Agonistic anti-CD40 Antibody Research Articles

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 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 2786: A FcgR IIB tumor enriching anti-CD137 novel antibody to avoid liver Immuno-toxicity

Abstract CD137 (4-1BB/TNFRSF9) is a costimulatory T-cell receptor belonging to the TNF receptor superfamily which is transiently expressed on T cell and NK cells. Targeting CD137 or its natural ligand 4-1BB ligand (4-1BBL) has important implications in many clinical conditions, including cancer. Anti-CD137 agonistic anti-CD137 antibodies have shown potent, often curative anti-tumor activity in preclinical models. In depth analysis revealed that 4-1BB-mediated anti-cancer effects are based on its ability to induce activation of cytotoxic T lymphocytes and high amounts of cytokines, including IFN-γ. However, this approach has suffered significant setback in the clinic due to either dose-dependent severe liver toxicity (e.g. Urelumab), or lack of single agent activity in the case of Utomilumab. Multiple therapeutics that attempt to restrict 4-1BB agonism to the tumor microenvironment and minimize systemic exposure have emerged. Here we present a novel antibody targeting 4-1BB receptor, named HLX25 with engineered Fc region to enhance the binding to FcγR IIB. In vitro, HLX25 induces comparable or superior CD137 activation and IL-2 release compared to Urelumab and Utomilumab, suggesting potentially a better safety profile. We showed that HLX25 binds to unique non-ligand competing epitope, which is different from both Urelumab and Utomilumab. In vivo, HLX25 shows potent dose-dependent single agent anti-tumor activity as low as 0.3mg/kg in MC38 model without sign of liver toxicity up to 20 mg/kg, suggesting superior efficacy and better safety profile. Citation Format: Jie Xue, Jen-Kuan Chang, Chen Dong, Yi-Ting Mao, Pallavi Raghavendra, Ou Li, Hassan Issafras, Wenfeng Xu, Weidong Jiang. A FcgR IIB tumor enriching anti-CD137 novel antibody to avoid liver Immuno-toxicity [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 2786.

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.

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.

Mechanisms of GVL-Resistance: T Cell Exhaustion and a Failure of Antigen Cross-Presentation

In MHC-matched allogeneic hematopoietic stem cell transplantation (alloSCT), minor histocompatibility antigens (miHAs) are the major targets of alloreactive CD8+ T cells that mediate the GVL effect. Despite the development of alloreactive CD8 cells in most if not all patients, relapsed leukemia is the greatest single cause of treatment failure. Why relapse occurs has been elusive. Previously we tested adoptive immunotherapy with bona fide CD8+ memory T cells (TM) from donors vaccinated against a single miHA expressed by leukemia cells. Vaccination against the Kb-restricted miHA H60 (LTFNYRNL) greatly augmented TM-mediated GVL against mouse chronic phase and blast crisis chronic myeloid leukemia (BC-CML). In TMH60 recipients, despite a rapid expansion of H60-reactive T cells (measured by tetramer staining and IFN-g production) and an impressive GVL effect, we consistently observed persistent leukemia and later death in a subset of mice. We used this system to test hypotheses on GVL failure as we could track and enumerate H60-reactive CD8 cells, the only cells that mediate GVL in this system, by tetramer staining, which is not possible in traditional polyclonal GVL models. We hypothesized that GVL resistance could be due to immune selection of GVL-resistant leukemia cells and/or an insufficient T cell response. However, we found no evidence of immune selection. H60+ leukemia cells harvested from mice with relapsed BC-CML after an initially effective TMH60 GVL response and H60+ leukemia cells from transplanted mice that did not receive donor T cells were similarly sensitive to in vivo killing by both H60-reactive T cells and by OT-1 effector T cells when leukemia cells were pulsed with SIINFEKL. An insufficient T cell response could have been due to insufficient presentation of H60, as H60 is largely restricted to host hematopoietic cells, which are eliminated by the transplant. Indeed, H60 presentation was inefficient as fresh congenically marked TMH60, transferred 14 days post initial transplantation with TMH60, proliferated poorly in mice with or without leukemia. However, infusion of an agonist anti-CD40 antibody, also on day 14, promoted the proliferation and GVL activity of fresh TMH60 but not by the TeffH60+ (H60-reactive CD8 effectors) derived from TMH60 infused on day 0. The activity of anti-CD40 was mostly abrogated when donor BM was CD40-/-. Hence, cross-presentation by donor-derived cells is limiting, but T cells that initially mediated GVL were also poorly responsive. Relative to progeny from TMH60 infused on day 14, these cells had higher levels of PD-1, TIM-3, and TIGIT and a lower level of IFN-g production; this was even more marked in mice with residual leukemia. The increased exhaustion in mice with residual leukemia required the leukemia cells to present H60, as it did not occur when leukemias were H60+ but Kb-/-. Using the H60+Kb-/- BC-CML we found little evidence for cross-presentation of leukemia-derived H60 indicating that T cell responses against leukemia-restricted antigens is inefficient. Consistent with this, the anti-CD40 effect promoted cross-presentation of recipient and not leukemia-derived H60. In vivo treatment with anti-PD-1, but not anti-TIM-3 mAb, greatly augmented TMH60 mediated GVL without causing clinical GVHD as the TMH60 only target host hematopoietic cells. Day 0 treatment with anti-CD40 also augmented TMH60 mediated GVL without an increase in GVHD; but in this case the activity was on host cells. Taken together, our data support both exhaustion and a lack of antigen presentation later post BMT as factors that contribute to an insufficient T cell response, thus leading to GVL resistance. DisclosuresNo relevant conflicts of interest to declare.

Phase I open-label, dose escalation of YH003, an anti-CD40 monoclonal antibody in combination with toripalimab (anti-PD-1 mAb) in patients with advanced solid tumors.

2580 Background: YH003, a recombinant, humanized agonistic anti-CD40 IgG2 monoclonal antibody (mAb) specifically recognizes and agonizes CD40 on the antigen-presenting cells to enhance immune responses. Preclinical data have shown potent anti-cancer activity when combined with anti-PD-1 antibodies. Methods: This is an ongoing phase 1 dose-escalation study. Patients with advanced solid tumors receive YH003 by IV administration Q3W as monotherapy at 0.03 to 3.0 mg/kg for the first cycle (21 days) then in combination with Toripalimab at 240 mg Q3W for the 4 subsequent cycles in an accelerated “3+3” design. The safety, tolerability and preliminary efficacy data will be analyzed. Results: As of 31 Dec 2020 data cutoff, 9 patients (pts) were enrolled and treated at 0.03 mg/kg (n = 3), 0.1mg/kg (n = 3), and 0.3mg/kg (n = 3). The median age was 63 years (range 33-68). Baseline ECOG scores were 0 (7 pts) and 1 (2 pts) with a median of 2 prior lines therapy (range 1-7). 5 pts had received prior immunotherapy (PD-1/PD-L1 or PD-1+CTLA-4). As of data cutoff, no dose limiting toxicities (DLT) were observed. No Serious Adverse Event (SAE) or AEs leading to treatment discontinuation were reported. Four drug related AEs were reported including one Grade 1 (G1) choroidal thickening (related to YH003) at 0.03 mg/kg, one G1 fatigue (related to YH003) at 0.1 mg/kg, two G1 febrile episodes (one related to YH003 and the other related to combination treatment) at 0.3 mg/kg. Among 5 patients assessable for response, there were 2 SD (one with anti-PDL1 refractory Merkel cell carcinoma at 0.03 mg/kg and one with anti-PD1 refractory NSCLC at 0.1 mg/kg) and 1 PR with anti-PD1/anti-CTLA4 refractory ocular melanoma at 0.1 mg/kg. Conclusions: YH003 was well tolerated up to 0.3 mg/kg dose levels when combined with Toripalimab and has shown encouraging antitumor activity in patients with advanced solid tumors. Clinical trial information: NCT04481009.

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.

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.

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.

CD40 Agonist Combined with Radiation and PD-1 Blockade Enhances Development Of Systemic Tumor-Specific B-Cells And B-Cell Memory

Checkpoint blockade immunotherapy (CBI) is approved for head and neck squamous cell carcinoma; however, the majority of patients do not have objective responses and combinatorial therapies are needed to improve outcomes. Radiation therapy (RT) can enhance presentation of tumor antigens, and multiple studies have investigated the immunological effects of combined RT and CBI, particularly focusing on T-cells and myeloid populations. B-cells, however, have been relatively understudied, but their contributions in mounting a highly antigen-specific adaptive immune response are being further elucidated. Wild type C3H mice were injected subcutaneously with 5x105 cells of AT-84-E7-OVA into the right flank. Mice were then treated intravenously with 2 doses of 100 μg agonistic anti-CD40 antibody, 3 doses of 200 μg anti-PD-L1 antibody, and 12 Gy of focused RT. Tumors and spleens were dissected and analyzed by flow cytometry. An OVA-antigen biotinylated multimer was generated inhouse for tracking of tumor specific B-cells. Data were collected using a BD LSRII and analyzed using FlowJo. Mice treated with the combination of RT, PD-1 blockade, and CD40 activation had the highest frequency of tumor specific B-cells recognizing OVA, (from 3.3 +/-0.8 events per 50K live cells in untreated mice to 8.5 +/- 2.4; P = 0.01). We also observed a significant increase in plasma cells (from 11.9 +/- 8.0 events per 50K live cells in untreated mice to 235.5 +/- 131.2; P < 0.001). Additionally, mice treated with triple combination therapy had the highest proportion of plasma cells in the spleen (from 0.42 +/- 0.12% in untreated mice to 0.76 +/- 0.38% of all B220+ cells; P = 0.01), indicating augmented systemic B-cell immune responses induced by local radiation. Furthermore, mice treated with triple combination therapy demonstrated increased differentiation into PD-L2+ memory B cells (from 0.61 +/- 0.37% to 3.2 +/- 1.2% of all B220+ cells; P < 0.0001). Addition of CD40 agonist to RT and CBI significantly increases the frequency of systemic antigen-specific B-cells and B-cell memory. Continued investigation into the roles of distinct B-cell subpopulations and their responses to novel therapeutic combinations are ongoing and will help detail mechanisms combinatory activity.

Targeting immune-checkpoint inhibitor resistance mechanisms by MEK inhibitor and agonist anti-CD40 antibody combination therapy.

The widespread application of immune-checkpoint blockade (ICB) has resulted in unprecedented response rates in patients with immunogenic cancers, such as melanoma and lung cancer. However, sub-groups of patients with these indications do not respond to ICB, and the same applies to patients with other cancer types. Mechanisms of resistance to ICB include low tumor immunogenicity associated with low T cell infiltration (‘cold' tumors), suppression of anti-tumor immunity by immunosuppressive cells in the tumor microenvironment (TME), lack of antigen-presentation and immune escape (e.g. by downregulation of MHC-I on tumor cells) as well as oncologic pathways that suppress immune responses. Combination strategies, involving cytostatic drugs, harbor the potential to overcome refractoriness to immunotherapy. However, suppression of immune cell function by cytostatic drugs may limit the efficacy. In our study, we show that combination treatment of targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) and agonist immunostimulatory anti-CD40 antibody (Ab) is particularly suitable in counteracting aforementioned ICB resistance mechanisms (Fig. 1).

Characteristics and clinical trial results of agonistic anti-CD40 antibodies in the treatment of malignancies.

Cluster of differentiation 40 (CD40) mediates many immune activities. Preclinical studies have shown that activation of CD40 can evoke massive antineoplastic effects in several tumour models in vivo, providing a rationale for using CD40 agonists in cancer immunotherapy. To date, several potential agonistic antibodies that target CD40 have been investigated in clinical trials. Early clinical trials have shown that the adverse events associated with agonists of CD40 thus far have been largely transient and clinically controllable, including storms of cytokine release, hepatotoxicity and thromboembolic events. An antitumour effect of targeting CD40 for monotherapy or combination therapy has been observed in some tumours. However, these antitumour effects have been moderate. The present review aimed to provide updated details of the clinical results of these agonists, and offer information to further investigate the strategies of combining CD40 activation with chemotherapy, radiotherapy, targeted therapy and immunomodulators. Furthermore, biomarkers should be identified for monitoring and predicting responses and informing resistance mechanisms.

Abstract 1073: A tumor-targeted CD40 agonistic DARPin® molecule leading to antitumor activity with limited systemic toxicity

Abstract CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily which can activate both innate and adaptive tumor immunity, making it an attractive target for cancer immunotherapy. Systemic administration of agonistic anti-CD40 antibodies has shown signs of activity in cancer patients, but dose-limiting toxicity has prevented the exploration of the full dose range and possibly has impaired clinical efficacy. To overcome this issue, we developed a multispecific DARPin® therapeutic candidate, MP0317, intended to activate CD40 receptor locally, rather than systemically, in the tumor microenvironment through cross-linking with fibroblast activation protein (FAP) that is abundantly present in the stroma of many solid tumors, but has limited expression in healthy adult tissues. Using primary human 1) B cells and 2) dendritic cells, MP0317 activated the CD40 pathway in vitro. Upregulation of CD80, CD86, CD69 and IL12 occurred exclusively in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x mCD40) was generated and tested in murine cell-based in vitro assays showing comparable results as MP0317 with a strict FAP-dependent activation of CD40. The mFAP x mCD40 was also active in vivo and inhibited substantially the progression of FAP-positive tumors. Moreover, in contrast to an anti-mouse CD40 antibody (clone FGK45), the antitumor activity of mFAP x mCD40 was neither associated with elevated blood cytokine levels nor with hepatotoxicity, both of which manifest as dose limiting toxicities of some of the clinical CD40 activating antibodies. These data support a mode of action, both in vitro and in vivo, that is dependent on FAP-mediated crosslinking of CD40 receptor resulting in a tumor-localized CD40 activation without peripheral or off-tumor organ toxicity. In conclusion, we have designed a tumor-targeted CD40 agonistic DARPin® therapeutic candidate that is able to activate the CD40 receptor locally in FAP-positive tumors producing impressive antitumor activity in the absence of systemic toxicity. Citation Format: Nicolo Rigamonti, Sarah Jetzer, Niels Van der Velden, Omar Abdelmotaleb, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Ralph Bessey, Andrea Valeri, Heidi Poulet, Christof Zitt, Pamela A. Trail, Victor Levitsky, Niina Veitonmäki, Clara Metz. A tumor-targeted CD40 agonistic DARPin® molecule leading to antitumor activity with limited systemic toxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1073.

Monocyte-derived APCs are central to the response of PD1 checkpoint blockade and provide a therapeutic target for combination therapy

BackgroundPD1 immune checkpoint blockade (αPD1 ICB) has shown unparalleled success in treating many types of cancer. However, response to treatment does not always lead to tumor rejection. While αPD1 ICB...

Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice

AbstractHemophagocytic syndromes comprise a cluster of hyperinflammatory disorders, including hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Overwhelming macrophage activation has long been considered a final common pathway in the pathophysiology of hemophagocytic syndromes leading to the characteristic cytokine storm, laboratory abnormalities, and organ injuries that define the clinical spectrum of the disease. So far, it is unknown whether primary macrophage activation alone can induce the disease phenotype. In this study, we established a novel mouse model of a hemophagocytic syndrome by treating mice with an agonistic anti-CD40 antibody (Ab). The response in wild-type mice is characterized by a cytokine storm, associated with hyperferritinemia, high soluble CD25, erythrophagocytosis, secondary endothelial activation with multiple organ vaso-occlusion, necrotizing hepatitis, and variable cytopenias. The disease is dependent on a tumor necrosis factor-α–interferon-γ–driven amplification loop. After macrophage depletion with clodronate liposomes or in mice with a macrophage-selective deletion of the CD40 gene (CD40flox/flox/LysMCre), the disease was abolished. These data provide a new preclinical model of a hemophagocytic syndrome and reinforce the key pathophysiological role of macrophages.

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.

FLT3 ligand (CDX-301) and stereotactic radiotherapy for advanced non-small cell lung cancer.

9618 Background: In a murine non-small cell lung cancer (NSCLC) model, we demonstrated synergy between localized radiotherapy and the dendritic cell growth factor fms-like tyrosine kinase 3 (FLT3) ligand. We now present results from a phase II study testing this combination in patients with advanced and treatment-refractory NSCLC. Methods: Advanced NSCLC patients with multifocal active disease after at least one line of systemic therapy and ECOG performance status 0-2 received 5 daily subcutaneous injections of CDX-301 (75 µg/kg) concurrent with stereotactic body radiotherapy (SBRT, 30-54 Gy in 1-5 fractions based on target size and location) directed at a single site of disease. Additional “cycles” of SBRT and CDX-301 could be administered at least four months after the initial study treatment, at the discretion of the treating physicians. The primary endpoint was progression-free survival four months after treatment initiation (PFS4), with a hypothesis that the PFS4 rate would exceed 40%. Secondary endpoints included overall survival (OS) duration, responses on PET (PERCIST criteria) and CT (RECIST criteria), and dose-limiting toxicities (grade ≥3 adverse events within 30 days). Lesions targeted with SBRT were excluded from response assessments. The intended sample size was 29 subjects. Blood samples were obtained for flow cytometry and other analyses of immune activation. Results: Twenty-nine subjects received study therapy between October 2016 and January 2020. Subjects received a median of 3 lines (range: 1-5) of systemic therapy prior to study enrollment, including immune checkpoint inhibitors targeting the PD-1/PD-L1 axis in 26 subjects (90%). At the time of this analysis, the actuarial PFS4 rate is 60%, which exceeds our pre-specified efficacy objective. With a median follow-up duration for living patients of 12 months, the actuarial 12-month OS rate is 55%. Partial response of lesions not targeted with SBRT (“abscopal effect”) was observed in 9 subjects (31%) using PET criteria and in 4 subjects (14%) using CT criteria. Seven subjects (24%) received a second course of SBRT and CDX-301 after initial study therapy. No dose-limiting toxicities have been observed. Only six subjects (21%) have received additional chemotherapy or immunotherapy after study treatment. Conclusions: The combination of CDX-301 and SBRT is well-tolerated and has activity as systemic therapy for advanced NSCLC. Additional studies to maximize the efficacy of this in situ vaccination approach with the addition of an agonist anti-CD40 antibody (CDX-1140) are planned. Clinical trial information: NCT02839265 .

CD40 signaling induces type I interferon and immune control in mouse pancreatic cancer lacking the CXCL12-coat

Abstract Pancreatic ductal adenocarcinoma (PDA) has a low 5 year survival rate of 8.5% because of its late diagnosis and resistance to the existing therapies. Patients with PDA who are microsatellite stable do not respond to check point blockade immunotherapy due to impaired T cell infiltration, which is mediated by the CXCL12-coat on cancer cells (Wang Z, et al. bioRχiv). In mouse hepatic metastases established with PDA cells from the KPC model of PDA, Krt19-CRISPR/Cas9-edited tumors lacking the CXCL12-coat showed a type I interferon (IFN) response as well as T cell infiltration and activation compared to the control scramble tumors. By RNA FISH, we found that IFNb1 is dominantly produced by CD40+ cells, and not by SiglecH+ plasmacytoid dendritic cells. RNA sequencing data showed up-regulation of Cd40lg in the Krt19-edited tumors compared to the scramble tumors. Administration of antagonistic anti-CD40 antibody prior cancer cells inoculation abolished IFNb1 production, T cell accumulation and activation. To confirm the role of CD40, administration of agonistic anti-CD40 antibody to mice bearing wild type PDA tumors elicited intra-tumoral IFNb1 production in CD11b+ CD11c+ myeloid cells. The T cell infiltration and activation induced by agonistic anti-CD40 antibody treatment sensitized both subcutaneous pancreatic tumors and hepatic metastases to anti-PD1 treatment. These findings indicate that the expression of CD40L elicits an intra-tumoral type I IFN response that is required for immune control of PDA tumors in which CXCR4 signaling is impaired.