In late 2020, Genmab and ADC Therapeutics announced the termination of clinical development of enapotamab vedotin and ADCT-601, respectively, because the two drugs did not achieve the desired results in early trials. Enapotamab vedotin and ADCT-601 are both ADC drugs targeting AXL. The difference is that enapotamab vedotin is conjugated to the microtubule inhibitor MMAE, and ADCT-601 is conjugated to PBD-dimeric cytotoxin. In January 2023, Bioatla announced the latest clinical data of BA-3011 (CAB-AXL-ADC), which did not show significant positive results. As soon as the data came out, Bioatla's stock price plunged 46%. BA-3011 is an ADC drug designed to target AXL based on CAB technology. According to incomplete statistics, there are currently 29 clinical drugs targeting AXL, 3 ADCs, 1 CAR-T, 1 antibody, and 24 small molecule drugs (including 2 marketed small molecule drugs). The successive failures of AXL-ADC drugs have put the immunity targeting AXL into a trough. So what is the mechanism of action of the AXL target?What is the current layout of immuno** targeting AXL?
AXL, also known as ARK, UFO, and Tyro-7, is a receptor tyrosine kinase that was first discovered by Liu et al. in 1988 while screening genes associated with chronic transacute transgression in myeloid leukemia (CML) [1]. The human axl gene is located on chromosome 19q132. Encode 20 exons. As shown in Figure 1, the AXL protein contains 894 amino acids with a molecular weight of 104 kDa and is divided into extracellular, transmembrane, and intracellular regions [2]. The extracellular region is encoded by exons 1-10 and includes a signal peptide, two immunoglobulin (Ig) domains, and two fibronectin-type (Fn) domainsThe entire transmembrane domain and a short extracellular region subject to proteolytic cleavage are encoded by exon 11;Exons 12-20 encode intracellular domains, including tyrosine kinase domains. The intracellular domain is essential for autophosphorylation and activated kinase activity, and is involved in the transmission of a variety of signals in both normal and tumor cells [3].
figure 1. the structure of axl [4]
Under normal physiological conditions, the expression of AXL is mostly confined to smooth muscle, lung, kidney, **, and blood in normal cells [5]. In the study of many cancer cells, it has been found that AXL is highly expressed in a variety of cancers such as non-small cell lung cancer, breast cancer, and ovarian cancer, which is closely related to the occurrence, development and drug resistance of tumors.
As mentioned earlier, AXL, like other TAM family receptors, induces efficient intracellular signaling in response to various biological processes such as inflammatory responses, generally through the integration of classical ligand binding. There are two modes of AXL activation, one is classical ligand-dependent activation (primary) and the other is non-GAS6-dependent activation. The classical ligand-dependent activation of AXL refers to the AXL GAS6 signaling pathway. The GAS6-independent activation of the AXL receptor refers to the interaction of the AXL with two other TAM receptors (MER and Tyro-3) and several other non-TAM family member proteins (e.g., EGFR, HER2, etc.) to activate. Here we focus on the main pathway of AXL GAS6.
As shown in the figure below, when the Ig domain of AXL binds to GA6 to form a complex, the complex dimerizes with another GAS6-AXL complex to form a 2:2 homodimerization complex. After the AXL receptor is dimerized, trans-autophosphorylation occurs with tyrosine residues in the intracellular domain of the AXL receptor. There are 6 phosphorylation sites in the intracellular kinase domain of AXL, of which 3 C-terminal phosphorylation sites (Y698, Y702, and Y703) are conserved in TAM, and the other 3 N-terminal sites (Y779, Y821, and Y866) are autophosphorylated and involved in docking and activation. Phosphorylation of tyrosine residues in the intracellular domain of AXL is required for the recruitment of intracellular linker molecules and effector proteins and ultimately the activation of downstream signaling pathways. Activation of AXLs initiates signaling in many downstream pathways, such as PI3K AKT, MAPK ERK, and PKC. The activation of these signaling plays a variety of roles in cellular activities, such as cell proliferation and survival, migration and invasion, epithelial-mesenchymal transition (EMT), angiogenesis, resistance, immunosuppression, and stem cell maintenance activation[6].
figure 2 axl signaling pathway[3
According to the clinical trail data, the current clinical drugs targeting AXL are still mainly small molecule compounds, and there are only 5 immune**-based drugs, 3 ADCs, 1 CAR-T, and 1 antibody.
axl-adc
enapotamab vedotin, also known as AXL-107-MMAE, is an antibody-drug conjugate (ADC) targeting AXL with a DAR of 4. Enapotamab Vedotin, wholly owned by Genmab, conjugates AXL-specific IgG1 to auristatin-methyl (MMAE) via a protease-cleavable valine-citrulline linker, which has been acquired by Seagen IncLicense. The current clinical indication is solid tumors, including ovarian cancer, non-small cell lung cancer (NSCLC), endometrial cancer, cervical cancer, and thyroid cancer. At present, the highest R&D stage is Phase II. Preclinical studies have shown that enapotamab vedotin exhibits significant antitumor activity in the NSCLC model[7]. Unfortunately, enapotamab vedotin did not meet the proof-of-concept criteria for Genmab's continued development in terms of efficacy, dose and biomarkers. On November 25, 2020, Genmab announced the termination of clinical development of enapotamab vedotin.
mipasetamab uzoptirine, also known as ADCT-601, is an AXL-targeted antibody-drug conjugate consisting of a humanized anti-AXL IgG1 antibody, a Val-ALA cleavable linker, and a potent pyrrolobenzodiazepine (PBD) dimer cytotoxin SG3199. The ADCT-601 was developed by ADC Therapeutics and has a DAR of 2. When ADCT-601 binds to cells expressing the AXL protein, ADCT-601 internalizes and releases PBD, which further enters the nucleus to bind to DNA. Preclinical studies of ADCT-601 have shown that in vivo, ADCT-601 exhibits significant and durable antitumor activity in a variety of human cancer xenograft mouse models[8]. At the 2019 ESMO meeting, ADC Therapeutics presented preliminary clinical results for ADCT-601 Phase I: in terms of safety, 13 patients received 50 150 g kg q3w**, including 3 patients with 50 g kg, 6 patients with 100 g kg, and 4 patients with 150 g kg. One patient receiving 100 g kg** developed a dose-limiting toxicity for grade 3 hematuria, all participants experienced an adverse event (TEAE) due to **, and seven of them developed a grade 3 TEAE. ADC Therapeutics announced the discontinuation of the ADCT-601 clinical trial in late 2020. However, according to clinical trial data, ADC Therapeutics initiated another clinical trial of ADCT-601 in 2022, CT05389462, which was previously terminated, and the main purpose of CT05389462 is to determine the Phase 2 recommended dose (RP2D) and maximum tolerated dose (MTD) of ADCT-601 to validate ADCT-601 single** and safety and tolerability in combination with gemcitabine**. The clinical trial is currently recruiting and there is no data available.
mecbotamab vedotin, also known as BA-3011, is an AXL receptor-targeted antibody conjugate designed by Bioatla, a San Diego-based biotechnology company, through the Conditionally Active Biologics (CAB) platform. After the antibody part is modified by CAB, it can selectively target the AXL receptor of tumor tissue, and is connected to the microscopic inhibitor drug MMAE, with a DAR value of 4. In the Phase II interim analysis of NSCLC released in January this year, 4 out of 10 patients with BA3011 monotherapy** responded, with an ORR of 40%. Of the 8 patients with BA3011 + Opdivo**, only 1 had PR and an ORR of 13%. As soon as the data came out, Bioatla's stock price plunged 46%.
axl-car-t
cct301-38 axl, also known as CCT301-38, is a CAR-T product for AXL developed by Exumabio (formerly F1 Oncology). The CCT301-38 AXL was processed using a third-generation suspension lentiviral system developed by F1 Oncology and a specialized T cell closure cell processing system. The current highest clinical stage is Phase II, which targets the indications of renal cell carcinoma and seasonal allergic rhinitis.
AXL antibodies
tilvestamabBGB-149, also known as BGB-149, is a fully humanized AXL-blocking antibody developed by Bergenbio that binds primarily to the extracellular IgG1 structure of AXL, thereby blocking the binding of GAS6 to AXL. Tilvestamab exhibits antitumor activity in AML, NSCLC, pancreatic cancer, and renal cancer models. Currently, a phase I clinical trial is underway for ovarian cancer.
DMA is a biotechnology company focused on providing preclinical R&D products and services for druggable targets, with innovative functional membrane protein development, single B cell lead antibody discovery, antibody engineering and functional validation platforms. DMA now offers a full range of products and services for AXL targets, including a variety of active proteins, flow-speciated monoclonal antibodies and reference antibodies. The service covers humanization, affinity maturation, and PTM risk site removal. In addition, in order to accelerate the research and development of AXL-targeted drugs, DMA has also prepared a B cell seed bank for AXL, which can complete the screening of lead antibody molecules in 20 days at the earliest.
A variety of recombinant proteins
More recombinant proteins:
human axl protein, mfc tag (pme100533)
human axl protein, hfc tag ( pme100532)
Flow cytometry validation of monoclonal antibodies
anti-axl antibody(dm158); rabbit mab (dme100158)
Flow cytometry validation of the reference antibody
anti-axl(mecbotamab biosimilar) mab ( bme100175)
More reference antibodies:
anti-axl (enapotamab biosimilar) mab (bme100033)
References: 1]Liu E, Hjelle B, Bishop JM transforming genes in chronic myelogenous leukemia. proc natl acad sci u s a. 1988 mar;85(6):1952-6.
2]o'bryan jp, frye ra, cogswell pc, neubauer a, kitch b, prokop c, espinosa r 3rd, le beau mm, earp hs, liu et. axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. mol cell biol. 1991 oct;11(10):5016-31.
3]tang y, zang h, wen q, fan s. axl in cancer: a modulator of drug resistance and therapeutic target. j exp clin cancer res. 2023 jun 16;42(1):148.
4]zhu c, wei y, wei x. axl receptor tyrosine kinase as a promising anti-cancer approach: functions, molecular mechanisms and clinical applications. mol cancer. 2019 nov 4;18(1):153.
5]uhlén m, fagerberg l, hallström bm, et al. tissue-based map of the human proteome. science. 2015 jan 23;347(6220):1260419.
6]ol**ito sa. axl as a target in breast cancer therapy. j oncol. 2020 feb 14;2020:5291952.
7]koopman la, terp mg, zom gg, et al. enapotamab vedotin, an axl-specific antibody-drug conjugate, shows preclinical antitumor activity in non-small cell lung cancer. jci insight. 2019 nov 1;4(21):e128199.
zammarchi f, h**enith ke, chivers s, et al. preclinical development of adct-601, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting axl-expressing cancers. mol cancer ther. 2022 apr 1;21(4):582-593.