Targeting AXL receptor kinase with a highly selective antibody presents a promising approach for inhibiting AXL and potentially improving cancer treatment. An essential step in antibody optimisation is the mapping of paratope residues to epitope residues. In the present study, we identify the residues of tilvestamab, a function-blocking anti-AXL monoclonal antibody, that are essential for its binding to the extracellular domain of AXL. A single-chain variable fragment (scFv) fused to osmotically inducible protein Y (osmY) was designed to enable the secretion of soluble scFv-osmY mutants, which could be directly subjected to high-throughput biolayer interferometry screening for binding to the AXL Ig1 domain. Each complementarity-determining region residue of scFv was mutated to Ala, while additional mutations were made on the basis of predicted contribution to binding. We generated AlphaFold3 predictions for the scFv (tilvestamab)-AXL Ig1 complex to gain insights into the molecular interactions of the essential residues, as determined by the experimental data. Our study reveals that tilvestamab binds to the Ig1 domain of AXL, with twelve residues on scFv (tilvestamab) contributing most to binding, likely being situated at the binding interface. Glu2 near the N-terminus of AXL is essential for binding. The data give a structural view into the AXL-tilvestamab complex and allow for further optimisation of the binding interface.

Tilvestamab is a highly selective humanised immunoglobulin G1 anti-AXL monoclonal antibody. This phase 1 study evaluated its optimal dose, safety, tolerability, immunogenicity and pharmacokinetics (PK) in relapsed platinum-resistant HGSOC patients. Patients received tilvestamab in three dose levels (1 mg/kg, 3 mg/kg and 5 mg/kg) via IV infusion every 2 weeks. Primary objectives included safety, tolerability and PK. Exploratory objectives included overall response, progression-free survival (PFS) and quality-of-life measures. Pharmacodynamic included AXL expression, gene and protein changes by transcriptomic and proteomic analysis. Between 25 February 2021 and 4 February 2022, 16 patients were enroled across 8 sites in Singapore, Korea, United Kingdom, and Norway. Median treatment duration was 6.1 weeks. Grade 3 or higher treatment-emergent adverse events occurred in 62.5% patients, but none were tilvestamab-related. Common events included fatigue (38%), anorexia (38%) infections (31%), anaemia (25%) and dyspnoea (25%). No objective responses were observed, but 7 (44%) had stable disease at 6 weeks. PK showed dose-proportional exposure and steady-state by the second dose. Pharmacodynamic analyses revealed reduced fibrosis-related gene signatures and AXL protein expression. Epithelial-mesenchymal transition reversal was seen in 2 patients. Tilvestamab was well-tolerated and further studies to examine the efficacy of AXL inhibition in other indications are required. This trial is registered at https://clinicaltrials.gov . Registration number: NCT04893551. EudraCT Number: 2020-001382-36

Beyond first line, the prognosis of relapsed/refractory (R/R) acute myeloid leukemia (AML) patients is poor with limited treatment options. Bemcentinib is an orally bioavailable, potent, highly selective inhibitor of AXL, a receptor tyrosine kinase associated with poor prognosis, chemotherapy resistance and decreased antitumor immune response. We report bemcentinib monotherapy and bemcentinib+low-dose cytarabine combination therapy arms from the completed BerGenBio-funded open-label Phase 1/2b trial NCT02488408 ( www.clinicaltrials.gov ), in patients unsuitable for intensive chemotherapy. The primary objective in the monotherapy arm was identification of maximum tolerated dose with secondary objectives to identify dose-limiting toxicities, safety and efficacy, and bemcentinib pharmacokinetic profile. In the combination arm, the primary objective was safety and tolerability, with efficacy and pharmacokinetics as secondary objectives. Safety and tolerability were based on standard clinical laboratory safety tests and Common Terminology Criteria for Adverse Events version 4. Bemcentinib monotherapy (32 R/R, 2 treatment-naïve AML and 2 myelodysplasia patients) was well-tolerated and a loading/maintenance dose of 400/200 mg was selected for combination treatment, comprising 30 R/R and 6 treatment-naïve AML patients. The most common grade 3/4 treatment-related adverse events were cytopenia, febrile neutropenia and asymptomatic QTcF prolongation, with no grade 5 events reported. In conclusion, bemcentinib+low-dose cytarabine was safe and well tolerated.

AXL expression in metastatic lung adenocarcinoma (LAC) promotes cancer cell survival, metastasis and engenders immune-suppressive tumor microenvironments. Immune cells (CD4+/CD8+ T cells, NK and NKT cells) become dysfunctional with reduced expression of granzyme B, resulting in impaired cytotoxicity. Bemcentinib (BEM), a selective oral AXL inhibitor, may enhance pembrolizumab (PEM) to restore cytotoxic T cell function in mouse models of LAC. We hypothesize that AXL-PD1 targeting may overcome immune exhaustion in LAC patients who previously failed chemoimmunotherapy. Peripheral blood monocytes (PBMC; n = 4,439,600 live cells) from 29 LAC patients enrolled in BGBC008 phase 2 open-label, single-arm study of BEM+PEM (NCT03184571) were collected at different treatment time points, analyzed by CyTOF using the Phenograph algorithm and visualized by tSNE plots. Immune cell type proportions and immune cell activation were calculated before and after treatment. Separately, we tested whether adding targeting of the JAK2/STAT3 pathway (an AXL bypass mechanism) with pacritinib, a selective JAK2 inhibitor with no JAK1 activity, can further inhibit tumor growth compared with targeting AXL-PD1 alone, in a C57BL/6 syngeneic mouse D19.1 LAC model. BEM + PEM led to immune cell responses following cycle 1 treatment in LAC patients previously treated with chemo-immunotherapy. Specifically, we observed a reduction in monocytes/macrophages (p=0.0134), CD123+ plasmacytoid dendritic cells (DC; p=0.0009), cDC1 (p=0.0009), and myeloid dendritic cell populations (p = 0.0073). Combination treatment led to a reduction in M2-polarized macrophages (reduced CD206 & CD163 levels), re-activation of cDC1 (high CD40 expression) and reduction in CD8+ T cell exhaustion (high granzyme B levels). Fold-change of granzyme B in CD8+ NKT cell populations positively correlated with progression free survival (PFS) (n = 29, Cor = 0.295). Additionally, high phospho-STAT3 expression in macrophages may correlate with poor PFS, supporting our previous research suggesting that pSTAT3 is a bypass mechanism of AXL signaling. In a syngeneic LAC mouse model, triple combination therapy targeting the JAK2/STAT3 pathway in addition to AXL and PD-1 resulted in 55% and 90% tumor size reduction and two-fold increase in CD8+ T cell infiltration when compared with AXL/PD-1 dual combination treatment and untreated control groups, respectively. CyTOF data suggest that AXL-PD1 drug targeting may overcome immune cell exhaustion and re-activate CD8+ T cell, cDC1 and NKT cells in treatment refractory LAC patients treated with BEM+PEM. Granzyme B expression in PBMCs may serve as a biomarker of treatment response. Furthermore, in vivo drug targeting of AXL-PD1-STAT3 axis demonstrates significant synergy in a syngeneic LAC mouse model, showing a strong potential for future clinical trials. Chia-Nung Hung, Daniel T. DeArmond, Jason M. Gauthier, Michael J. McGinity, Catherine Limboy, Meizhen Chen, Yajaira Esmeralda Mendoza-Plata, Jordy Urbina, Jacobo Louis Garza, Joel E. Michalek, David Micklem, Akil Jackson, Michael Chisamore, Tim H.M. Huang, Josephine A. Taverna. Bemcentinib and pembrolizumab may overcome exhaustive immune phenotypes in patients with treatment refractory lung adenocarcinoma and reveals a potential STAT3 bypass mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB401.

AXL is a receptor tyrosine kinase with a significant role in various biological processes and important medical implications, particularly in cancer. AXL transduces signals from the extracellular environment into the cytoplasm by binding to its ligand, growth arrest-specific protein 6 (GAS6). Activation of AXL leads to autophosphorylation of its intracellular domain and subsequent activation of downstream signaling pathways involved in cell proliferation, migration, differentiation, and survival. Tilvestamab (also known as BGB149) is a first-in-class, humanized, therapeutic anti-AXL function-blocking monoclonal antibody. We carried out a structural characterization of the AXL-tilvestamab complex using both negative-stain and cryogenic transmission electron microscopy as well as synchrotron small-angle X-ray scattering. While AXL-Fc was highly elongated and formed large heterogeneous complexes with the full antibody, homogeneous samples for structural studies could be made using the monomeric soluble AXL extracellular domain, the Fab fragment of tilvestamab, and an anti-Fab nanobody. Both SAXS and cryo-EM confirmed successful complex formation between the three proteins, and a low-resolution 3D model for the tilvestamab-AXL complex is presented. The data allow for sample optimization for high-resolution structural biology, as well as designing mutations that could alter binding affinity and specificity.

The receptor tyrosine kinase AXL promotes tumor progression, metastasis, and therapy resistance through the induction of epithelial-mesenchymal transition (EMT). Here, we found that activation of AXL resulted in the phosphorylation of TANK-binding kinase 1 (TBK1) and the downstream activation of AKT3 and Snail, a transcription factor critical for EMT. Mechanistically, we showed that TBK1 directly bound to and phosphorylated AKT3 in a manner dependent on the multiprotein complex mTORC1. Upon activation, AKT3 interacted with and promoted the nuclear accumulation of Snail, which led to increased EMT as assessed by marker abundance. In human pancreatic ductal adenocarcinoma tissue, nuclear AKT3 colocalized with Snail and correlated with worse clinical outcomes. Primary mouse pancreatic cancer cells deficient in AKT3 showed reduced metastatic spread in vivo, suggesting selective AKT3 inhibition as a potential therapeutic avenue for targeting EMT in aggressive cancers.

Soluble biomarkers are paramount to personalized medicine. However, the in vivo turnover and biodistribution of soluble proteins is seldom characterized. The cleaved extracellular domain of the AXL receptor (sAXL) is a prognostic biomarker in several diseases and a predictive marker of AXL targeting agents. Plasma sAXL reflects a balance between production in tissues with lymphatic transport into the circulation and removal from blood by degradation or excretion. It is unclear how this transport cycle affects plasma sAXL levels that are the metric for biomarker development. Radiolabeled mouse sAxl was monitored after intravenous injection to measure degradation and urinary excretion of sAxl, and after intradermal injection to mimic tissue or tumor production. sAxl was rapidly taken-up and degraded by the liver and kidney cortex. Surprisingly, intact sAxl was detectable in urine, indicating passage through the glomerular filter and a unique sampling opportunity. The structure of sAxl showed an elongated, flexible molecule with a length of 18 nm and a thickness of only 3 nm, allowing passage through the glomerulus and excretion into the urine. Intradermally injected sAxl passed through local and distant lymph nodes, followed by uptake in liver and kidney cortex. Low levels of sAxl were seen in the plasma, consistent with an extended transit time from local tissue to circulation. The rapid plasma clearance of sAxl suggests that steady-state levels in blood will sensitively and dynamically reflect the rate of production of sAxl in the tissues but will be influenced by perturbations of liver and kidney function.

IntroductionAXL receptor expression is proposed to confer immune-checkpoint inhibitor (ICI)-resistance in non-small cell lung cancer (NSCLC) patients. We sought to interrogate AXL expression in conjunction with mutational and tumor-microenvironmental features to uncover predictive mechanisms of resistance in ICI-treated NSCLC patients.MethodsTumor samples from 111 NSCLC patients treated with ICI-monotherapy were analyzed by immunohistochemistry for tumor- and immune-AXL expression. Subsets of patients were analyzed by whole-exome sequencing (n = 44) and imaging mass cytometry (n = 14). Results were related to ICI-outcome measurements.ResultsTumor-cell AXL expression correlated with aggressive phenotypic features including reduced OS in patients treated with ICIs (P = 0.04) after chemotherapy progression, but conversely associated with improved disease control (P = 0.045) in ICI-treated, PD-L1 high first-line patients. AXL+ immune-cell infiltration correlated with total immune-cell infiltration and improved overall outcomes (PFS: P = 0.044, OS: P = 0.054). Tumor-cell AXL-upregulation showed enrichment in mutations associated with PD-L1-upregulation and ICI-response such as MUC4 and ZNF469, as well as adverse mutations including CSMD1 and LRP1B which associated with an immune-suppressed tumor phenotype and poor ICI prognosis particularly within chemotherapy-treated patients. Tumor mutational burden had no effect on ICI-outcomes and was associated with a lack of tumor-infiltrating immune cells. Spatial-immunophenotyping provided evidence that tumor-cell AXL-upregulation and adverse mutations modulate the tumor microenvironment in favor of infiltrating, activated neutrophils over anti-tumor immune-subsets including CD4 and CD8 T-cells.ConclusionTumor-cell AXL-upregulation correlated with distinct oncotypes and microenvironmental immune-profiles that define chemotherapy-induced mechanisms of ICI-resistance, which suggests the combination of AXL inhibitors with current chemoimmunotherapy regimens can benefit NSCLC patients.

Treatment options in 2L metastatic NS NSCLC remain limited with modest outcomes to date. AXL, a receptor tyrosine kinase, mediates resistance to immune- and chemotherapy (CT) and is a poor prognostic biomarker in NSCLC. Bemcentinib (bem), a highly selective oral AXL inhibitor, potentiates the immune-checkpoint inhibitor (ICI) effect in pre-clinical models, providing a strong scientific rationale for the bem+pembro combination in this patient population (PP). BGBC008 is a ph2 open-label, single-arm study of bem+pembro in 2L NS NSCLC: ICI-naïve patients (pts) (cohort A), CT-naïve pts (cohort B) and pts with prior CT+ICI (cohort C). Primary endpoint was objective response rate (ORR); secondary endpoints included safety, progression-free survival (PFS) and overall survival (OS). Exploratory analyses assessed PD-L1 (by TPS) and AXL (by H-score) status impact on outcomes. The study enrolled 99 safety-evaluable pts; median age 65 years (range 39-86); 90 pts were efficacy-evaluable (EE). Most common adverse events were gastrointestinal disorders (G ≤2) in >20% of pts and transient liver enzyme increase (G ≤3) in 10% pts. Median (m) OS and mPFS were 13 and 6.2 months (mths), respectively. AXL status impacted mOS: 14.8 vs 9.9 mths (p=0.029) for AXL >5 vs AXL ≤5, respectively. No difference in mOS (10.6 vs 12.4 mths) or mPFS (6 vs 7 mths) in PD-L1+ (TPS≥1) vs PD-L1- (TPS<1) pts, respectively. The ORR of 11.1% in all EE pts and 21.9% in pts with AXL >5 favourably aligns with therapies in 2L NSCLC. Median OS and mPFS in KRAS mutated (KRASMT) (n=20) and KRAS wild-type (KRASWT)(n=36) were 14.1 vs 10.0 mths (p=0.49) and 9.8 vs 3.8 mths (p=0.009), respectively. The mOS and mPFS for the PD-L1- (n=6) and PD-L1+ (n=11) KRASMT pts were 18 vs 11.4 mths (p=0.017) and 17.3 vs 6.1 mths (p=0.09), respectively. Bem+pembro was well tolerated and efficacious compared to historical controls. Survival benefit was observed in pts with AXL >5, regardless of prior therapy or PD-L1 status. The promising efficacy signal observed in KRASMT pts warrants further validation.

PDF file - 99K, Materials and methods of CytoCy5S synthesis, purification, spectral and structural characterization in addition to NTR/CytoCy5S enzymatic reaction conditions and western blots.

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

PDF file - 402K, Synthesis, mechanism and spectral characteristics of CytoCy5S.

Acute myeloid leukemia can be treated by intensive chemotherapy. However, this treatment is unsuitable for a subset of patients because of age and/or co-morbidities. Such patients benefit from hypomethylating agents and venetoclax but frequently develop resistance to this treatment regimen. This calls for the development of novel drugs that can prolong survival in AML patients unfit for intensive chemotherapy. The receptor tyrosine kinase AXL is a negative prognostic factor in AML. Its activation promotes survival, chemoresistance and proliferation of AML blasts. In addition, it is also present on innate immune cells and contributes to the formation of immunosuppressive environments. Together, this makes AXL a promising target for the treatment of AML. Bemcentinib is a first-in-class, orally available, selective inhibitor of AXL. Here, we report a single-cell translational sub-study in a sub-cohort of patients unfit for intensive chemotherapy (B2+B5) from the phase Ib/II clinical trial BGBC003 (NCT02488408). This sub-cohort was treated by a combination of bemcentinib and low-dose cytarabine (LDAC). To gain first insights into the mechanisms underlying treatment response, we conducted a translational study evaluating 13 participating patients. Among 32 patients evaluated for efficacy, 8 patients responded to their treatment (objective response rate: 25%). Among patients who received previous therapy ( i.e. relapsed/refractory patients), objective response rate was 18.5% (5/27). The median overall survival was 8.0 months, with a notable survival benefit (median overall survival of 24.8 months) in responders. Relapsed/refractory patients exhibited a median overall survival of 7.8 months. These data highlight that bemcentinib-LDAC benefits a considerable subset of AML patients unfit for intensive chemotherapy. In order to gain mechanistic insights into the efficacy of bemcentinib-LDAC, we profiled cells from bone marrows of 13 participating patients (6 responders, 7 non-responders, according to best response) using single-cell transcriptomics and multi-omics (CITE-seq). Cell type annotation highlighted various immune cell populations next to AML blasts. Successful treatment was associated with stronger TNFα signaling in blasts before treatment. A tight link between TNFα and AXL could subsequently be established in vitro as several AML cell lines up-regulated expression of AXL upon exposure to TNFα. Inhibiting AXL in these cell lines using bemcentinib increased expression of TNFα. This indicates a potential negative feedback loop between these two players. Furthermore, cytotoxic immune cells (CD8 + effector T cells, γδ T cells and natural killer cells) from responders displayed evidence for increased pro-inflammatory signaling upon bemcentinib-LDAC treatment. This included TNFα signaling programs next to IFNα and IFNγ signaling programs. Thus, our data indicate that bemcentinib-LDAC promotes the activity of such cytotoxic cells. In line with this, we observed increasing crosstalk between immune cells upon bemcentinib-LDAC treatment in responders. Importantly, cells from non-responders generally exhibited the opposite trend, highlighting the aforementioned cell types and pathways as factors that distinguish responders from non-responders. Together, our results indicate a potential role of TNFα and cytotoxic immune cells in the successful application of bemcentinib-LDAC. In conclusion, our findings warrant further clinical development of bemcentinib-LDAC for AML patients unfit for intensive chemotherapy. Additionally, extended research on TNFα and cytotoxic immune cells may lead to a better understanding of what mechanisms cause a treatment response. This may ultimately enable an accurate selection of patients who will benefit from bemcentinib-LDAC.

Supplementary Tables S1-S2. Metastatic events in preclinical models of PDA (S1); Warfarin enhances the activity of gemcitabine in an Axl-dependent manner (S2).

Supplementary Figures S1-S6. Axl is required for the growth of Mia PaCa-2 tumors (S1); Characterization of 10C9 (S2); Warfarin inhibits autocrine activation of Axl in pancreatic tumor cells (S3); In vivo warfarin effects (S4); Maintenance of mesenchymal phenotype is Axl dependent and inhibited by warfarin (S5); Acquisition of Axl expression sensitizes Capan-EMT cells to warfarin (S6).

PDF file - 314K, In vivo characterization of NTR/CytoCy5S fluorescence and fluorescence lifetime distributions in NB4NTR+GFP+ subcutaneous tumors, NCI-H460NTR+Luc+GFP+ orthotopic xenografts and gastrointestinal tract.

STK11 (LKB1) is a tumor suppressor, and loss-of-function mutations contribute to tumorigenesis. Mutations in the STK11 gene (STK11m) are present in ~ 20% of NSCLCs and are associated with poor response to chemotherapy and immune checkpoint inhibition (ICI) resulting in inferior survival outcomes. STK11m tumors are characterized by high oxidative stress/ROS, EMT, enhanced replication stress tolerance, resistance to DNA damage and a highly immunosuppressive tumor microenvironment with limited activation and expansion of anti-tumor CD8 T-cells. AXL, a member of the TAM family of receptor tyrosine kinases, is activated in response to cellular stress such as ROS and hypoxia. AXL expression and activation is associated with EMT and drug resistance, tumor tolerance towards oxidative stress and apoptosis, as well as an immunosuppressed tumor microenvironment. We have previously shown that selective AXL inhibition by bemcentinib potentiated ICI in STK11m NSCLC preclinical models and led to objective clinical response in individuals with STK11m NSCLC receiving bemcentinib and pembrolizumab (NCT03184571). Data from this clinical study, an independent real-world cohort of patients studied at the Haukeland Hospital in Bergen, Norway (REC 45562) and published results1, suggest AXL is expressed in ~ 80% of NSCLCs harboring a STK11m, indicating AXL expression is a characteristic of STK11m NSCLC and confirming that AXL is an attractive target in STK11m NSCLC. Inhibition of AXL in dendritic cells has been shown to increase ICI responses in preclinical models of STK11m NSCLC1. The relative contribution of targeting AXL in STK11m tumor cells vs the tumor microenvironment, and the impact on tumor cells after tumor cell targeting needs further exploration. Transcriptional analysis of STK11m and STK11wt sequences from public datasets, NCT03184571 patients and NSCLC cell lines identified transcriptional signatures consistent with the known roles of STK11 in DNA damage response and immunosuppression. Treatment of STK11m NSCLC cell lines with the AXL inhibitor bemcentinib led to a reduction in the STK11-associated DDR signature and increase in inflammatory signatures demonstrating the impact of AXL targeting on tumor cells. Due to the high unmet medical need in individuals harboring a STK11 mutation, the encouraging efficacy in the NCT03184571 clinical trial and the high incidence of AXL protein expression in STK11m tumors, a global, open-label Phase 1b/2a trial to determine the safety, tolerability and anti-tumor activity of bemcentinib with SOC (pembrolizumab, pemetrexed and carboplatin) in 1L advanced/metastatic non-squamous NSCLC patients with STK11 mutations and no actionable mutations is currently enrolling. The Phase 1b part of the study will evaluate the safety and tolerability of bemcentinib regardless of STK11 status, whereas the Phase 2a part will assess the efficacy in NSCLC patients with STK11 mutations. 1 Li et al., 2022; Cell Rep Med., PMID: 35492873 Citation Format: Magnus Blø, Austin Rayford, Noëlly Madeleine, Fabian Gärtner, Dana Bohan, Natalie Ruggio, Huiyu Li, Luc Girard, Rolf Brekken, John Minna, Marianne Ånerud, Wendy Maury, Claudia Gorcea-Carson, Gro Gausdal, David R. Micklem, Nigel McCracken. AXL as a therapeutic target in STK11 mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3245.

Abstract The ability to visualize reporter gene expression in vivo has revolutionized all facets of biologic investigation and none more so than imaging applications in oncology. Near-infrared reporter gene imaging may facilitate more accurate evaluation of chemotherapeutic response in preclinical models of orthotopic and metastatic cancers. We report the development of a cell permeable, quenched squarine probe (CytoCy5S), which is reduced by Escherichia coli nitroreductase (NTR), resulting in a near-infrared fluorescent product. Time-domain molecular imaging of NTR/CytoCy5S reporter platform permitted noninvasive monitoring of disease progression in orthotopic xenografts of disseminated leukemia, lung, and metastatic breast cancer. This methodology facilitated therapeutic evaluation of NTR gene–directed enzymatic prodrug therapy with conventional metronidazole antibiotics. These studies show NTR/CytoCy5S as a near-infrared gene reporter system with broad preclinical and prospective clinical applications within imaging, and gene therapy, of cancer. Cancer Res; 73(4); 1276–86. ©2012 AACR.