H60 vaccinated mice (TMH60), as compared to TM from unvaccinated mice, modestly improved engraftment. We next lowered the dose of TBI further. At 5 Gy, recipients of TMH60 had multilineage engraftment of donor-derived hematopoietic cells in bone marrow (BM), spleen and lymph nodes (LN) nine weeks after transplantation. In contrast, mice with TM failed to engraft. Importantly, mice did not develop clinical GVHD. At a TBI dose of 4 Gy, recipients with TMH60 had poor donor engraftment, while TMH60 recipients treated with an anti-CD40 agonist antibody (FGK45) on day 0 achieved full multilineage engraftment in spleen, LN and BM. In addition we achieved mixed chimerism in some TMH60 and anti-CD40 treated mice conditioned with 3 Gy. We also tested the utility of TM targeting the Kb-restricted SIINFEKL peptide derived from OVA (TM-OVA) in a mouse model wherein OVA expression is restricted to hematopoietic cells. We used B6.OVA mice, which express OVA ubiquitously, as BM donors and created B6.OVA–>B6 BM chimeras (H-2b, Ly9.1-, OVA+). We retransplanted these chimeras with C3H.SW (H-2b, Ly9.1+, OVA-) BM with C3H.SW TM-OVA, containing 3500 tetramer+ cells, or TM from unvaccinated mice following 5 Gy or 4 Gy of TBI. TM-OVA, but not TM promoted full multilineage engraftment in mice conditioned with 5 Gy in blood of mice analyzed two weeks after transplantation. At dose of 4 Gy, TM-OVA induced full donor engraftment, but only with the addition of anti-CD40. This experiment is ongoing. In sum, our data demonstrate that TM reactive against a miHA expressed on hematopoietic cells can dramatically promote donor multilineage engraftment without GVHD, and that the effectiveness of these TM can be augmented by stimulating CD40. Future experiments will test miHA-reactive TM with chemotherapy-based conditioning, in haploidentical models and the utility of blocking PD-1, which we have found greatly augments GVL mediated by miHA-reactive TM. This approach holds the promise of achieving consistent donor engraftment without GVHD, a prerequisite for the widespread use of alloSCT in treatment of autoimmune diseases and for tolerance induction in tandem with organ transplantation. DisclosuresNo relevant conflicts of interest to declare.

Identification of a predictive biomarker and two pharmacodynamic biomarkers to ADG106 treatment, a novel anti-CD137 agonist antibody, in phase I clinical trials.

e14505 Background: ADG106 is a fully human agonistic anti-CD137 monoclonal IgG4 antibody that mediates anti-tumor activities via unique mechanisms of action. Here we provide safety and efficacy updates from our phase I trials and report the findings of a predictive biomarker and two pharmacodynamic biomarkers which correlate with patients’ clinical responses to ADG106 treatment and demonstrate target engagement, respectively. Methods: Formalin fixed and paraffin embedded (FFPE), blood and plasma specimens were collected from 92 patients enrolled in our phase I trials. We measured expression across a panel of protein biomarkers in FFPE specimens using three highly sensitive detection technologies: multiple immunohistochemical (IHC) staining of protein expression, the BD Multitest 6-color TBNK reagent for profiling immune cell subpopulations, and the MSD-ECL electrochemiluminescence assay for detection of soluble CD137. Objective tumor responses were determined using RECIST v1.1 for solid tumor patients and Lugano classification for lymphoma patients. Results: As of November 30, 2020, ADG106 has demonstrated a favorable safety profile and efficacy in the phase I clinical trials with a disease control rate of 56%. From a retrospective analysis of 28 pretreatment FFPE specimens, we identified a predictive biomarker that correlated with tumor shrinkage upon ADG106 treatment. We identified four biomarker positive specimens from two patients with lymphoma and two with solid tumors. Three out of four biomarker positive patients achieved greater than 30% tumor shrinkage after 3mg/kg or 5mg/kg ADG106 treatment. One biomarker positive patient with stable disease received a low dose ADG106 treatment at 0.5mg/kg during dose escalation. None of the 24 biomarker negative patients showed significant clinical response. A tissue microarray study confirmed expression of this predictive biomarker in a variety of tumor types suggesting a broad indication for ADG106 therapy. Our biomarker studies also demonstrated target engagement with increased NK cell proliferation and soluble CD137 upon ADG106 treatment. Analysis of safety, efficacy, PK and PD data allowed us to select a recommended dose for the upcoming phase II study. Conclusions: We identified a biomarker predictive of response to antitumor CD137 blockade by ADG106, as well as demonstrated the involvement of NK cells in ADG106 mediated anti-tumor activities. In upcoming phase II trials, we plan to enrich for populations expressing this predictive biomarker to demonstrate a clinical benefit to ADG106 therapy further validating early biomarker-based patient stratification. We will also explore the potential of selecting patients for combination treatment with anti-PD-1 therapies. Clinical trial information: NCT03802955.

Dendritic cell targeting with Fc-enhanced CD40 antibody agonists induces durable antitumor immunity in humanized mouse models of bladder cancer.

Intravesical immunotherapy using Bacille Calmette-Guérin (BCG) attenuated bacteria delivered transurethrally to the bladder has been the standard of care for patients with high-risk non-muscle-invasive bladder cancer (NMIBC) for several decades. BCG therapy continues to be limited by high rates of disease recurrence and progression, and patients with BCG-unresponsive disease have few effective salvage therapy options besides radical cystectomy, highlighting a need for new therapies. We report that the immune-stimulatory receptor CD40 is highly expressed on dendritic cells (DCs) within the bladder tumor microenvironment of orthotopic bladder cancer mouse models, recapitulating CD40 expression by DCs found in human disease. We demonstrate that local CD40 agonism in mice with orthotopic bladder cancer through intravesical delivery of anti-CD40 agonist antibodies drives potent antitumor immunity and induces pharmacodynamic effects in the bladder tumor microenvironment, including a reduction in CD8+ T cells with an exhausted phenotype. We further show that type 1 conventional DCs (cDC1) and CD8+ T cells are required for both bladder cancer immune surveillance and anti-CD40 agonist antibody responses. Using orthotopic murine models humanized for CD40 and Fcγ receptors, we demonstrate that intravesical treatment with a fully human, Fc-enhanced anti-CD40 agonist antibody (2141-V11) induces robust antitumor activity in both treatment-naïve and treatment-refractory settings, driving long-term systemic antitumor immunity with no evidence of systemic toxicity. These findings support targeting CD40-expressing DCs in the bladder cancer microenvironment through an intravesical agonistic antibody approach for the treatment of NMIBC.

Activation of CD137 signaling promotes macrophage apoptosis dependent on p38 MAPK pathway-mediated mitochondrial fission.

CD137 signaling is an essential factor in cell fate and atherosclerosis. An increase in the number of apoptotic macrophages accelerates atherosclerotic development involving mitochondrial dynamics.However, the role of CD137 signaling in macrophage apoptosis and changes in mitochondria has not been demonstrated clearly. Here, we used ApoE-/- mice as a model of atherosclerotic plaques. Mouse agonist anti-CD137 L and inhibitory anti-CD137 antibody were used to activate or block the CD137 signaling, respectively. Treatment of ApoE-/- mice with agonist anti-CD137 L promoted the formation of necrotic cores and macrophage apoptosis in plaques. Further, activation of CD137 signaling caused macrophage apoptosis in vitro, with upregulation of caspase-9 and caspase-3 expression and an increase in the Bax/Bcl-2 ratio. Meanwhile, CD137 signaling promoted mitochondrial fission observed by mitochondrial fragmentation. Interestingly, inhibition of mitochondrial dynamin-related protein 1 (Drp1) using Mdivi-1 reduced the expression of pro-apoptotic proteins and the amounts of apoptotic macrophages induced by CD137 signaling. Finally, we also found that the p38 MAPK pathway activated by CD137 signaling increased the expression of Drp1 expression and number of mitochondrial fragmented structures. Inhibition of the p38 MAPK pathway by SB203580 attenuated mitochondrial dysfunction through reducing mitochondrial membrane potential loss, cytochrome c release, and mitochondrial reactive oxygen species (ROS) generation. Overall, we identify a novel mechanism whereby CD137 signaling induces macrophage apoptosis through promoting mitochondrial fission dependent on the p38 MAPK pathway.

The human anti-CD40 agonist antibody mitazalimab (ADC-1013; JNJ-64457107) activates antigen-presenting cells, improves expansion of antigen-specific T cells, and enhances anti-tumor efficacy of a model cancer vaccine in vivo

Non-responders to checkpoint inhibitors generally have low tumor T cell infiltration and could benefit from immunotherapy that activates dendritic cells, with priming of tumor-reactive T cells as a result. Such therapies may be augmented by providing tumor antigen in the form of cancer vaccines. Our aim was to study the effects of mitazalimab (ADC-1013; JNJ-64457107), a human anti-CD40 agonist IgG1 antibody, on activation of antigen-presenting cells, and how this influences the priming and anti-tumor potential of antigen-specific T cells, in mice transgenic for human CD40. Mitazalimab activated splenic CD11c+ MHCII+ dendritic cells and CD19+ MHCII+ B cells within 6 h, with a return to baseline within 1 week. This was associated with a dose-dependent release of proinflammatory cytokines in the blood, including IP-10, MIP-1α and TNF-α. Mitazalimab administered at different dose regimens with ovalbumin protein showed that repeated dosing expanded ovalbumin peptide (SIINFEKL)-specific CD8+ T cells and increased the frequency of activated ICOS+ T cells and CD44hi CD62L− effector memory T cells in the spleen. Mitazalimab prolonged survival of mice bearing MB49 bladder carcinoma tumors and increased the frequency of activated granzyme B+ CD8+ T cells in the tumor. In the ovalbumin-transfected tumor E.G7-OVA lymphoma, mitazalimab administered with either ovalbumin protein or SIINFEKL peptide prolonged the survival of E.G7-OVA tumor-bearing mice, as prophylactic and therapeutic treatment. Thus, mitazalimab activates antigen-presenting cells, which improves expansion and activation of antigen-specific T cells and enhances the anti-tumor efficacy of a model cancer vaccine.

Agonistic Anti-CD40 Antibody Triggers an Acute Liver Crisis With Systemic Inflammation in Humanized Sickle Cell Disease Mice.

Sickle cell disease (SCD) is an inherited hemolytic disorder, defined by a point mutation in the β-globin gene. Stress conditions such as infection, inflammation, dehydration, and hypoxia trigger erythrocyte sickling. Sickled red blood cells (RBCs) hemolyze more rapidly, show impaired deformability, and increased adhesive properties to the endothelium. In a proinflammatory, pro-coagulative environment with preexisting endothelial dysfunction, sickled RBCs promote vascular occlusion. Hepatobiliary involvement related to the sickling process, such as an acute sickle hepatic crisis, is observed in about 10% of acute sickle cell crisis incidents. In mice, ligation of CD40 with an agonistic antibody leads to a macrophage activation in the liver, triggering a sequence of systemic inflammation, endothelial cell activation, thrombosis, and focal ischemia. We found that anti-CD40 antibody injection in sickle cell mice induces a systemic inflammatory and hemodynamic response with accelerated hemolysis, extensive vaso-occlusion, and large ischemic infarctions in the liver mimicking an acute hepatic crisis. Administration of the tumor necrosis factor-α (TNF-α) blocker, etanercept, and the heme scavenger protein, hemopexin attenuated end-organ damage. These data collectively suggest that anti-CD40 administration offers a novel acute liver crisis model in humanized sickle mice, allowing for evaluation of therapeutic proof-of-concept.

Therapeutic vaccination targeting CD40 and TLR3 controls melanoma growth through existing intratumoral CD8 T cells without new T cell infiltration.

Dendritic cells are potently activated by the synergistic action of CD40 stimulation in conjunction with signaling through toll like receptors, subsequently priming T cells. Cancer vaccines targeting the activation of dendritic cells in this manner show promise in murine models and are being developed for human patients. While the efficacy of vaccines based on CD40 and toll like receptor stimulation has been established, further investigation is needed to understand the mechanism of tumor control and how vaccination alters tumor infiltrating immune cells. In this study we vaccinated mice bearing established murine melanoma tumors with agonistic anti-CD40, polyI:C, and tumor antigen. Vaccination led to increased intratumoral T cell numbers and delayed tumor growth, yet did not require trafficking of T cells from the periphery. Pre-existing intratumoral T cells exhibited an acute burst in proliferation but became less functional in response to vaccination. However, the increased intratumoral T cell numbers yielded increased numbers of effector T cells per tumor. Together, our data indicate that the existing T cell response and intratumoral dendritic cells are critical for vaccination efficacy. It also suggests that circulating T cells responding to vaccination may not be an appropriate biomarker for vaccine efficacy.

Boiling histotripsy and in-situ CD40 stimulation improve the checkpoint blockade therapy of poorly immunogenic tumors.

Background: Advanced stage cancers with a suppressive tumor microenvironment (TME) are often refractory to immune checkpoint inhibitor (ICI) therapy. Recent studies have shown that focused ultrasound (FUS) TME-modulation can synergize ICI therapy, but enhancing survival outcomes in poorly immunogenic tumors remains challenging. Here, we investigated the role of focused ultrasound based boiling histotripsy (HT) and in-situ anti-CD40 agonist antibody (αCD40) combinatorial therapy in enhancing therapeutic efficacy against ICI refractory murine melanoma.Methods: Unilateral and bilateral large (~330-400 mm3) poorly immunogenic B16F10 melanoma tumors were established in the flank regions of mice. Tumors were exposed to single local HT followed by an in-situ administration of αCD40 (HT+ αCD40: HT40). Inflammatory signatures post treatment were assessed using pan-cancer immune profiling and flow cytometry. The ability of HT40 ± ICI to enhance local and systemic effects was determined by immunological characterization of the harvested tissues, and by tumor growth delay of local and distant untreated tumors 4-6 weeks post treatment.Results: Immune profiling revealed that HT40 upregulated a variety of inflammatory markers in the tumors. Immunologically, HT40 treated tumors showed an increased population of granzyme B+ expressing functional CD8+ T cells (~4-fold) as well as an increased M1 to M2 macrophage ratio (~2-3-fold) and CD8+ T: regulatory T cell ratio (~5-fold) compared to the untreated control. Systemically, the proliferation rates of the melanoma-specific memory T cell population were significantly enhanced by HT40 treatment. Finally, the combination of HT40 and ICI therapy (anti-CTLA-4 and anti-PD-L1) caused superior inhibition of distant untreated tumors, and prolonged survival rates compared to the control.Conclusions: Data suggest that HT40 reprograms immunologically cold tumors and sensitizes them to ICI therapy. This approach may be clinically useful for treating advanced stage melanoma cancers.

CD137 agonist-based combination immunotherapy enhances activated, effector memory T cells and prolongs survival in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma(PDAC) is resistant to the PD-1/PD-L1 blockade therapy. Previously, the combination of PD-1 blockade and vaccine therapy was shown to have a modest antitumor activity in murine models of PDAC. We used a murine syngeneic model of metastatic PDAC to identify, among multiple T cell modulators tested, which therapeutic agents in combination with the GVAX cancer vaccine and an anti-PD-1 antagonist antibody(αPD-1) are able to improve the survival. We found that an anti-CD137 agonist antibody(αCD137) most significantly improved survival in the mouse PDAC model. Moreover, αPD-1 and αCD137 together in combination with vaccine therapy more significantly increased the expression of costimulatory molecules CD137 and OX40 on CD4+PD-1+ and CD8+PD-1+ T cells comparing to αPD-1 or αCD137, respectively, suggesting that T cell activation within PDACs were enhanced by a synergy of αCD137 and αPD-1. On another hand, αCD137 treatment led to an increase in effector memory T cells independent of αPD-1. Although αCD137 does not increase the cytotoxic effector T cell function, the addition of αCD137 to GVAX+αPD-1 increased expression of IFNγ in EOMES+exhausted tumor-infiltrating T cells. Taken together, this preclinical study established the mechanism of targeting CD137 to enhance effector memory and activated T cells in PDAC. Immunohistochemistry analysis of resected human PDACs following the neo-adjuvant GVAX treatment showed increased levels of CD8+ T cells in those with high levels of CD137 expression, supporting an ongoing clinical trial of testing CD137 as a potential target in treating PDACs that are inflamed with T cells by vaccine therapy.

Priming the Abscopal Effect in Cervical Cancer

Current treatment options for advanced cervical cancer are limited, especially for patients in low-resource settings. The purpose of this study is to investigate a potential therapeutic approach by priming the abscopal effect, leading to regression of both locally treated and distant untreated tumors. We hypothesize that radiotherapy combined with locally delivered anti-CD40 will boost the abscopal effect, with initial investigation in mice. Naïve healthy female mice C57BL/6 were injected with mice cervical cancer cells TC-1 subcutaneously on contralateral flanks, mimicking primary and metastatic tumors. The animals were then randomized into different cohorts. In the treatment cohort, only the primary tumor was treated and response of both tumors was monitored. The treatment consisted of image-guided radiotherapy (IGRT) with a single 6 Gy dose with 10x10 mm2 field size using the Small Animal Radiation Research Platform (SARRP), followed by intratumoral injection of 20 μg anti-CD40. The study was repeated while also varying different treatment parameters including radiotherapy dose (6 Gy, 10 Gy, and 15 Gy) and dosing schedule (one versus three times administration over 2 weeks) of immunoadjuvant anti-CD40. For each study, the size of both treated and untreated tumors was measured with a caliper and SARRP computed tomography imaging and responses compared to those in appropriate control cohorts. Statistical analyses for tumor volume and Kaplan–Meier survival assay were achieved using ANOVA and Log-rank test. The results consistently showed that one 6 Gy compared to 10 Gy or 15 Gy fraction doses of RT with a single dose of agonistic anti-CD40 can generate highly effective abscopal responses after 14 days, with a significant increase in animal survival (p = 0.0004). 60% of the mice treated with this combination showed long term survival with complete tumor regression, where tumors of mice in other cohorts continued to grow (see Table below). Moreover, re-challenged responders to the treatment developed vitiligo suggesting developed immune memory for this cancer. The findings corroborate the hypothesis and show that a single RT fraction dose optimized at 6 Gy in combination with locally delivered anti-CD40 can generate effective abscopal responses. Further investigations to optimize and advance mechanistic understanding of the abscopal effect associated with cervical cancer could lead to innovative treatment methods offering a new more accessible therapy option for cervical cancer care, with a major potential impact, especially in resource-poor settings.Tabled 1Abstract 3022; TableGroupsShrinkage Rate: Treated Tumors [responders/mice in group]Shrinkage Rate: Untreated Tumors: [responders/mice in groups]Complete Tumors Regression [%]Control0/150/1506 Gy2/100/100mAb (1 dose)1/102/100mAb (3 doses)1/51/506 Gy + mAb10/159/156010 Gy + mAb2/51/5015 Gy + mAb4/51/506 Gy + mAb (3 doses)3/53/50 Open table in a new tab

CD40 Agonist Restores the Antitumor Efficacy of Anti-PD1 Therapy in Muscle-Invasive Bladder Cancer in an IFN I/II-Mediated Manner.

Bladder cancer is one of the most common malignancies and has poor prognosis for patients with locally advanced, muscle-invasive, disease despite the efficacy of immune checkpoint blockade. To develop more effective immunotherapy strategies, we studied a genetic mouse model carrying deletion of Tp53 and Pten in the bladder, which recapitulates bladder cancer tumorigenesis and gene expression patterns found in patients. We discovered that tumor cells became more malignant and the tumor immune microenvironment evolved from an inflammatory to an immunosuppressive state. Accordingly, treatment with anti-PD1 was ineffective, but resistance to anti-PD1 therapy was overcome by combination with a CD40 agonist (anti-CD40), leading to strong antitumor immune responses. Mechanistically, this combination led to CD8+ T-cell recruitment from draining lymph nodes. CD8+ T cells induced an IFNγ-dependent repolarization toward M1-like/IFNβ-producing macrophages. CD8+ T cells, macrophages, IFN I, and IFN II were all necessary for tumor control, as demonstrated in vivo by the administration of blocking antibodies. Our results identify essential cross-talk between innate and adaptive immunity to control tumor development in a model representative of anti-PD1-resistant human bladder cancer and provide scientific rationale to target CD40 in combination with blocking antibodies, such as anti-PD1/PD-L1, for muscle-invasive bladder cancer.

Abstract NG12: Mechanisms governing efficacy of combination CD40 agonist and anti-PD-L1 in pancreatic ductal adenocarcinoma

Abstract NG12: Mechanisms governing efficacy of combination CD40 agonist and anti-PD-L1 in pancreatic ductal adenocarcinoma

An Agonistic Anti-CD137 Antibody Disrupts Lymphoid Follicle Structure and T-Cell-Dependent Antibody Responses

SummaryCD137 is a costimulatory receptor expressed on natural killer cells, T cells, and subsets of dendritic cells. An agonistic monoclonal antibody (mAb) against CD137 has been used to reduce tumor burden or reverse autoimmunity in animal models and clinical trials. Here, we show that mice treated with an agonistic anti-CD137 mAb have reduced numbers of germinal center (GC) B cells and follicular dendritic cells (FDCs) in lymphoid tissues, which impair antibody responses to multiple T-cell-dependent antigens, including infectious virus, viral proteins, and conjugated haptens. These effects are not due to enhanced apoptosis or impaired proliferation of B cells but instead correlate with changes in lymphoid follicle structure and GC B cell dispersal and are mediated by CD137 signaling in CD4+ and CD8+ T cells. Our experiments in mice suggest that agonistic anti-CD137 mAbs used in cancer and autoimmunity therapy may impair long-term antibody and B cell memory responses.