Immunological effects of amivantamab in EGFR or MET-expressing non-small cell lung cancer

Immunological effects of amivantamab in EGFR or MET-expressing non-small cell lung cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immunological effects of amivantamab in EGFR or MET-expressing non-small cell lung cancer Ryo Yoshichika, Fumiaki Mukohara, Kotaro Yamada, Joji Nagasaki, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8141506/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2026 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Epidermal growth factor receptor ( EGFR ) mutations represent one of the most frequent oncogenic driver in non-small cell lung cancer (NSCLC). Amivantamab, a bispecific antibody targeting EGFR and MNNG HOS Transforming (MET), has demonstrated clinical benefit in EGFR -mutant NSCLC through dual blockade, but its immunological role in human clinical specimens, especially tumor-infiltrating lymphocytes (TILs), has not been directly evaluated. Methods: We analyzed surgically resected tumor samples from 40 patients with NSCLC to investigate immune responses and their associations with EGFR and MET expression. TILs were characterized by flow cytometry (FCM) and immunohistochemistry (IHC). To assess the immunomodulatory potential of amivantamab, fresh tumor digests containing live tumor cells and TILs were cultured ex vivo with CD3 and CD28 stimulation in the absence or presence of amivantamab, followed by FCM. EGFR and MET expression were also evaluated by IHC. Results: EGFR mutations and high EGFR protein expression were associated with a trend toward reduced CD8⁺ T-cell and dendritic cell (DC) infiltration. In ex vivo TIL assays, exposure to amivantamab significantly activated CD8⁺ T cells, such as programmed cell death-1 expression and cytokine production, and promoted DC maturation. These effects were most pronounced in tumors with high EGFR or MET protein expression rather than EGFR mutations. Conclusions: This study provides the first direct evidence from ex vivo fresh TIL assays using human NSCLC clinical specimens that amivantamab can activate immune responses. EGFR and MET expression may serve as potential biomarkers for amivantamab-induced immune responses. Non-small cell lung cancer amivantamab antitumor immunity EGFR MET Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Lung cancer is the leading cause of cancer-related mortality worldwide [1]. Although smoking is a well-known major risk factor, environmental factors also contribute to disease pathogenesis. Indeed, a proportion of cases occur in never-smokers [2]. Along with changes in smoking prevalence, the epidemiology of lung cancer has changed, with increasing incidence observed particularly among young women and never-smokers. Therefore, lung cancer remains a life-threatening disease for a broad population [2]. Recent advances in the sequencing technologies have revealed the more presence of oncogenic driver mutations in such never-smoker populations [3]. Among them, the mutations in the epidermal growth factor receptor ( EGFR ), such as exon 19 deletions and L858R point mutation in exon 21, are the most commonly observed. In particular, approximately half of non-small cell lung cancer (NSCLC) cases in East Asian populations are known to have EGFR mutations [3]. EGFR is a receptor tyrosine kinase (RTK) which belongs to the erythroblastic leukemia viral oncogene homolog (ERBB) family. Activation of EGFR signaling leads to the induction of various downstream signaling pathways that promote cell proliferation and survival [4]. Because EGFR mutations result in ligand-independent activation of the downstream signaling, they are considered key oncogenic driver mutations. Therefore, various EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed, leading to a paradigm shift in NSCLC treatment because of their high response rate and long progression-free survival (PFS) compared to conventional cytotoxic chemotherapies [3]. In addition, recent studies have revealed the involvement of the EGFR signaling pathway in the establishment of an immunosuppressive tumor microenvironment (TME) [5, 6]. Thus, EGFR-TKIs are considered to not only inhibit cancer cell proliferation but also enhance antitumor immune responses [5, 6]. However, even with osimertinib, a third-generation EGFR-TKI, PFS for EGFR -mutant NSCLC remains limited to approximately two years due to acquired resistance [7, 8]. Activation of MNNG HOS Transforming (MET) signaling is known as one of the representative resistance mechanisms to EGFR-TKIs [6, 8]. MET is an RTK that uses hepatocyte growth factor (HGF) as a ligand and serves as a bypass signaling pathway for EGFR signaling, contributing to resistance against EGFR-TKIs. Therefore, MET has been considered a therapeutic target, leading to recent success of amivantamab, a bispecific antibody targeting EGFR and MET, in treating EGFR-mutant NSCLC [9-11]. Amivantamab inhibits EGFR and MET signaling pathways by internalizing and degrading EGFR and MET, as well as by inhibiting binding to epidermal growth factor (EGF) and HGF ligands [12]. In addition, amivantamab has been reported to induce antitumor immune responses through antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) activity, and trogocytosis by recruiting macrophages and natural killer cells via the Fc region [12, 13]. However, evidence about antitumor immunity is limited [12, 13]. In this study, we analyzed 40 NSCLC clinical samples to evaluate the infiltration and functions of CD8⁺ T cells, regulatory T cells (Tregs), and dendritic cells (DCs) as well as the expression of EGFR and MET. We demonstrated that EGFR and MET expression could be involved in the suppression of antitumor immune responses. In addition, we established an original experimental system applying amivantamab to the digest of these fresh living tumor cells and tumor-infiltrating lymphocytes (TILs) with CD3 and CD28 stimulation, showing its potential to activate antitumor immune responses, particularly in cases with high EGFR or MET expression. These findings highlight the immunological role of amivantamab in human clinical samples, suggesting potential biomarkers of EGFR and MET expression for amivantamab-induced immune responses. Materials and Methods Patient samples and ethical approval Fresh tumor tissues were obtained from 40 patients with NSCLC who underwent surgical resection at Okayama University Hospital between December 2023 and December 2024 ( Table S1 ). Clinical data including EGFR mutation status and MET exon 14 skipping mutation, for which mutation tests were performed according to the physician’s request as part of routine clinical care, were extracted from clinical records. All patients provided written informed consent, and the study protocol was approved by the Institutional Review Board of Okayama University. TIL assay Fresh tumor specimens were mechanically and enzymatically dissociated to generate single-cell suspensions with Tumor & Tissue Dissociation Reagent (BD Biosciences, Franklin Lakes, NJ, Cat# 661563). Tumor digests containing tumor cells and TILs were cultured in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, Cat# 189-02025) supplemented with 10% AB serum (MP Biomedicals, Irvine, CA, Cat# 092930949) for 72 hours in the presence of anti-CD3 (3 μg/mL) (OKT-3, BD Biosciences, RRID: AB_2869821)/CD28 antibodies (5 μg/mL) (CD28.2, Thermo Fisher Scientific, RRID: AB_468926) with or without amivantamab (Janssen Pharmaceutical, JNJ-61186372) (0.5 μg/mL). Following culture, immune cell phenotypes were assessed by flow cytometry (FCM) ( Fig. 1A ). FCM FCM was performed as previously described [14]. Briefly, cells were washed with phosphate-buffered saline (PBS) (FUJIFILM Wako Pure Chemical Corporation, Cat# 048-29805) containing 2% fetal bovine serum (FBS) (Cytiva, Tokyo, Japan, Cat# SH30070.03) and subjected to staining with surface antibodies. Intracellular staining was performed with specific antibodies and the Fixation/Permeabilization Buffer Set (Thermo Fisher Scientific, Waltham, MA, Cat# 88-8824-00), according to the manufacturer's instructions. Antibodies were diluted according to the manufacturer's instructions before staining. Detailed information on the antibodies used is summarized in Table S2. Effector Tregs (eTregs) were identified as CD45RA⁻FoxP3 high CD4⁺ T cells. DCs were defined as Lin (CD3/14/19/56)⁻HLA-DR⁺CD11c + cells. For cytokine production, cells were suspended in 1 μL/mL of GolgiStop™ Protein Transport Inhibitor (BD Biosciences, RRID: 2869012), and cultured for 6 hours at 37°C. After incubation, cells were collected and stained. FCM was performed on a BD LSRFortessa X-20 (BD Biosciences, RRID: SCR_025285), and data were analyzed using FlowJo v10 software (BD Biosciences, RRID: SCR_008520). Immunohistochemistry (IHC) Two samples were unavailable for IHC because of insufficient tissue volume, resulting in 38 samples. Formalin-fixed, paraffin-embedded tissue sections were cut into 4 μm slices, deparaffinized, and rehydrated. Antigen retrieval was carried out. After blocking endogenous peroxidase activity and nonspecific binding, sections were incubated overnight at 4°C with an anti–human CD8 monoclonal antibody (mAb) (Cell Signaling Technology, Danvers, MA, RRID: AB_2800052), an anti–human CD11c mAb (Cell Signaling Technology, RRID: AB_2799286), an anti–human EGFR mAb (Cell Signaling Technology, RRID: AB_2246311), or an anti–human cMET mAb (Cell Signaling Technology, RRID: AB_10858224), diluted in 5% BSA (Albumin, Bovine Serum, F-Ⅴ, pH5.2; Nacalai tesque, Cat# 01863-48). The slides were then incubated with the SignalStain® Boost IHC Detection Reagent (Cell Signaling Technology, RRID: AB_10966207) and detected using the SignalStain ® DAB Substrate Kit (Cell Signaling Technology, RRID: AB_10796606). All images were acquired using a BZ-X800 all-in-one fluorescence microscope (Keyence, Osaka, Japan, RRID:SCR_023617). CD8 and CD11c positive cells were counted in five randomly selected high-power fields (×200 magnification). For semi-quantitative evaluation of EGFR and MET expression, membranous staining intensity was classified into weak, moderate, and strong as previously reported [15, 16]. EGFR expression was scored by IHC as follows: 1+ for weak membrane staining in ≥10% of tumor cells, 2+ for moderate staining in ≥10%, and 3+ for strong staining in ≥10% as previously reported [15]. MET expression was scored by IHC as follows: 1+ for weak membrane staining in ≥50% of tumor cells, 2+ for moderate staining in ≥50%, and 3+ for strong staining in ≥50% as previously reported [16]. Tumors with 0–1+ were defined as “low expression” and those with 2–3+ as “high expression.” Cell counts and expression scores were independently evaluated by two investigators blinded to mutation status. Statistical analysis GraphPad Prism 9 (GraphPad Software, San Diego, CA, RRID: SCR_002798) was used for statistical analyses. Recurrence-free survival was defined as the time from the date of surgery to the date of the first documented recurrence or death from any cause, and was estimated using a Kaplan–Meier curve. Continuous variables were compared using the two-tailed Student’s t-test. Categorical variables were analyzed with Fisher’s exact test. P-values < 0.05 were considered statistically significant. All statistical details are provided in the figure legends. Results Antitumor immune responses appear to be impaired in EGFR -mutant NSCLC tumors. We analyzed tumor samples from 40 patients with NSCLC who underwent surgical resection and the patient characteristics are summarized in Table S1 . All patients had good performance status (0 or 1) with median age of 75 years. Twenty-nine patients (73%) were male and 27 (68%) had a history of smoking. Adenocarcinoma was the predominant histological subtype (32 patients, 80%). Only two patients (5%) received neoadjuvant chemotherapy, both of which consisted of combination immunotherapy. Postoperative pathological staging revealed stage I in 23 patients (58%), stage II in 12 (30%), and stage III in 4 (10%). Genome analyses were performed at the request of the attending physician, which identified EGFR mutations in 35% (13/37) and MET exon 14 skipping mutation in 13% (2/16) ( Table S1 ). During the observation period, postoperative recurrence was observed in only one patient, with no deaths observed ( Table S1 and Fig. S1A ). Because amivantamab is clinically indicated for EGFR -mutant NSCLC, we first compared the immune microenvironment between EGFR -mutant and wild-type (WT) tumors. CD8⁺ T cells and DCs, which play central roles in antitumor immune responses, were evaluated by IHC. Two samples were unavailable for IHC because of insufficient tissue volume. As a result, compared with WT tumors, EGFR -mutant tumors exhibited a trend toward lower CD8⁺ T-cell infiltration ( Fig. S1B ) and a significant reduction in DC infiltration ( Fig. S1C ). We also analyzed TILs by FCM, evaluating CD8⁺ T cells, DCs, and eTregs. Consequently, the CD8⁺ T cell to eTreg ratio, which has been reported to reflect antitumor immune responses [5], was lower in EGFR -mutant cases in our cohort, although the difference was not statistically significant ( Fig. S1D ). In addition, programmed cell death-1 (PD-1) expression in tumor-infiltrating CD8⁺ T cells, a widely used activation marker that reflects antitumor immune responses [17-19], also tended to be lower in EGFR -mutant cases ( Fig. S1E ). Conversely, CD80 and CD86 expression, representative DC maturation markers [20], were not correlated with EGFR mutation status ( Fig. S1F and G ). We also evaluated expression of PD-1 and inducible T-cell co-stimulator (ICOS) in tumor-infiltrating eTregs and observed no significant differences regardless of EGFR mutation status ( Fig. S1H and I ). Amivantamab can activate immune responses in NSCLC We investigated whether antitumor immune responses can be affected by amivantamab using fresh TIL assay. In brief, the fresh tumor digests containing tumor cells and TILs from each sample were cultured for 72 hours in the presence of anti-CD3/CD28 antibodies with or without amivantamab. Subsequently, immune cells were analyzed by FCM ( Fig. 1A ). As a result, amivantamab promoted CD8⁺ TIL activation with increasing PD-1 expression and cytokine production such as interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) ( Fig. 1B-D ). Amivantamab also promoted tumor-infiltrating DC maturation ( Fig. 1E and F ), whereas little effect on eTregs was observed ( Fig. 1G and H ). We also examined the relationship between immune cell infiltration and the effects of amivantamab on antitumor immune responses. Tumors were stratified into two groups based on the median values of CD8 + T cell counts assessed by IHC. As a result, tumors with high CD8⁺ T cell infiltration tended to exhibit greater DC infiltration ( Fig. S2A ) and showed a significantly higher CD8⁺ T cell to eTreg ratio ( Fig. S2B ). Regarding the effects of amivantamab, it significantly promoted CD8⁺ T cell activation ( Fig. S2C-E ) and exhibited a trend of increased DC maturation in tumors with high CD8⁺ T cell infiltration ( Fig. S2F and G ). Conversely, no significant changes in tumor-infiltrating eTregs were observed in both groups ( Fig. S2H and I ). In addition, we performed the same analysis based on DC infiltration counts. The tumors with high DC infiltration had significantly higher CD8⁺ T cell infiltration compared to low infiltration tumors ( Fig. S3A ), while the CD8⁺ T cell to eTreg ratio was comparable between the groups ( Fig. S3B ). When evaluating the effects of amivantamab, tumor-infiltrating CD8⁺ T cell activation and DC maturation were induced regardless of DC infiltration level ( Fig. S3E-G ), whereas eTregs showed no significant changes ( Fig. S3H and I ). These results suggest that amivantamab can activate immune responses in NSCLC tumors, particularly in those with high CD8⁺ T cell infiltration. Amivantamab potentially activates immune responses in NSCLC regardless of EGFR mutation Because amivantamab is used clinically for EGFR -mutant NSCLC patients, we next evaluated the effect of amivantamab according to EGFR mutation status. In EGFR -mutant tumors, amivantamab promoted CD8⁺ TIL activation, as indicated by PD-1 expression and cytokine production ( Fig. 2A-C ). However, amivantamab also promoted cytokine production even in EGFR-WT tumors ( Fig. 2A-C ). In addition, the maturation of tumor-infiltrating DCs was also promoted by amivantamab regardless of EGFR mutation status ( Fig. 2D and E ), whereas eTregs were unaffected in both groups ( Fig. S1J and K ). These findings suggest that amivantamab can activate immune responses not only in EGFR -mutant but also in EGFR -WT tumors. Amivantamab activates immune responses in tumors with high EGFR or MET expression. Given the targets of amivantamab, we evaluated the relationship between EGFR expression and antitumor immunity. Based on previous reports [15], EGFR expression was classified into 4 categories (0-3+) ( Fig. 3A ), with 0 or 1+ defined as EGFR-low and 2+ or 3+ as EGFR-high. In the entire cohort, 34% (13/38) of the tumors were classified as EGFR-low and 66% (25/38) were EGFR-high ( Fig. 3A ). As previously reported [21], the majority of the EGFR -mutant tumors highly expressed EGFR (85%, 11/13). However, more than half of EGFR -WT tumors also expressed high levels of EGFR (59%, 13/22) ( Fig. 3B ). There was a trend toward an association between EGFR mutation and EGFR expression, although not statistically significant (Fisher’s exact test: P = 0.150). We next compared the immune microenvironment and immune cell phenotypes according to EGFR expression. As a result, infiltration of CD8⁺ T cells and DCs was significantly reduced in EGFR-high tumors ( Fig. S4B and C ). In contrast, the ratio of CD8⁺ T cells to eTregs, as well as the activation status of CD8⁺ T cells, DCs, and eTregs, was comparable irrespective of EGFR expression levels ( Fig. S4D-I ). We further analyzed the relationship between EGFR expression and amivantamab-mediated immune activation. Amivantamab increased PD-1 expression and cytokine production in CD8⁺ TILs from EGFR-high tumors, whereas these effects were limited in EGFR-low tumors ( Fig. 3C-E ). Similarly, tumor-infiltrating DC maturation was significantly promoted by amivantamab in EGFR-high tumors but remained limited in EGFR-low tumors ( Fig. S4J and K ). In contrast, tumor-infiltrating eTregs were unaffected by amivantamab regardless of EGFR expression ( Fig. S4L and M ). These findings suggest that high EGFR expression is associated with an immunosuppressive TME, but amivantamab can promote immune activation in such a TME. Similar to EGFR, we also examined MET, another target of amivantamab, by classifying its expression in tumors into four categories (0-3+) based on the previous report [16]. In the entire cohort, MET was highly expressed (2+ or 3+) in half of the cases (19/38) ( Fig. 3F and Fig. S5A ). The comparison of immune features between MET-high and MET-low tumors revealed no significant differences in CD8⁺ T-cell or DC infiltration ( Fig. S5B and C ) and in the CD8⁺ T-cell to eTreg ratio ( Fig. S5D ). In addition, the activation status of CD8⁺ T cells, DCs, and eTregs were also comparable between the two groups ( Fig. S5E-I ). However, in MET-high tumors, amivantamab increased PD-1 expression and cytokine production in CD8⁺ TILs ( Fig. 3G-I ), whereas such effects were limited in MET-low tumors ( Fig. 4B-D ). Tumor-infiltrating DC maturation tended to be promoted by amivantamab regardless of MET expression ( Fig. S5J and K ), and tumor-infiltrating eTregs remained unchanged in either group ( Fig. S5L and M ). Taken together, these findings suggest that, similar to EGFR, MET expression can be associated with immune-activating effects of amivantamab. Amivantamab promotes immune responses in EGFR or MET high tumors, even with WT EGFR . Given the individual importance of EGFR and MET expression in amivantamab-mediated immune activation, we next analyzed their combined association. The cohort was divided into four groups (EGFR/MET, Low/Low; Low/High; High/Low; High/High) based on EGFR and MET expression, with a significant association between EGFR and MET expression levels (Fisher’s exact test: P = 0.038) ( Fig. 4A ). Although EGFR and MET expression were positively correlated, 32% (12/38) of the tumors exhibited high expression of either EGFR or MET ( Fig. 4A ). We subsequently divided the tumors into two groups, low expression of both EGFR and MET (EGFR/MET Low/Low) and those with high EGFR or MET expression (the others), and compared their immune microenvironment and immune cell phenotypes. The results revealed that tumors with high EGFR or MET expression exhibited a trend toward lower CD8⁺ T cell infiltration ( Fig. S6A ) and significantly reduced DC infiltration ( Fig. S6B ). In contrast, the CD8⁺ T cell to eTreg ratio and the activation status of immune cells were comparable between the two groups ( Fig. S6C–H ). We also examined the association of EGFR and MET expression with the immune-activating effects of amivantamab. In tumors with high EGFR or MET expression, amivantamab increased PD-1 expression and cytokine production in CD8⁺ TILs. In contrast, in tumors with low expression of both EGFR and MET, amivantamab-induced CD8⁺ TIL activation was limited ( Fig. 4B-D ). Similarly, tumor-infiltrating DC maturation was promoted by amivantamab in the group with high EGFR or MET expression, but not in the group with both low ( Fig. S6I and J ). Tumor-infiltrating eTregs were unaffected by amivantamab, irrespective of EGFR and MET expression levels (Fig. S6K and L ). Collectively, these findings indicate that high expression of EGFR or MET is important for amivantamab-induced immune responses. We further hypothesized that EGFR and MET expression might influence the efficacy of amivantamab even in EGFR -WT tumors. EGFR -mutant and EGFR -WT tumors were each stratified into four groups (EGFR/MET, Low/Low; Low/High; High/Low; High/High) based on EGFR and MET expression, and 72.7% (16/22) exhibited high EGFR or MET expression in EGFR -WT tumors ( Fig. 5A ). We classified EGFR -mutant and EGFR -WT tumors into two groups based on EGFR and MET expression; EGFR/MET Low/Low and the others, showing no correlation between EGFR mutation status and the expression of EGFR or MET (Fisher’s exact test: P = 0.680). We examined the association of EGFR and MET expression with the immune-activating effects of amivantamab in EGFR -WT tumors. In EGFR or MET–high tumors, amivantamab significantly increased PD-1 expression and cytokine production in CD8⁺ TILs ( Fig. 5B-D ). In contrast, amivantamab induce significant changes in tumor-infiltrating DCs or eTregs in neither group ( Fig. S7A-D ). These findings suggest that even EGFR -WT tumors, the presence of high expression of EGFR or MET can allow amivantamab to promote immune responses. Conclusion EGFR -mutant NSCLCs are characterized by the immunosuppressive TME and poor responses to immune checkpoint inhibitors (ICIs) [22, 23]. Although preclinical studies have suggested that amivantamab may activate antitumor immunity, direct evidence in human clinical specimens has been lacking [12, 13]. In this study, we provide the first direct evidence from ex vivo fresh TIL assays using human NSCLC clinical specimens that amivantamab can activate antitumor immune responses. Notably, these immune-activating effects were more strongly associated with high expression of EGFR or MET protein rather than EGFR mutation status, suggesting that EGFR/MET expression levels may serve as biomarkers for amivantamab-induced antitumor immunity. Given the limited effectiveness of ICIs in NSCLC, particularly in EGFR -mutant one, our findings highlight the potential of amivantamab, especially in combination with immunotherapy, as a promising strategy to overcome this major clinical challenge. However, due to the unpredictable nature of cancer biology and human therapeutics, clinical studies would be required to determine the effectiveness of such a strategy. EGFR mutations induce ligand-independent phosphorylation of RTK and activate EGFR signaling pathways, which contribute not only to cancer cell proliferation but also to the establishment of the immunosuppressive TME. Specifically, EGFR activation regulates chemokine production, leading to reduced infiltration of cytotoxic CD8⁺ T cells while promoting the recruitment of Tregs [5, 6]. This pathway also increases tumor-promoting cytokines and EGFR ligands, suppressing CD8⁺ T-cell function and DC maturation while enhancing the immunosuppressive activity of Tregs and tumor-associated macrophages [5, 6]. Consistently, in our cohort, EGFR -mutant tumors exhibited reduced infiltration of CD8⁺ T cells and DCs, reinforcing the concept of the immunosuppressive TME in these tumors. Blockade of the EGFR pathway therefore could potentially counteract this immunosuppressive TME. Amivantamab, a bispecific antibody targeting both EGFR and MET, has been shown to activate antitumor immune responses in vitro and in mouse models, and its clinical activity includes durable responses with a tail plateau pattern reminiscent of immunotherapy [11-13, 24]. In our study, we provide the first demonstration in fresh human TILs from NSCLC that amivantamab activates antitumor immunity, particularly through T-cell-mediated responses. Although amivantamab is currently approved only for EGFR -mutant NSCLC, previous reports have suggested efficacy in EGFR -WT tumors [25]. Consistently, we observed that amivantamab promoted TIL activation in tumors with high EGFR or MET expression, regardless of mutation status. This finding is aligned with its pharmacological mechanism and underscores the potential of EGFR/MET expression as biomarkers for amivantamab-induced antitumor immunity. Indeed, several ongoing clinical trials are already including EGFR-WT cohorts [26-28], and our results support such trial designs. Importantly, these findings suggest that tumors with high EGFR/MET expression may benefit from amivantamab even without EGFR mutations. The mechanisms underlying amivantamab-induced immune activation remain incompletely understood. Because EGFR signaling is involved in immune suppression, its blockade could contribute to immune activation [29, 30]. In addition, activation of T-cell immunity through innate immune stimulation mediated by ADCC and ADCP activity is considered another process [13, 31]. We also observed DC maturation following amivantamab treatment, although its correlation with EGFR or MET expression was less clear than that observed for T cells. Furthermore, because tumor-infiltrating natural killer (NK) cells are generally small, their potential contribution was not evaluated in our cohort, highlighting the need for further investigation. Mouse models frequently fail to predict human clinical outcomes, and peripheral blood–based assays cannot recapitulate the chronic antigen exposure and suppressive cues that shape TIL function in the TME. In contrast, our fresh TIL assay preserves the viability of tumor and immune cells, enabling real-time assessment of therapeutic antibody activity within a physiologically relevant context. Unlike assays relying on TIL expansion, organoid generation, or cryopreservation, our approach is very simple, minimizing artifacts and reflecting in vivo conditions more faithfully [32]. Voabil et al. also developed a similar tumor fragment platform, showing the advantages [33]. This approach not only provides a more faithful reflection of the in vivo TME but also holds broader implications for drug development. Moreover, with further development, this approach could evolve into a patient-specific tool, enabling clinicians to project therapeutic responses to treatment. In summary, this study provides the first ex vivo human evidence that amivantamab activates TILs together with the unique strengths of our fresh TIL assay. These effects extended beyond EGFR -mutant tumors to EGFR -WT tumors with high EGFR or MET expression, underscoring the potential of EGFR/MET expression as biomarkers for amivantamab-induced antitumor immunity. Further basic and translational research is warranted to validate these findings and guide future clinical applications. Declarations Funding This study was supported by Janssen Pharmaceutical K.K. Competing Interests S. Toyooka received research grants from TAIHO PHARMA, Eli Lilly, Chugai Pharmaceutical and Astellas Pharma, and honoraria from TAIHO PHARMA, Eli Lilly, Chugai Pharmaceutical, GUARDANT, AstraZeneca, Illumina, MERCK, CSL Behring, Nippon Kayaku, Daiichi-Sankyo, Ono Pharmaceutical, Medtronic, Ziosoft, NOVARTIS, Sysmex and Riken Genesis outside of this study. Y. Togashi received a research grant from Janssen Pharmaceutical K.K. for this study. Y. Togashi received research grants from AstraZeneca, TAIHO PHARMA, Takeda, Chugai Pharmaceutical, Daiichi-Sankyo, and KORTUC, and honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, AstraZeneca, Chugai Pharmaceutical, Eisai and MSD outside of this study. The other authors declare that they have no research support relevant to financial competing interests. Author Contributions RY: Data curation, formal analysis, investigation, methodology, writing – original draft. FM: Data curation, formal analysis, investigation, methodology. KY: Formal analysis and investigation. JN: Conceptualization, data curation, formal analysis, methodology. HW: Data curation, formal analysis, investigation. YU: Methodology. K. Suzawa: Investigation. K. Shien: Investigation. ST: Supervision. TI: Data curation, formal analysis, investigation, visualization, methodology, writing – original draft, writing – review and editing. YT: Conceptualization, data curation, funding acquisition, methodology, writing – original draft, project administration, writing-review and editing. Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. Additional data are accessible from the corresponding author upon reasonable request. Ethical Approval The study protocol was approved by the Institutional Review Board of Okayama University. Consent to participate All patients provided written informed consent. Consent to publish All patients provided written informed consent. References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Toyooka received research grants from TAIHO PHARMA, Eli Lilly, Chugai Pharmaceutical and Astellas Pharma, and honoraria from TAIHO PHARMA, Eli Lilly, Chugai Pharmaceutical, GUARDANT, AstraZeneca, Illumina, MERCK, CSL Behring, Nippon Kayaku, Daiichi-Sankyo, Ono Pharmaceutical, Medtronic, Ziosoft, NOVARTIS, Sysmex and Riken Genesis outside of this study. Y. Togashi received a research grant from Janssen Pharmaceutical K.K. for this study. Y. Togashi received research grants from AstraZeneca, TAIHO PHARMA, Takeda, Chugai Pharmaceutical, Daiichi-Sankyo, and KORTUC, and honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, AstraZeneca, Chugai Pharmaceutical, Eisai and MSD outside of this study. The other authors declare that they have no research support relevant to financial competing interests. Supplementary Files YoshichikaetalSupple251118.pdf Cite Share Download PDF Status: Published Journal Publication published 24 Mar, 2026 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 21 Dec, 2025 Reviews received at journal 15 Dec, 2025 Reviews received at journal 06 Dec, 2025 Reviewers agreed at journal 26 Nov, 2025 Reviewers agreed at journal 26 Nov, 2025 Reviewers agreed at journal 25 Nov, 2025 Reviewers invited by journal 23 Nov, 2025 Editor assigned by journal 19 Nov, 2025 Submission checks completed at journal 19 Nov, 2025 First submitted to journal 18 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":4632582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEx vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eactivation of tumor-infiltrating lymphocytes (TILs) by amivantamab in fresh non-small cell lung cancer (NSCLC) clinical specimens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental schema. Freshly resected NSCLC tumor digests containing living tumor cells and TILs were cultured for 72 hours with anti-CD3/CD28 stimulation in the absence or presence of amivantamab, which were subsequently analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e(B–H) TILs activation by amivantamab. Summaries of programmed cell death-1 (PD-1) expression (B), interferon-γ (IFNγ) production (C), and tumor necrosis factor-α (TNFα) production (D) in tumor-infiltrating CD8⁺ T cells, CD80 (E) and CD86 expression (F) in tumor-infiltrating dendritic cells (DCs), and PD-1 (G) and inducible T-cell co-stimulator (ICOS) expression (H) in tumor-infiltrating effector regulatory T cells (eTregs) are shown.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed by t-tests in (B-H). In box-and-whisker plots, the box spans from the first to the third quartile with a line at the median and the whiskers extend from the minimum to the maximum. NS: not significant; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003cem\u003e\u003cbr\u003e\n\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-8141506/v1/c1306819284c6ab2309c31a3.png"},{"id":97120183,"identity":"fcb4c430-6317-4807-927d-2098dfe344e2","added_by":"auto","created_at":"2025-12-01 07:48:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3667090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEx vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eactivation of tumor-infiltrating lymphocytes (TILs) by amivantamab according to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eepidermal growth factor receptor\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEGFR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) mutation status\u003c/strong\u003e\u003cbr\u003e\n\u003cem\u003eEx vivo\u003c/em\u003e TIL assays were performed as described in \u003cstrong\u003eFigure 1A\u003c/strong\u003e. Summaries of programmed cell death-1 (PD-1) expression (A), interferon-γ (IFNγ) production (B), and tumor necrosis factor-α (TNFα) production (C) in tumor-infiltrating CD8⁺ T cells, and CD80 (D) and CD86 expression (E) in tumor-infiltrating dendritic cells (DCs) according to \u003cem\u003eEGFR\u003c/em\u003e mutation status (left, wild-type (WT) ; right, mutant) are shown.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Statistical analyses were performed by t-tests. In box-and-whisker plots, the box spans from the first to the third quartile with a line at the median and the whiskers extend from the minimum to the maximum. NS: not significant; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8141506/v1/541b7fd55751c008dc699e05.png"},{"id":97142168,"identity":"1daca1b3-2e70-4eb2-a2a6-0f1b40f53893","added_by":"auto","created_at":"2025-12-01 10:07:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6367350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEx vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activation of tumor-infiltrating lymphocytes (TILs) by amivantamab according to epidermal growth factor receptor (EGFR) or MNNG HOS Transforming (MET) expression level\u003c/strong\u003e\u003cbr\u003e\n(A) Distribution of EGFR protein expression levels in tumor samples. EGFR expression was scored by immunohistochemistry (IHC) as follows: 1+ for weak membrane staining in ≥10% of tumor cells, 2+ for moderate staining in ≥10%, and 3+ for strong staining in ≥10%. A pie chart is shown.\u003cbr\u003e\n(B) Association between \u003cem\u003eEGFR \u003c/em\u003emutation status and EGFR protein expression. A bar graph is shown.\u003cbr\u003e\n(C–E) Tumor-infiltrating CD8⁺ T cell activation\u003cstrong\u003e \u003c/strong\u003estratified by EGFR expression level. \u003cem\u003eEx vivo\u003c/em\u003e TIL assays were performed as described in \u003cstrong\u003eFigure 1A.\u003c/strong\u003e Summaries of programmed cell death-1 (PD-1) expression (C), interferon-γ (IFNγ) production (D), and tumor necrosis factor-α (TNFα) production (E) in tumor-infiltrating CD8⁺ T cells according to EGFR expression level (left, low; right, high) are shown.\u003cbr\u003e\n(F) Distribution of MET protein expression levels in tumor samples. MET expression was scored by IHC as follows: 1+ for weak membrane staining in ≥50% of tumor cells, 2+ for moderate staining in ≥50%, and 3+ for strong staining in ≥50%. A pie chart is shown.\u003cbr\u003e\n(G–I) Tumor-infiltrating CD8⁺ T cell activation stratified by MET expression level categorized as low (0–1+) or high (2–3+). \u003cem\u003eEx vivo\u003c/em\u003e TIL assays were performed as described in \u003cstrong\u003eFigure 1A.\u003c/strong\u003e Summaries of PD-1 expression (G), IFNγ production (H), and TNFα production (I) in tumor-infiltrating CD8⁺ T cells according to MET expression level (left, low; right, high) are shown.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe correlation between\u003cem\u003e \u003c/em\u003eEGFR expression levels classified into low (0–1+) and high (2–3+) and \u003cem\u003eEGFR \u003c/em\u003emutation status was analyzed using Fisher’s exact test in (B). T-tests were performed in (C-E) and (G-I). In box-and-whisker plots, the box spans from the first to the third quartile with a line at the median and the whiskers extend from the minimum to the maximum. NS: not significant; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003cem\u003e\u003cbr\u003e\n\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8141506/v1/2be269de14bf4e17ef79f932.png"},{"id":97120195,"identity":"5eea75ce-b283-4bbb-9ff9-203a09dc9946","added_by":"auto","created_at":"2025-12-01 07:48:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2610180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEx vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activation of tumor-infiltrating lymphocytes (TILs) by amivantamab according to epidermal growth factor receptor (EGFR)/MNNG HOS Transforming (MET) expression level.\u003c/strong\u003e\u003cbr\u003e\n(A) Distribution of EGFR/MET protein expression levels in tumor samples. The EGFR/MET expression was stratified as described in \u003cstrong\u003eFigure 3\u003c/strong\u003e. A pie chart is shown.\u003cbr\u003e\n(B–D) Tumor-infiltrating CD8⁺ T cell activation stratified by EGFR/MET expression level.\u003cem\u003e Ex vivo\u003c/em\u003e TIL assays were performed as described in \u003cstrong\u003eFigure 1A.\u003c/strong\u003e Summaries of programmed cell death-1 (PD-1) expression (B), interferon-γ (IFNγ) production (C), and tumor necrosis factor-α (TNFα) production (D) in tumor-infiltrating CD8⁺ T cells according to EGFR/MET expression level (left, both low; right, the others) are shown.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Statistical analyses were performed by t-tests in (B-D). In box-and-whisker plots, the box spans from the first to the third quartile with a line at the median and the whiskers extend from the minimum to the maximum. NS: not significant; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8141506/v1/5ce69d42521b5591e13b60c1.png"},{"id":97120189,"identity":"f0488d25-3edf-46a3-8b8f-b69e4db9eec0","added_by":"auto","created_at":"2025-12-01 07:48:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3118204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEx vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activation of tumor-infiltrating lymphocytes (TILs) by amivantamab based on epidermal growth factor receptor (EGFR)/MNNG HOS Transforming (MET) expression level in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEGFR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-wild type (WT) tumors.\u003c/strong\u003e\u003cbr\u003e\n(A) Association between \u003cem\u003eEGFR \u003c/em\u003emutation status and EGFR/MET protein expression. The EGFR/MET expression was stratified as described in \u003cstrong\u003eFigure 3\u003c/strong\u003e. A bar graph is shown.\u003cbr\u003e\n(B–D) Tumor-infiltrating CD8⁺ T cell activation stratified by EGFR/MET expression level in \u003cem\u003eEGFR\u003c/em\u003e-WT tumors. \u003cem\u003eEx vivo\u003c/em\u003e TIL assays were performed as described in \u003cstrong\u003eFigure 1A.\u003c/strong\u003e Summaries of programmed cell death-1 (PD-1) expression (B), interferon-γ (IFNγ) production (C), and tumor necrosis factor-α (TNFα) production (D) in tumor-infiltrating CD8⁺ T cells according to EGFR/MET expression level (left, both low; right, the others) are shown.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe correlation between \u003cem\u003eEGFR \u003c/em\u003emutation status and EGFR/MET expression classified into Low/Low and the others was analyzed using Fisher’s exact test in (A). T-tests were performed in (B-D). In box-and-whisker plots, the box spans from the first to the third quartile with a line at the median and the whiskers extend from the minimum to the maximum. NS: not significant; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8141506/v1/e0fbef349b237f186e1b27b2.png"},{"id":105755012,"identity":"2f343a32-d521-49c7-8ccd-5afb47e63cd1","added_by":"auto","created_at":"2026-03-30 16:24:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16024526,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8141506/v1/449e0b47-896b-4ef5-b1b7-f0fae6065882.pdf"},{"id":97120191,"identity":"c54e0e3d-4d1c-4ca4-b7e7-f19ada0a1383","added_by":"auto","created_at":"2025-12-01 07:48:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2377961,"visible":true,"origin":"","legend":"","description":"","filename":"YoshichikaetalSupple251118.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8141506/v1/042af41b85d3b3421710c661.pdf"}],"financialInterests":"Competing interest reported. S. Toyooka received research grants from TAIHO PHARMA, Eli Lilly, Chugai Pharmaceutical and Astellas Pharma, and honoraria from TAIHO PHARMA, Eli Lilly, Chugai Pharmaceutical, GUARDANT, AstraZeneca, Illumina, MERCK, CSL Behring, Nippon Kayaku, Daiichi-Sankyo, Ono Pharmaceutical, Medtronic, Ziosoft, NOVARTIS, Sysmex and Riken Genesis outside of this study. Y. Togashi received a research grant from Janssen Pharmaceutical K.K. for this study. Y. Togashi received research grants from AstraZeneca, TAIHO PHARMA, Takeda, Chugai Pharmaceutical, Daiichi-Sankyo, and KORTUC, and honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, AstraZeneca, Chugai Pharmaceutical, Eisai and MSD outside of this study. The other authors declare that they have no research support relevant to financial competing interests.","formattedTitle":"Immunological effects of amivantamab in EGFR or MET-expressing non-small cell lung cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is the leading cause of cancer-related mortality worldwide [1]. Although smoking is a well-known major risk factor, environmental factors also contribute to disease pathogenesis. Indeed, a proportion of cases occur in never-smokers [2]. Along with changes in smoking prevalence, the epidemiology of lung cancer has changed, with increasing incidence observed particularly among young women and never-smokers. Therefore, lung cancer remains a life-threatening disease for a broad population [2]. Recent advances in the sequencing technologies have revealed the more presence of oncogenic driver mutations in such never-smoker populations [3]. Among them, the mutations in the \u003cem\u003eepidermal growth factor receptor\u003c/em\u003e (\u003cem\u003eEGFR\u003c/em\u003e), such as exon 19 deletions and L858R point mutation in exon 21, are the most commonly observed. In particular, approximately half of non-small cell lung cancer (NSCLC) cases in East Asian populations are known to have \u003cem\u003eEGFR\u003c/em\u003e mutations [3].\u003c/p\u003e\n\u003cp\u003eEGFR is a receptor tyrosine kinase (RTK) which belongs to the erythroblastic leukemia viral oncogene homolog (ERBB) family. Activation of EGFR signaling leads to the induction of various downstream signaling pathways that promote cell proliferation and survival [4]. Because \u003cem\u003eEGFR\u003c/em\u003e mutations result in ligand-independent activation of the downstream signaling, they are considered key oncogenic driver mutations. Therefore, various EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed, leading to a paradigm shift in NSCLC treatment because of their high response rate and long progression-free survival (PFS) compared to conventional cytotoxic chemotherapies [3]. In addition, recent studies have revealed the involvement of the EGFR signaling pathway in the establishment of an immunosuppressive tumor microenvironment (TME) [5, 6]. Thus, EGFR-TKIs are considered to not only inhibit cancer cell proliferation but also enhance antitumor immune responses [5, 6]. However, even with osimertinib, a third-generation EGFR-TKI, PFS for \u003cem\u003eEGFR\u003c/em\u003e-mutant NSCLC remains limited to approximately two years due to acquired resistance [7, 8]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eActivation of MNNG HOS Transforming (MET) signaling is known as one of the representative resistance mechanisms to EGFR-TKIs [6, 8]. MET is an RTK that uses hepatocyte growth factor (HGF) as a ligand and serves as a bypass signaling pathway for EGFR signaling, contributing to resistance against EGFR-TKIs. Therefore, MET has been considered a therapeutic target, leading to recent success of amivantamab, a bispecific antibody targeting EGFR and MET, in treating EGFR-mutant NSCLC [9-11]. Amivantamab inhibits EGFR and MET signaling pathways by internalizing and degrading EGFR and MET, as well as by inhibiting binding to epidermal growth factor (EGF) and HGF ligands [12]. In addition, amivantamab has been reported to induce antitumor immune responses through antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) activity, and trogocytosis by recruiting macrophages and natural killer cells via the Fc region [12, 13]. However, evidence about antitumor immunity is limited [12, 13].\u003c/p\u003e\n\u003cp\u003eIn this study, we analyzed 40 NSCLC clinical samples to evaluate the infiltration and functions of CD8⁺ T cells, regulatory T cells (Tregs), and dendritic cells (DCs) as well as the expression of EGFR and MET. We demonstrated that EGFR and MET expression could be involved in the suppression of antitumor immune responses. In addition, we established an original experimental system applying amivantamab to the digest of these fresh living tumor cells and tumor-infiltrating lymphocytes (TILs) with CD3 and CD28 stimulation, showing its potential to activate antitumor immune responses, particularly in cases with high EGFR or MET expression. These findings highlight the immunological role of amivantamab in human clinical samples, suggesting potential biomarkers of EGFR and MET expression for amivantamab-induced immune responses.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePatient samples and ethical approval\u003cbr\u003e\u003c/strong\u003eFresh tumor tissues were obtained from 40 patients with NSCLC who underwent surgical resection at Okayama University Hospital between December 2023 and December 2024 (\u003cstrong\u003eTable S1\u003c/strong\u003e). Clinical data including\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eEGFR\u0026nbsp;\u003c/em\u003emutation status and \u003cem\u003eMET\u003c/em\u003e exon 14 skipping mutation, for which mutation tests were performed according to the physician\u0026rsquo;s request as part of routine clinical care, were extracted from clinical records. All patients provided written informed consent, and the study protocol was approved by the Institutional Review Board of Okayama University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTIL assay\u003cbr\u003e\u003c/strong\u003eFresh tumor specimens were mechanically and enzymatically dissociated to generate single-cell suspensions with Tumor \u0026amp; Tissue Dissociation Reagent (BD Biosciences, Franklin Lakes, NJ, Cat# 661563). Tumor digests containing tumor cells and TILs were cultured in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, Cat# 189-02025) supplemented with 10% AB serum (MP Biomedicals, Irvine, CA, Cat# 092930949) for 72 hours in the presence of anti-CD3 (3 \u0026mu;g/mL) (OKT-3, BD Biosciences, RRID: AB_2869821)/CD28 antibodies (5 \u0026mu;g/mL) (CD28.2, Thermo Fisher Scientific, RRID: AB_468926) with or without amivantamab (Janssen Pharmaceutical, JNJ-61186372) (0.5 \u0026mu;g/mL). Following culture, immune cell phenotypes were assessed by flow cytometry (FCM) (\u003cstrong\u003eFig. 1A\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFCM\u003cbr\u003e\u003c/strong\u003eFCM was performed as previously described [14]. Briefly, cells were washed with phosphate-buffered saline (PBS) (FUJIFILM Wako Pure Chemical Corporation, Cat# 048-29805) containing 2% fetal bovine serum (FBS) (Cytiva, Tokyo, Japan, Cat# SH30070.03) and subjected to staining with surface antibodies. Intracellular staining was performed with specific antibodies and the Fixation/Permeabilization Buffer Set (Thermo Fisher Scientific, Waltham, MA, Cat# 88-8824-00), according to the manufacturer\u0026apos;s instructions. Antibodies were diluted according to the manufacturer\u0026apos;s instructions before staining. Detailed information on the antibodies used is summarized in \u003cstrong\u003eTable S2.\u003c/strong\u003e Effector Tregs (eTregs) were identified as CD45RA⁻FoxP3\u003csup\u003ehigh\u003c/sup\u003eCD4⁺ T cells. DCs were defined as Lin (CD3/14/19/56)⁻HLA-DR⁺CD11c\u003csup\u003e+\u003c/sup\u003e cells. For cytokine production, cells were suspended in 1 \u0026mu;L/mL of GolgiStop\u0026trade; Protein Transport Inhibitor (BD Biosciences, RRID: 2869012), and cultured for 6 hours at 37\u0026deg;C. After incubation, cells were collected and stained. FCM was performed on a BD LSRFortessa X-20 (BD Biosciences, RRID: SCR_025285), and data were analyzed using FlowJo v10 software (BD Biosciences, RRID: SCR_008520).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC)\u003cbr\u003e\u003c/strong\u003eTwo samples were unavailable for IHC because of insufficient tissue volume, resulting in 38 samples. Formalin-fixed, paraffin-embedded tissue sections were cut into 4 \u0026mu;m slices, deparaffinized, and rehydrated. Antigen retrieval was carried out. After blocking endogenous peroxidase activity and nonspecific binding, sections were incubated overnight at 4\u0026deg;C with an anti\u0026ndash;human CD8 monoclonal antibody (mAb) (Cell Signaling Technology, Danvers, MA, RRID: AB_2800052), an anti\u0026ndash;human CD11c mAb (Cell Signaling Technology, RRID: AB_2799286), an anti\u0026ndash;human EGFR mAb (Cell Signaling Technology, RRID: AB_2246311), or an anti\u0026ndash;human cMET mAb (Cell Signaling Technology, RRID: AB_10858224), diluted in 5% BSA (Albumin, Bovine Serum, F-Ⅴ, pH5.2; Nacalai tesque, Cat#\u0026nbsp;01863-48). The slides were then incubated with the SignalStain\u0026reg; Boost IHC Detection Reagent (Cell Signaling Technology, RRID: AB_10966207) and detected using the SignalStain\u003csup\u003e\u0026reg;\u003c/sup\u003e DAB Substrate Kit (Cell Signaling Technology, RRID: AB_10796606). All images were acquired using a BZ-X800 all-in-one fluorescence microscope (Keyence, Osaka, Japan, RRID:SCR_023617). CD8 and CD11c positive cells were counted in five randomly selected high-power fields (\u0026times;200 magnification). For semi-quantitative evaluation of EGFR and MET expression, membranous staining intensity was classified into weak, moderate, and strong as previously reported [15, 16]. EGFR expression was scored by IHC as follows: 1+ for weak membrane staining in \u0026ge;10% of tumor cells, 2+ for moderate staining in \u0026ge;10%, and 3+ for strong staining in \u0026ge;10% as previously reported [15]. MET expression was scored by IHC as follows: 1+ for weak membrane staining in \u0026ge;50% of tumor cells, 2+ for moderate staining in \u0026ge;50%, and 3+ for strong staining in \u0026ge;50% as previously reported [16]. Tumors with 0\u0026ndash;1+ were defined as \u0026ldquo;low expression\u0026rdquo; and those with 2\u0026ndash;3+ as \u0026ldquo;high expression.\u0026rdquo; Cell counts and expression scores were independently evaluated by two investigators blinded to mutation status.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003cbr\u003e\u003c/strong\u003eGraphPad Prism 9 (GraphPad Software, San Diego, CA, RRID: SCR_002798) was used for statistical analyses. Recurrence-free survival was defined as the time from the date of surgery to the date of the first documented recurrence or death from any cause, and was estimated using a Kaplan\u0026ndash;Meier curve. Continuous variables were compared using the two-tailed Student\u0026rsquo;s t-test. Categorical variables were analyzed with Fisher\u0026rsquo;s exact test. P-values \u0026lt; 0.05 were considered statistically significant. All statistical details are provided in the figure legends.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAntitumor immune responses appear to be impaired in \u003cem\u003eEGFR\u003c/em\u003e-mutant NSCLC tumors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed tumor samples from 40 patients with NSCLC who underwent surgical resection and the patient characteristics are summarized in \u003cstrong\u003eTable S1\u003c/strong\u003e. All patients had good performance status (0 or 1) with median age of 75 years. Twenty-nine patients (73%) were male and 27 (68%) had a history of smoking. Adenocarcinoma was the predominant histological subtype (32 patients, 80%). Only two patients (5%) received neoadjuvant chemotherapy, both of which consisted of combination immunotherapy. Postoperative pathological staging revealed stage I in 23 patients (58%), stage II in 12 (30%), and stage III in 4 (10%). Genome analyses were performed at the request of the attending physician, which identified \u003cem\u003eEGFR\u003c/em\u003e mutations in 35% (13/37) and \u003cem\u003eMET\u003c/em\u003e exon 14 skipping mutation in 13% (2/16) (\u003cstrong\u003eTable S1\u003c/strong\u003e). During the observation period, postoperative recurrence was observed in only one patient, with no deaths observed (\u003cstrong\u003eTable S1\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;Fig. S1A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eBecause amivantamab is clinically indicated for \u003cem\u003eEGFR\u003c/em\u003e-mutant NSCLC, we first compared the immune microenvironment between \u003cem\u003eEGFR\u003c/em\u003e-mutant and wild-type (WT) tumors. CD8⁺ T cells and DCs, which play central roles in antitumor immune responses, were evaluated by IHC. Two samples were unavailable for IHC because of insufficient tissue volume. As a result, compared with WT tumors, \u003cem\u003eEGFR\u003c/em\u003e-mutant tumors exhibited a trend toward lower CD8⁺ T-cell infiltration (\u003cstrong\u003eFig. S1B\u003c/strong\u003e) and a significant reduction in DC infiltration (\u003cstrong\u003eFig. S1C\u003c/strong\u003e). We also analyzed TILs by FCM, evaluating CD8⁺ T cells, DCs, and eTregs. Consequently, the CD8⁺ T cell to eTreg ratio, which has been reported to reflect antitumor immune responses [5], was lower in \u003cem\u003eEGFR\u003c/em\u003e-mutant cases in our cohort, although the difference was not statistically significant (\u003cstrong\u003eFig. S1D\u003c/strong\u003e). In addition, programmed cell death-1 (PD-1) expression in tumor-infiltrating CD8⁺ T cells, a widely used activation marker that reflects antitumor immune responses [17-19], also tended to be lower in \u003cem\u003eEGFR\u003c/em\u003e-mutant cases (\u003cstrong\u003eFig. S1E\u003c/strong\u003e). Conversely, CD80 and CD86 expression, representative DC maturation markers [20], were not correlated with \u003cem\u003eEGFR\u003c/em\u003e mutation status (\u003cstrong\u003eFig. S1F\u003c/strong\u003e and\u003cstrong\u003e\u0026nbsp;G\u003c/strong\u003e). We also evaluated expression of PD-1 and inducible T-cell co-stimulator (ICOS) in tumor-infiltrating eTregs and observed no significant differences regardless of \u003cem\u003eEGFR\u0026nbsp;\u003c/em\u003emutation status (\u003cstrong\u003eFig. S1H\u003c/strong\u003e and \u003cstrong\u003eI\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmivantamab can activate immune responses in NSCLC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated whether antitumor immune responses can be affected by amivantamab using fresh TIL assay. In brief, the fresh tumor digests containing tumor cells and TILs from each sample were cultured for 72 hours in the presence of anti-CD3/CD28 antibodies with or without amivantamab. Subsequently, immune cells were analyzed by FCM (\u003cstrong\u003eFig. 1A\u003c/strong\u003e). As a result, amivantamab promoted CD8⁺ TIL activation with increasing PD-1 expression and cytokine production such as interferon-\u0026gamma; (IFN\u0026gamma;) and tumor necrosis factor-\u0026alpha; (TNF\u0026alpha;) (\u003cstrong\u003eFig. 1B-D\u003c/strong\u003e). Amivantamab also promoted tumor-infiltrating DC maturation (\u003cstrong\u003eFig. 1E\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;F\u003c/strong\u003e), whereas little effect on eTregs was observed (\u003cstrong\u003eFig. 1G and H\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe also examined the relationship between immune cell infiltration and the effects of amivantamab on antitumor immune responses. Tumors were stratified into two groups based on the median values of CD8\u003csup\u003e+\u003c/sup\u003e T cell counts assessed by IHC. As a result, tumors with high CD8⁺ T cell infiltration tended to exhibit greater DC infiltration (\u003cstrong\u003eFig. S2A\u003c/strong\u003e) and showed a significantly higher CD8⁺ T cell to eTreg ratio (\u003cstrong\u003eFig. S2B\u003c/strong\u003e). Regarding the effects of amivantamab, it significantly promoted CD8⁺ T cell activation (\u003cstrong\u003eFig. S2C-E\u003c/strong\u003e) and exhibited a trend of increased DC maturation in tumors with high CD8⁺ T cell infiltration (\u003cstrong\u003eFig. S2F\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;G\u003c/strong\u003e). Conversely, no significant changes in tumor-infiltrating eTregs were observed in both groups (\u003cstrong\u003eFig. S2H\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;I\u003c/strong\u003e). In addition, we performed the same analysis based on DC infiltration counts. The tumors with high DC infiltration had significantly higher CD8⁺ T cell infiltration compared to low infiltration tumors (\u003cstrong\u003eFig. S3A\u003c/strong\u003e), while the CD8⁺ T cell to eTreg ratio was comparable between the groups (\u003cstrong\u003eFig. S3B\u003c/strong\u003e). When evaluating the effects of amivantamab, tumor-infiltrating CD8⁺ T cell activation and DC maturation were induced regardless of DC infiltration level (\u003cstrong\u003eFig. S3E-G\u003c/strong\u003e), whereas eTregs showed no significant changes (\u003cstrong\u003eFig. S3H\u003c/strong\u003e and \u003cstrong\u003eI\u003c/strong\u003e). These results suggest that amivantamab can activate immune responses in NSCLC tumors, particularly in those with high CD8⁺ T cell infiltration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmivantamab potentially activates immune responses in NSCLC regardless of \u003cem\u003eEGFR\u003c/em\u003e mutation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause amivantamab is used clinically for \u003cem\u003eEGFR\u003c/em\u003e-mutant NSCLC patients, we next evaluated the effect of amivantamab according to \u003cem\u003eEGFR\u003c/em\u003e mutation status. In \u003cem\u003eEGFR\u003c/em\u003e-mutant tumors, amivantamab promoted CD8⁺ TIL activation, as indicated by PD-1 expression and cytokine production (\u003cstrong\u003eFig. 2A-C\u003c/strong\u003e). However, amivantamab also promoted cytokine production even in EGFR-WT tumors (\u003cstrong\u003eFig. 2A-C\u003c/strong\u003e). In addition, the maturation of tumor-infiltrating DCs was also promoted by amivantamab regardless of \u003cem\u003eEGFR\u003c/em\u003e mutation status (\u003cstrong\u003eFig. 2D\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;E\u003c/strong\u003e), whereas eTregs were unaffected in both groups (\u003cstrong\u003eFig. S1J\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eK\u003c/strong\u003e). These findings suggest that amivantamab can activate immune responses not only in \u003cem\u003eEGFR\u003c/em\u003e-mutant but also in \u003cem\u003eEGFR\u003c/em\u003e-WT tumors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmivantamab activates immune responses in tumors with high EGFR or MET expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the targets of amivantamab, we evaluated the relationship between EGFR expression and antitumor immunity. Based on previous reports [15], EGFR expression was classified into 4 categories (0-3+) (\u003cstrong\u003eFig. 3A\u003c/strong\u003e), with 0 or 1+ defined as EGFR-low and 2+ or 3+ as EGFR-high. In the entire cohort, 34% (13/38) of the tumors were classified as EGFR-low and 66% (25/38) were EGFR-high (\u003cstrong\u003eFig. 3A\u003c/strong\u003e). As previously reported [21], the majority of the \u003cem\u003eEGFR\u003c/em\u003e-mutant tumors highly expressed EGFR (85%, 11/13). However, more than half of \u003cem\u003eEGFR\u003c/em\u003e-WT tumors also expressed high levels of EGFR (59%, 13/22) (\u003cstrong\u003eFig. 3B\u003c/strong\u003e). There was a trend toward an association between \u003cem\u003eEGFR\u003c/em\u003e mutation and EGFR expression, although not statistically significant (Fisher\u0026rsquo;s exact test: P = 0.150).\u003c/p\u003e\n\u003cp\u003eWe next compared the immune microenvironment and immune cell phenotypes according to EGFR expression. As a result, infiltration of CD8⁺ T cells and DCs was significantly reduced in EGFR-high tumors (\u003cstrong\u003eFig. S4B\u003c/strong\u003e and\u003cstrong\u003e\u0026nbsp;C\u003c/strong\u003e). In contrast, the ratio of CD8⁺ T cells to eTregs, as well as the activation status of CD8⁺ T cells, DCs, and eTregs, was comparable irrespective of EGFR expression levels (\u003cstrong\u003eFig. S4D-I\u003c/strong\u003e). We further analyzed the relationship between EGFR expression and amivantamab-mediated immune activation. Amivantamab increased PD-1 expression and cytokine production in CD8⁺ TILs from EGFR-high tumors, whereas these effects were limited in EGFR-low tumors (\u003cstrong\u003eFig. 3C-E\u003c/strong\u003e). Similarly, tumor-infiltrating DC maturation was significantly promoted by amivantamab in EGFR-high tumors but remained limited in EGFR-low tumors (\u003cstrong\u003eFig. S4J\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;K\u003c/strong\u003e). In contrast, tumor-infiltrating eTregs were unaffected by amivantamab regardless of EGFR expression (\u003cstrong\u003eFig. S4L\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;M\u003c/strong\u003e). These findings suggest that high EGFR expression is associated with an immunosuppressive TME, but amivantamab can promote immune activation in such a TME.\u003c/p\u003e\n\u003cp\u003eSimilar to EGFR, we also examined MET, another target of amivantamab, by classifying its expression in tumors into four categories (0-3+) based on the previous report [16]. In the entire cohort, MET was highly expressed (2+ or 3+) in half of the cases (19/38) (\u003cstrong\u003eFig. 3F\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eFig. S5A\u003c/strong\u003e). The comparison of immune features between MET-high and MET-low tumors revealed no significant differences in CD8⁺ T-cell or DC infiltration (\u003cstrong\u003eFig. S5B\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;C\u003c/strong\u003e) and in the CD8⁺ T-cell to eTreg ratio (\u003cstrong\u003eFig. S5D\u003c/strong\u003e). In addition, the activation status of CD8⁺ T cells, DCs, and eTregs were also comparable between the two groups (\u003cstrong\u003eFig. S5E-I\u003c/strong\u003e). However, in MET-high tumors, amivantamab increased PD-1 expression and cytokine production in CD8⁺ TILs (\u003cstrong\u003eFig. 3G-I\u003c/strong\u003e), whereas such effects were limited in MET-low tumors (\u003cstrong\u003eFig. 4B-D\u003c/strong\u003e). Tumor-infiltrating DC maturation tended to be promoted by amivantamab regardless of MET expression (\u003cstrong\u003eFig. S5J\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;K\u003c/strong\u003e), and tumor-infiltrating eTregs remained unchanged in either group (\u003cstrong\u003eFig. S5L\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eM\u003c/strong\u003e). Taken together, these findings suggest that, similar to EGFR, MET expression can be associated with immune-activating effects of amivantamab.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmivantamab promotes immune responses in EGFR or MET high tumors, even with WT \u003cem\u003eEGFR\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the individual importance of EGFR and MET expression in amivantamab-mediated immune activation, we next analyzed their combined association. The cohort was divided into four groups (EGFR/MET, Low/Low; Low/High; High/Low; High/High) based on EGFR and MET expression, with a significant association between EGFR and MET expression levels (Fisher\u0026rsquo;s exact test: P = 0.038) (\u003cstrong\u003eFig. 4A\u003c/strong\u003e). Although EGFR and MET expression were positively correlated, 32% (12/38) of the tumors exhibited high expression of either EGFR or MET (\u003cstrong\u003eFig. 4A\u003c/strong\u003e). We subsequently divided the tumors into two groups, low expression of both EGFR and MET (EGFR/MET Low/Low) and those with high EGFR or MET expression (the others),\u0026nbsp;and compared their immune microenvironment and immune cell phenotypes. The results revealed that tumors with high EGFR or MET expression exhibited a trend toward lower CD8⁺ T cell infiltration (\u003cstrong\u003eFig. S6A\u003c/strong\u003e) and significantly reduced DC infiltration (\u003cstrong\u003eFig. S6B\u003c/strong\u003e). In contrast, the CD8⁺ T cell to eTreg ratio and the activation status of immune cells were comparable between the two groups (\u003cstrong\u003eFig. S6C\u0026ndash;H\u003c/strong\u003e). We also examined the association of EGFR and MET expression with the immune-activating effects of amivantamab. In tumors with high EGFR or MET expression, amivantamab increased PD-1 expression and cytokine production in CD8⁺ TILs. In contrast, in tumors with low expression of both EGFR and MET, amivantamab-induced CD8⁺ TIL activation was limited (\u003cstrong\u003eFig. 4B-D\u003c/strong\u003e). Similarly, tumor-infiltrating DC maturation was promoted by amivantamab in the group with high EGFR or MET expression, but not in the group with both low (\u003cstrong\u003eFig. S6I\u003c/strong\u003e and\u003cstrong\u003e\u0026nbsp;J\u003c/strong\u003e). Tumor-infiltrating eTregs were unaffected by amivantamab, irrespective of EGFR and MET expression levels \u003cstrong\u003e(Fig. S6K\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;L\u003c/strong\u003e). Collectively, these findings indicate that high expression of EGFR or MET is important for amivantamab-induced immune responses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe further hypothesized that EGFR and MET expression might influence the efficacy of amivantamab even in \u003cem\u003eEGFR\u003c/em\u003e-WT tumors.\u003cem\u003e\u0026nbsp;EGFR\u003c/em\u003e-mutant and \u003cem\u003eEGFR\u003c/em\u003e-WT tumors were each stratified into four groups (EGFR/MET, Low/Low; Low/High; High/Low; High/High) based on EGFR and MET expression, and 72.7% (16/22) exhibited high EGFR or MET expression in \u003cem\u003eEGFR\u003c/em\u003e-WT tumors (\u003cstrong\u003eFig. 5A\u003c/strong\u003e). We classified \u003cem\u003eEGFR\u003c/em\u003e-mutant and \u003cem\u003eEGFR\u003c/em\u003e-WT tumors into two groups based on EGFR and MET expression; EGFR/MET Low/Low and the others, showing no correlation between \u003cem\u003eEGFR\u003c/em\u003e mutation status and the expression of EGFR or MET (Fisher\u0026rsquo;s exact test: P = 0.680). We examined the association of EGFR and MET expression with the immune-activating effects of amivantamab in \u003cem\u003eEGFR\u003c/em\u003e-WT tumors. In EGFR or MET\u0026ndash;high tumors, amivantamab significantly increased PD-1 expression and cytokine production in CD8⁺ TILs (\u003cstrong\u003eFig. 5B-D\u003c/strong\u003e). In contrast, amivantamab induce significant changes in tumor-infiltrating DCs or eTregs in neither group (\u003cstrong\u003eFig. S7A-D\u003c/strong\u003e). These findings suggest that even \u003cem\u003eEGFR\u003c/em\u003e-WT tumors, the presence of high expression of EGFR or MET can allow amivantamab to promote immune responses.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eEGFR\u003c/em\u003e-mutant NSCLCs are characterized by the immunosuppressive TME and poor responses to immune checkpoint inhibitors (ICIs) [22, 23]. Although preclinical studies have suggested that amivantamab may activate antitumor immunity, direct evidence in human clinical specimens has been lacking [12, 13]. In this study, we provide the first direct evidence from \u003cem\u003eex vivo\u003c/em\u003e fresh TIL assays using human NSCLC clinical specimens that amivantamab can activate antitumor immune responses. Notably, these immune-activating effects were more strongly associated with high expression of EGFR or MET protein rather than \u003cem\u003eEGFR\u003c/em\u003e mutation status, suggesting that EGFR/MET expression levels may serve as biomarkers for amivantamab-induced antitumor immunity. Given the limited effectiveness of ICIs in NSCLC, particularly in \u003cem\u003eEGFR\u003c/em\u003e-mutant one, our findings highlight the potential of amivantamab, especially in combination with immunotherapy, as a promising strategy to overcome this major clinical challenge.\u0026nbsp;However, due to the unpredictable nature of cancer biology and human therapeutics, clinical studies would be required to determine the effectiveness of such a strategy.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEGFR\u003c/em\u003e mutations induce ligand-independent phosphorylation of RTK and activate EGFR signaling pathways, which contribute not only to cancer cell proliferation but also to the establishment of the immunosuppressive TME. Specifically, EGFR activation regulates chemokine production, leading to reduced infiltration of cytotoxic CD8⁺ T cells while promoting the recruitment of Tregs [5, 6]. This pathway also increases tumor-promoting cytokines and EGFR ligands, suppressing CD8⁺ T-cell function and DC maturation while enhancing the immunosuppressive activity of Tregs and tumor-associated macrophages [5, 6]. Consistently, in our cohort, \u003cem\u003eEGFR\u003c/em\u003e-mutant tumors exhibited reduced infiltration of CD8⁺ T cells and DCs, reinforcing the concept of the immunosuppressive TME in these tumors. Blockade of the EGFR pathway therefore could potentially\u0026nbsp;counteract this immunosuppressive TME. Amivantamab, a bispecific antibody targeting both EGFR and MET, has been shown to activate antitumor immune responses \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand in mouse models, and its clinical activity includes durable responses with a tail plateau pattern reminiscent of immunotherapy\u0026nbsp;[11-13, 24]. In our study, we provide the first demonstration in fresh human TILs from NSCLC that amivantamab activates antitumor immunity, particularly through T-cell-mediated responses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough amivantamab is currently approved only for \u003cem\u003eEGFR\u003c/em\u003e-mutant NSCLC, previous reports have suggested efficacy in \u003cem\u003eEGFR\u003c/em\u003e-WT tumors [25]. Consistently, we observed that amivantamab promoted TIL activation in tumors with high EGFR or MET expression, regardless of mutation status. This finding is aligned with its pharmacological mechanism and underscores the potential of EGFR/MET expression as biomarkers for amivantamab-induced antitumor immunity. Indeed, several ongoing clinical trials are already including EGFR-WT cohorts [26-28], and our results support such trial designs. Importantly, these findings suggest that tumors with high EGFR/MET expression may benefit from amivantamab even without \u003cem\u003eEGFR\u003c/em\u003e mutations.\u003c/p\u003e\n\u003cp\u003eThe mechanisms underlying amivantamab-induced immune activation remain incompletely understood. Because EGFR signaling is involved in immune suppression, its blockade could contribute to immune activation [29, 30]. In addition, activation of T-cell immunity through innate immune stimulation mediated by ADCC and ADCP activity is considered another process [13, 31]. We also observed DC maturation following amivantamab treatment, although its correlation with EGFR or MET expression was less clear than that observed for T cells. Furthermore, because tumor-infiltrating natural killer (NK) cells are generally small, their potential contribution was not evaluated in our cohort, highlighting the need for further investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMouse models frequently fail to predict human clinical outcomes, and peripheral blood\u0026ndash;based assays cannot recapitulate the chronic antigen exposure and suppressive cues that shape TIL function in the TME. In contrast, our fresh TIL assay preserves the viability of tumor and immune cells, enabling real-time assessment of therapeutic antibody activity within a physiologically relevant context. Unlike assays relying on TIL expansion, organoid generation, or cryopreservation, our approach is very simple, minimizing artifacts and reflecting \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003econditions more faithfully [32]. Voabil et al. also developed a similar tumor fragment platform, showing the advantages [33]. This approach not only provides a more faithful reflection of the \u003cem\u003ein vivo\u003c/em\u003e TME but also holds broader implications for drug development. Moreover, with further\u0026nbsp;development, this approach could evolve into a patient-specific\u0026nbsp;tool, enabling clinicians to\u0026nbsp;project\u0026nbsp;therapeutic responses\u0026nbsp;to treatment.\u003c/p\u003e\n\u003cp\u003eIn summary, this study provides the first ex vivo human evidence that amivantamab activates TILs together with the unique strengths of our fresh TIL assay. These effects extended beyond \u003cem\u003eEGFR\u003c/em\u003e-mutant tumors to \u003cem\u003eEGFR\u003c/em\u003e-WT tumors with high EGFR or MET expression, underscoring the potential of EGFR/MET expression as biomarkers for amivantamab-induced antitumor immunity. Further basic and translational research is warranted to validate these findings and guide future clinical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Janssen Pharmaceutical K.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS. Toyooka received research grants from TAIHO PHARMA, Eli Lilly, Chugai Pharmaceutical and Astellas Pharma, and honoraria from TAIHO PHARMA, Eli Lilly, Chugai Pharmaceutical, GUARDANT, AstraZeneca, Illumina, MERCK, CSL Behring, Nippon Kayaku, Daiichi-Sankyo, Ono Pharmaceutical, Medtronic, Ziosoft, NOVARTIS, Sysmex and Riken Genesis outside of this study. Y. Togashi received a research grant from Janssen Pharmaceutical K.K. for this study. Y. Togashi received research grants from AstraZeneca, TAIHO PHARMA, Takeda, Chugai Pharmaceutical, Daiichi-Sankyo, and KORTUC, and honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, AstraZeneca, Chugai Pharmaceutical, Eisai and MSD outside of this study. The other authors declare that they have no research support relevant to financial competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRY: Data curation, formal analysis, investigation, methodology, writing \u0026ndash; original draft. FM: Data curation, formal analysis, investigation, methodology. KY: Formal analysis and investigation. JN: Conceptualization, data curation, formal analysis, methodology. HW: Data curation, formal analysis, investigation. YU: Methodology. K. Suzawa: Investigation. K. Shien: Investigation. ST: Supervision. TI: Data curation, formal analysis, investigation, visualization, methodology, writing \u0026ndash; original draft, writing \u0026ndash; review and editing. YT: Conceptualization, data curation, funding acquisition, methodology, writing \u0026ndash; original draft, project administration, writing-review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information. Additional data are accessible from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Institutional Review Board of Okayama University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients provided written informed consent.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Nat Med 27:1250-1261. https://doi.org/10.1038/s41591-021-01398-3\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Non-small cell lung cancer, amivantamab, antitumor immunity, EGFR, MET","lastPublishedDoi":"10.21203/rs.3.rs-8141506/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8141506/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003eEpidermal growth factor receptor\u003c/em\u003e (\u003cem\u003eEGFR\u003c/em\u003e) mutations represent one of the most frequent oncogenic driver in non-small cell lung cancer (NSCLC). Amivantamab, a bispecific antibody targeting EGFR and MNNG HOS Transforming (MET), has demonstrated clinical benefit in \u003cem\u003eEGFR\u003c/em\u003e-mutant NSCLC through dual blockade, but its immunological role in human clinical specimens, especially tumor-infiltrating lymphocytes (TILs), has not been directly evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe analyzed surgically resected tumor samples from 40 patients with NSCLC to investigate immune responses and their associations with EGFR and MET expression. TILs were characterized by flow cytometry (FCM) and immunohistochemistry (IHC). To assess the immunomodulatory potential of amivantamab, fresh tumor digests containing live tumor cells and TILs were cultured \u003cem\u003eex vivo\u003c/em\u003e with CD3 and CD28 stimulation in the absence or presence of amivantamab, followed by FCM. EGFR and MET expression were also evaluated by IHC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e\u003cem\u003eEGFR\u003c/em\u003e mutations and high EGFR protein expression were associated with a trend toward reduced CD8⁺ T-cell and dendritic cell (DC) infiltration. In \u003cem\u003eex vivo\u003c/em\u003e TIL assays, exposure to amivantamab significantly activated CD8⁺ T cells, such as programmed cell death-1 expression and cytokine production, and promoted DC maturation. These effects were most pronounced in tumors with high EGFR or MET protein expression rather than \u003cem\u003eEGFR\u003c/em\u003e mutations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis study provides the first direct evidence from \u003cem\u003eex vivo\u003c/em\u003e fresh TIL assays using human NSCLC clinical specimens that amivantamab can activate immune responses. EGFR and MET expression may serve as potential biomarkers for amivantamab-induced immune responses.\u003c/p\u003e","manuscriptTitle":"Immunological effects of amivantamab in EGFR or MET-expressing non-small cell lung cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 07:48:01","doi":"10.21203/rs.3.rs-8141506/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-22T04:04:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T09:37:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-06T19:24:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284188500811517618020083874110069853375","date":"2025-11-26T19:17:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118780872707151431660257563649418078555","date":"2025-11-26T09:48:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223179193773905672890523009898858803876","date":"2025-11-26T01:07:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-24T00:57:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-19T05:38:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-19T05:36:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2025-11-18T05:58:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"23007b5a-5b78-4a33-b245-70e7f668dbe9","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:18:58+00:00","versionOfRecord":{"articleIdentity":"rs-8141506","link":"https://doi.org/10.1007/s00262-026-04369-0","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2026-03-24 16:11:21","publishedOnDateReadable":"March 24th, 2026"},"versionCreatedAt":"2025-12-01 07:48:01","video":"","vorDoi":"10.1007/s00262-026-04369-0","vorDoiUrl":"https://doi.org/10.1007/s00262-026-04369-0","workflowStages":[]},"version":"v1","identity":"rs-8141506","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8141506","identity":"rs-8141506","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}