Abstract
Background
SAR443216 is an engineered human trispecific antibody that targets human epidermal growth factor receptor 2 (HER2)-positive (HER2+) cancer cells and activates T cells via co-engagement of cluster of differentiation (CD)3 and CD28. This first-in-human, dose-escalation study evaluated the safety, efficacy, pharmacokinetics (PK) and pharmacodynamics of SAR443216 in participants with relapsed/refractory (R/R) HER2-expressing solid tumors.
Methods
In this multicenter, open-label, non-randomized Phase 1 study (NCT05013554), SAR443216 was administered intravenously at dose levels (DLs) of 18–900 µg. Dose escalation occurred within participants using intraparticipant lead-in dosing (2-week and 3-week lead-in cohorts). The primary objective was to determine the maximum tolerated dose (MTD); secondary objectives included PK, immunogenicity, and preliminary clinical activity.
Results
40 participants (n≥3 at each DL) were treated with SAR443216. The median treatment duration was ~8 weeks in both 2-week and 3-week lead-in cohorts. Nearly all participants (97.5%) had at least one treatment-emergent adverse event (TEAE), of which 45% were grade ≥3. Most frequent TEAEs were cytokine release syndrome (CRS, 50%), fever (35%), alanine aminotransferase elevation (32.5%), aspartate aminotransferase elevation (27.5%), and infusion-related reactions (IRRs, 27.5%). No severe CRS, IRRs, fever, or pulmonary and cardiac toxicities were observed. Disease control rates were 34.5% in the 2-week and 36.4% in the 3-week lead-in cohorts. Average duration of disease stabilization was 10.48 weeks. Median follow-up time was 3.43 weeks. No objective responses were observed. The MTD was not reached. Dose-dependent PK showed overall consistent PK profiles across DLs. SAR443216 induced serum proinflammatory cytokines and increased multiple T-cell activation markers in peripheral blood mononuclear cells, indicating T-cell activation and target engagement. However, no clear trend in T-cell abundance or activation was observed among tumor-infiltrating T cells or other immune cells.
Conclusion
These findings indicate that SAR443216 treatment is feasible and well tolerated in participants with R/R HER2+solid tumors. Further evaluation is warranted to fully characterize the efficacy and safety of SAR443216.
Trial registration number
Keywords: Solid tumor, T cell, Immunotherapy, co-stimulatory molecules, Tri-specific antibody, HER2
WHAT IS ALREADY KNOWN ON THIS TOPIC
Despite therapeutic advancements, human epidermal growth factor receptor 2 (HER2)+solid tumors often show disease progression due to primary or acquired resistance.
Co-stimulatory modulation of immune cells has strong potential as antitumor therapy.
WHAT THIS STUDY ADDS
SAR443216 is a human IgG4-based trispecific antibody engineered to simultaneously target HER2 on tumor cells and activate T cells via co-engagement of cluster of differentiation (CD)3 and CD28.
In this first-in-human study, SAR443216 demonstrated a manageable safety profile following intravenous administration. Dual T-cell stimulation via CD3 and CD28 did not increase the severity of adverse events related to cytokine release syndrome or infusion-related reaction compared with T-cell stimulation via CD3 alone. Compared with other HER2-targeting treatments, no serious pulmonary and cardiac toxicities were observed.
Dose-dependent pharmacokinetics (PK) with overall consistent profiles was observed. Post-dose increases in serum interleukins, along with peripheral T-cell activation indicated T-cell activation and target engagement.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
SAR443216 treatment is feasible and well tolerated, with manageable toxicities in participants with relapsed/refractory HER2+solid tumors.
Introduction
Human epidermal growth factor receptor 2 (HER2), a member of the human epidermal growth factor receptor tyrosine kinase family, plays an important role in cancer pathogenesis by acting on multiple signaling pathways.1 2 It is overexpressed in various types of cancers, including breast, gastric, non-small cell lung, biliary, bladder, colorectal, and other malignancies.3–9 Clinically, HER2 is a validated tumor target, which is evidenced by approved HER2-targeting treatments such as monoclonal antibodies (eg, trastuzumab and pertuzumab), small-molecule tyrosine kinase inhibitors (eg, lapatinib, neratinib, and tucatinib), and antibody drug-conjugates (eg, trastuzumab emtansine and trastuzumab deruxtecan) directed against HER2 overexpressing tumors.10–12 Participants with HER2-positive (HER2+) solid tumors often experience disease progression due to primary or acquired resistance after HER2 targeted treatments.13 14 Despite various advances in HER2-targeting treatments, a significant unmet need exists for novel approaches that can improve participant outcomes by engaging durable antitumor immune responses.
T-cell engagers (TCEs) represent an advancement in the treatment of B cell and plasma cell malignancies and are emerging as a promising therapeutic approach for solid tumors.15 TCEs activate T cells primarily by binding the T-cell receptor (TCR)/cluster of differentiation (CD)3 complex in a major histocompatibility complex-independent manner,16 and some TCEs can also incorporate costimulatory domains, for example, CD28, to enhance T-cell proliferation, differentiation, and survival.17 18 CD28 is one of the most well-known co-stimulatory molecules; its interaction with B7 family ligands CD80 (B7-1) and CD86 (B7-2), along with TCR-mediated signaling, is required for complete activation of T cells.19 20
SAR443216 is an engineered human immunoglobulin 4 (IgG4)-based trispecific antibody that contains three distinct binding sites: one targeting HER2-expressing cancer cells, one CD3 binding site for T-cell activation, and one CD28 binding site for providing co-stimulatory signals to T cells (figure 1). SAR443216 primarily acts against cancer cells through co-engagement of CD3 and CD28 on T cells and HER2 on tumor cells, forming an immunological synapse between HER2-expressing tumor cells and T cells; this leads to T-cell activation and subsequent cytolytic activity against tumor cells.
Figure 1. Structure of SAR443216 FALA, F234A and L235A mutations. IgG4, immunoglobulin G subclass 4; HER2, human epidermal growth factor receptor 2.
Preclinical data demonstrate that both CD3 and CD28 contribute to significant cytokine production, T-cell activation, and proliferation. Furthermore, CD28 signaling enhances T-cell survival by inhibiting programmed cell death.21 22 In preclinical studies, SAR443216 demonstrated potent activity by activating CD4 and CD8 T cells, inducing T-cell proliferation, secretion of cytokines and granzyme B, and exhibiting strong T cell-dependent cellular cytotoxicity in HER2-expressing cancer cell lines, across both HER2-high and HER2-low models.21 23
This first-in-human, dose-escalation study with SAR443216 aimed to evaluate its safety, preliminary efficacy, pharmacokinetics (PK), and biomarker data in participants with relapsed/refractory (R/R) HER2-expressing solid tumors.
Methods
Study design and treatment
In this multicenter, open-label, non-randomized Phase 1/1b single-agent study, participants with solid tumors were administered SAR443216 intravenously or subcutaneously (SC) (NCT05013554). The study comprised two parts: dose escalation and dose expansion (figure 2). Herein, we report findings from the intravenously and SC cohorts of the dose-escalation part.
Figure 2. Study design. D, day; DL, dose level; DoR, duration of response; ECOG PS, Eastern Cooperative Oncology Group performance status; HER2, human epidermal growth factor receptor 2; ILD, interstitial lung disease; IHC, immunohistochemistry; RECIST, Response Evaluation Criteria in Solid Tumors.
The participants received SAR443216 intravenous infusions on day (D)1 and D4 during Cycle (C)1 (each cycle consists of 4 weeks), followed by weekly administrations starting at week 2 of C1 until the end of treatment (EOT). Response assessment occurred every two cycles (ie, 8 weeks) ±7 days after the date of the first SAR443216 administration. A premedication regimen comprising dexamethasone (20 mg intravenous, which could be switched to oral administration from C2 onward) and montelukast (10 mg oral administration) was implemented to manage potential cytokine release syndrome (CRS) or infusion-related reactions (IRRs). All participants were hospitalized for the first two infusions and in the absence of severe IRRs or CRS, treatment was continued in an outpatient setting. A total infusion duration of approximately 4 hours was recommended during C1 with an option to reduce the duration in subsequent cycles. To reduce the risk of severe CRS, intraparticipant dose escalation (lead-in doses) was implemented in C1. A 2-week lead-in period (intravenous-administration cohort) and an extended 3-week lead-in period (intravenous-ext cohort) were investigated. After the lead-in period, participants continued with weekly administration of the target dose level (DL) for which they were enrolled for the remainder of treatment, unless safety adjustments were needed (online supplementary table S1). Dose-limiting toxicities (DLTs) were observed until 1 week after the first two consecutive target dose administrations (defined as DLT observation period). In the SC cohort, participants received SAR443216 SC on D1 and D4 of C1, followed by weekly administrations after week 1 of C1 until EOT (Protocol available at online supplemental file 2).
The study was conducted in compliance with the principles of international ethics guidelines, including the Declaration of Helsinki; the International Council for Harmonization guidelines for Good Clinical Practice; and all applicable laws, rules, and regulations. The study was approved by the Institutional Review Board at each site, and written informed consent was obtained from all study participants prior to enrollment.
Patient population
Approximately 54 DLT-evaluable participants were planned for the dose-escalation part of the study, with cohorts of ≥4 participants to ensure ≥3 DLT-evaluable participants. The dose-escalation part included participants aged ≥18 years and weighing 45–150 kg, who had advanced R/R solid tumors histologically or cytologically confirmed as metastatic, exhausted all standard-of-care therapies, HER2 expression in tumor tissue (1+, 2+, or 3+) as assessed by immunohistochemistry (IHC) and/or activating mutations detected in tumor or blood, measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1, and an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0–1. Additional inclusion and exclusion criteria are detailed in online supplemental table S2.
Study objectives
The primary objectives were to determine the maximum tolerated dose (MTD) or maximum administered dose based on the incidence of DLTs and to evaluate the safety of SAR443216 during the dose-escalation part of the study in participants with HER2+solid tumors. Secondary objectives included assessment of the PK profile, immunogenicity, and preliminary clinical activity of SAR443216. Exploratory objectives included monitoring of immune cell dynamics; modulation of immune biomarkers in tumor microenvironment (TME); and assessment of cytokine kinetics in blood, HER2 expression in tumors, and biomarker-response associations (online supplemental table S3).
PK analysis
Plasma samples were collected from the participants at various time points (pre-dose, end of infusion, and after the end of infusion) during each cycle, with additional sampling time points following C1D1 and C1D15 dosing for 48 hours after infusion. SAR443216 concentrations in the samples were measured using a bi-active (CD3 for detection/CD28 for capture) assay via a validated Meso Scale Discovery electrochemiluminescence immunoassay, with no or limited interference from soluble HER2. The lower and upper limit of quantitation for SAR443216 were 1 ng/mL and 1,000 ng/mL, respectively.
Immunogenicity assessments
A standard tiered approach (screening, confirmation, and titration) via a fully validated assay was used to assess the immunogenicity of SAR443216. Anti-SAR443216 antibodies in plasma samples were quantified using a PandA-based immunoassay combined with electrochemiluminescence detection.
Biomarker analysis
Blood samples were collected from participants at specified time intervals. Serum cytokine levels were measured (PPD BioA Richmond, VA, 23230, USA) and the kinetics of various immune cell populations were assessed via flow cytometry using frozen peripheral blood mononuclear cells isolated from peripheral whole blood (Sanofi, Montpellier, France). HER2 expression in tumor tissue along with changes in infiltrating immune cells and immune biomarkers (eg, programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and antigen Ki67) was evaluated at baseline and at C2D15 (approximately 42 days after the first SAR443216 administration) using multiplex immunofluorescence (Navigate Biopharma, Carlsbad, California, USA). Three validated biomarker panels (CD8/GZMB/PD-1/PD-L1/TIM-3/CK, CD68/CD163/HLA-DR/IDO1/PD-L1/CK-PanMel, and CD3/CD4/CD8/FoxP3/Ki67/CK) were used to assess the TME. Additionally, an HER2/CK assay was used to assess HER2 levels in the tumor sections. Multiplex fluorescent IHC staining was performed on specimens fixed in 10% neutral buffered formalin. Fields were imaged per sample, based on the pathologist review of the H&E-stained samples. Spectrally unmixed images were analyzed using proprietary software (AQUAnalysis, Navigate Biopharma), which is designed to quantify the expression of protein biomarkers within a specified subcellular compartment (typically the nucleus or cytoplasm), based on pixel intensity. The software generated a quantitative AQUA score for biomarker expression within the expected cellular compartment for each tissue. It was also used to determine frequency (%) of biomarker-positive cells of interest by dividing the total area (in pixels) positive for the biomarker of interest by the cellular areas co-localized with 4′,6-diamidino-2-phenylindole (DAPI) positivity, which is used to identify all nucleated cells.
Statistical analysis
All participants who received ≥1 dose of SAR443216 were included in the safety analysis. The DLT-evaluable population included all participants who completed the DLT observation period and received ≥75% of the planned doses. DLTs were summarized by DL with worst-grade events listed, using the National Cancer Institute Common Terminology Criteria for Adverse Events V.5.0. Adverse events (AEs) were coded according to the Medical Dictionary for Regulatory Activities (MedDRA 26.1). Efficacy was evaluated by the investigator according to RECIST V.1.1, based on the safety population. The disease control rate (DCR) was defined as the proportion of participants achieving complete response (CR), partial response (PR), or stable disease (SD) per RECIST V.1.1. The PK profile was assessed in the PK population, which included all participants from the safety population with at least one measurable SAR443216 concentration after the first dose. PK parameters were summarized using descriptive statistics. Immunogenicity analyses were performed in the antidrug antibody (ADA)-evaluable population, which included SAR443216-treated participants with at least one ADA result (positive, negative, or inconclusive).
Results
Patient disposition and baseline characteristics
This study was conducted at 10 centers in the USA, France, Spain, Taiwan, and the Republic of Korea between August 16, 2021, and January 15, 2024. A total of 41 participants were enrolled across the intravenous cohorts, comprising the 2-week (n=29) and 3-week (n=11) lead-in cohorts. In the 2-week-lead-in cohort, 29 participants were treated, of whom 25 were DLT-evaluable. Four received 18 µg (DL1), three received 60 µg (DL2), nine received 180 µg (DL3), four received 240 µg (DL3b), six received 360 µg (DL4), and three received 720 µg (DL5). In the 3-week lead-in cohort, 12 participants were enrolled, of whom 1 participant discontinued due to exclusion criteria; the remaining 11 participants were treated, with 8 being DLT-evaluable. Among these participants, four received a target dose of 480 µg, four received 720 µg, and three participants received 900 µg (online supplemental figure S1).
The median age of the treated population (N=40) was 56 years (range: 46–63), and the majority of participants were female (62.5%). In terms of racial distribution, most participants were white (55%) or Asian (40%). The most frequently reported primary tumor types were breast cancer (17.5%), stomach cancer (12.5%), and lung, rectal, or ovarian cancers (7.5% each). 21 participants (52.5%) had an ECOG PS of 1 (table 1), and 52.5% of all participants had received prior HER2-targeting treatment (online supplemental table S4). The proportion of participants who had received ≥5 prior treatments was higher in the 2-week lead-in cohort than in the 3-week lead-in cohort (44.8% vs 9.1%) (table 1). Further details by DLs are provided in online supplemental table S5. The median duration of SAR443216 treatment was 8.1 (3–41) weeks in the 2-week lead-in cohort and 7.93–17 weeks in the 3-week lead-in cohort.
Table 1. Patient demographics and disease characteristics.
| 2-week lead-in (IV administration cohort) (n=29) | 3-week lead-in (IV-ext cohort) (n=11) | Total (N=40) | |
|---|---|---|---|
| Age (years), median (range) | 52 (46–62) | 60 (53–67) | 56 (46–63) |
| Weight (kg) mean (SD) | 67.71 (16.79) | 68.40 (18.70) | 67.90 (17.09) |
| Sex, n (%) | |||
| Male | 9 (31.0) | 6 (54.5) | 15 (37.5) |
| Female | 20 (69.0) | 5 (45.5) | 25 (62.5) |
| Race n (%) | |||
| White | 17 (58.6) | 5 (45.5) | 22 (55.0) |
| Black or African American | 2 (6.9) | 0 | 2 (5.0) |
| Asian | 10 (34.5) | 6 (54.5) | 16 (40.0) |
| ECOG PS, n (%) | |||
| 0 | 15 (51.7) | 4 (36.4) | 19 (47.5) |
| 1 | 14 (48.3) | 7 (63.6) | 21 (52.5) |
| No. of prior regimens, n (%) | |||
| 1 | 4 (13.8) | 0 | 4 (10.0) |
| 2 | 4 (13.8) | 2 (18.2) | 6 (15.0) |
| 3 | 6 (20.7) | 4 (36.4) | 10 (25.0) |
| 4 | 2 (6.9) | 4 (36.4) | 6 (15.0) |
| ≥5 | 13 (44.8) | 1 (9.1) | 14 (35.0) |
| Primary tumor location, n (%) | |||
| Breast | 6 (20.7) | 1 (9.1) | 7 (17.5) |
| Stomach | 4 (13.8) | 1 (9.1) | 5 (12.5) |
| Lung | 2 (6.9) | 1 (9.1) | 3 (7.5) |
| Ovary | 3 (10.3) | 0 | 3 (7.5) |
| Rectum | 2 (6.9) | 1 (9.1) | 3 (7.5) |
| Colon | 2 (6.9) | 0 | 2 (5.0) |
| Gallbladder | 2 (6.9) | 0 | 2 (5.0) |
| Others | 8 (27.6) | 7 (54.5) | 15 (37.5) |
| Tumor histology – IHC, n (%) | |||
| n* | 22 | 9 | 31 |
| IHC3+ | 12 (41.4) | 5 (45.5) | 17 (42.5) |
| IHC2+ | 7 (24.1) | 3 (27.3) | 10 (25.0) |
| IHC1+ | 3 (10.3) | 1 (9.1) | 4 (10.0) |
| HER2 status**, n (%) | |||
| Abnormality/alteration detected by NGS | 15 (51.7) | 7 (63.6) | 22 (55.0) |
| Mutation | 15 (51.7) | 4 (36.4) | 19 (47.5) |
| Amplification | 4 (13.8) | 4 (36.4) | 8 (20.0) |
| Translocation | 1 (3.4) | 0 | 1 (2.5) |
Nine participants who did not have IHC results were included in the study with HER2 mutation/aberration.
DL, dose level; ECOG PS, Eastern Cooperative Oncology Group performance status; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; IV, intravenous; NGS, Next-Generation Sequencing.
Safety
Almost all participants (98%) across both 2-week lead-in and 3-week lead-in cohorts experienced ≥1 TEAEs of any grade. Among these participants, 35 (87.5%) reported SAR443216-related AEs, 18 (45%) experienced grade ≥3 TEAEs and 16 (40%) had serious TEAEs. The most frequently reported TEAEs of any grade were CRS in 20 (50%), fever in 14 (35%), increased alanine aminotransferase (ALT) levels in 13 (32.5%), increased aspartate aminotransferase (AST) levels in 11 (27.5%) and IRRs in 11 (27.5%) participants. No severe or life-threatening (≥3 grade) CRS, IRRs, or fever were observed (table 2). Further details by DLs are provided in online supplemental table S6.
Table 2. Overview of safety profile.
| Incidence, n (%) | 2-week lead-in (IV administration cohort) (n=29) | 3-week lead-in (IV-ext cohort) (n=11) | Total (N=40) | |
|---|---|---|---|---|
| DLTs | 3 (12.0) | 0 | 3 (12.0) | |
| Any TEAE | 28 (96.6) | 11 (100) | 39 (97.5) | |
| Grade ≥3 TEAE | 15 (51.7) | 3 (27.3) | 18 (45.0) | |
| Most common TEAEs (≥10% population) | ||||
| CRS | Any grade | 11 (37.9) | 9 (81.8) | 20 (50.0) |
| Grade ≥3 | 0 | 0 | 0 | |
| Fever | Any grade | 9 (31.0) | 5 (45.5) | 14 (35.0) |
| Grade ≥3 | 0 | 0 | 0 | |
| Increased ALT levels | Any grade | 10 (34.5) | 3 (27.3) | 13 (32.5) |
| Grade ≥3 | 5 (17.2) | 1 (9.1) | 6 (15.0) | |
| Infusion-related reaction | Any grade | 7 (24.1) | 4 (36.4) | 11 (27.5) |
| Grade ≥3 | 0 | 0 | 0 | |
| Increased AST levels | Any grade | 8 (27.6) | 3 (27.3) | 11 (27.5) |
| Grade ≥3 | 4 (13.8) | 1 (9.1) | 5 (12.5) | |
| Fatigue | Any grade | 7 (24.1) | 2 (18.2) | 9 (22.5) |
| Grade ≥3 | 1 (3.4) | 0 | 1 (2.5) | |
| Dyspnea | Any grade | 6 (20.7) | 2 (18.2) | 8 (20.0) |
| Grade ≥3 | 0 | 0 | 0 | |
| Headache | Any grade | 6 (20.7) | 1 (9.1) | 7 (17.5) |
| Grade ≥3 | 0 | 0 | 0 | |
| Nausea | Any grade | 6 (20.7) | 1 (9.1) | 7 (17.5) |
| Grade ≥3 | 0 | 0 | 0 | |
| Anemia | Any grade | 7 (24.1) | 0 | 7 (17.5) |
| Grade ≥3 | 1 (3.4) | 0 | 1 (2.5) | |
| Decreased appetite | Any grade | 5 (17.2) | 1 (9.1) | 6 (15.0) |
| Grade ≥3 | 0 | 0 | 0 | |
| Asthenia | Any grade | 5 (17.2) | 1 (9.1) | 6 (15.0) |
| Grade ≥3 | 1 (3.4) | 0 | 1 (2.5) | |
| Chills | Any grade | 5 (17.2) | 1 (9.1) | 6 (15.0) |
| Grade ≥3 | 0 | 0 | 0 | |
| Any treatment-emergent SAE | 14 (48.3) | 2 (18.2) | 16 (40.0) | |
| Any TEAE leading to permanent discontinuation | 1 (3.4)* | 0 | 1 (2.5) | |
| Any treatment-emergent AESI | 9 (31.0) | 3 (27.3) | 12 (30.0) | |
| Any TEAE related to SAR443216 | 24 (82.8) | 11 (100) | 35 (87.5) | |
| Deaths during the treatment period† | 3 (10.3) | 0 | 3 (10.3) | |
| Deaths post-treatment period‡ | 4 (13.8) | 0 | 4 (13.8) | |
Cardiac failure.
Due to disease progression.
Two deaths were due to disease progression, one from metastatic colon cancer and one from unknown a reason.
AESI, adverse event of special interest; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRS, cytokine release syndrome; DL, dose level; DLT, dose limiting toxicities; IV, intravenous; SAE, serious adverse event; TEAE, treatment-emergent adverse event.
All reported CRS and IRR events were grade 1 or 2 (online supplemental table S6), consistent with the symptomatology of cytokine release, including fever, chills, in addition to hypotension or hypoxia in grade 2 CRS cases. There was no real-time cytokine measurement to distinguish between CRS and other types of hypersensitivity reactions at the time of AE reporting, but based on the symptomatology and retrospective cytokine data (figure 3), IRRs likely represented low-grade CRSs. Fever and/or chills were reported in all affected participants, with or without other symptoms including nausea, myalgia, headache, dizziness or hypotension. These events usually occurred during or shortly after the infusion; however, delayed onset (>24 hours) was reported in few cases. All reactions were reversible and resolved quickly with conventional treatment (ie, anti-pyretic, antihistamine, or steroid). No participant with CRS in the intravenous cohort required tocilizumab treatment. Asymptomatic, transient increase in transaminase (TA) (ALT and/or AST) levels were reported in a high proportion of participants (>70%). Six participants experienced grade 3 or higher TA elevations. Two of the six participants experienced grade 4 ALT and/or AST elevations: one participant had both ALT and AST grade 4 elevations lasting 2 days, and one participant had a grade 4 ALT elevation lasting 1 day before decreasing to grade 3. All these TA elevations were isolated (ie, without concurrent bilirubin elevation), asymptomatic, transient and reversible following drug interruption and steroid treatment (systemic steroids were administered to three out of the six participants). Of the six participants, three had concurrent grade 2 CRS; both participants who experienced a short episode of grade 4 elevations had concurrent grade 2 CRS, while the remaining three participants reporting grade 3 TA elevations had no concurrent CRS. All grade ≥3 TA elevations occurred during C1, typically after D4, D8 or D15 ramp-up, at DL3 or higher DLs. Recovery time to grade ≤1 TA elevation was between 3 and 14 days. Treatment with systemic steroid did not appear to have an effect on recovery time; however, longer recovery time was observed with higher grade TA elevations. Owing to the small number of participants with grade ≥3 TA elevation, no definitive conclusions can be drawn regarding the effectiveness of steroid treatment on normalization of liver functions in this study. All TA elevations subsided after pausing the treatment and did not lead to permanent discontinuation of study drug.
Figure 3. Modulation of serum cytokines by SAR443216. a. IL-2, b. IL-6, c. TNF-α, d. IFN-γ.
A semi-log plot using standard hash-marks. ADA, antidrug antibody; C, cycle; D, day; IFN-γ, interferon-gamma; IL-2, interleukin-2; IV, intravenous; T, time; TNF-α, tumor necrosis factor α.
Three DLTs including grade 2 cardiac failure (n=1) and grade 4 ALT elevations (n=2) were observed in the 2-week lead-in cohort, whereas no DLTs were observed in the 3-week lead-in cohort. One participant with DLT of grade 2 cardiac failure with preserved left ventricular ejection fraction (LVEF) discontinued the study. The patient case was confounded by concurrent high cardiovascular risk, pre-existing cardiac ischemia (elevated troponin at screening and baseline with specific ECG changes) and asymptomatic heart failure based on significantly elevated pro-B-type natriuretic peptide levels at screening (>8 × upper limit of normal). The investigator assessed the event as related to SAR443216. Cardiotoxicity is a recognized on-target toxicity associated with all HER2-targeting anticancer treatments. In this study, no other cardiac events were reported. In the 2-week lead-in cohort, 14 (48.3%) participants reported serious adverse events (SAEs), and 6 (20.7%) participants reported treatment-related SAEs, including CRS, TA elevations, and cardiac failure. In the 3-week lead-in cohort, three SAEs (urinary tract infection, lung opacity, and biliary obstruction) were reported, and none were related to SAR443216 (online supplemental table S7). Details on treatment-related AEs and AEs of special interest are presented in online supplemental tables S8 and S9, respectively. Three (10.3%) deaths occurred during the treatment period (15, 24 and 27 days after the last SAR443216 administration) due to disease progression, whereas four (13.8%) occurred post-treatment, with three related to disease progression and one due to unknown causes. None of the seven reported deaths were related to SAR443216.
The extended lead-in period cohort showed a more favorable safety profile. Only 1 of 11 participants (9.1%) experienced grade 2 CRS. Similarly, 1 of 11 participants (9.1%) had grade 3 TA elevation, with no grade >3 in the extended lead-in cohort. No treatment-related SAE was reported in the extended cohort. In summary, based on the limited exposure, the extension of the lead-in period decreased the risk of >1 grade CRS and severe liver function test (LFT) elevations.
The subcutaneous cohort was terminated predominantly due to low bioavailability before the conclusion of the study. All three participants enrolled in the initial DL reported maximum grade 2 injection site reactions and two out of three participants had grade 2 CRS. More detailed safety data have been provided in the online supplemental table S10.
Efficacy
A total of 14 participants achieved SD as their best overall response, for a DCR of 35.0% (95% CI 20.63 to 51.68) of which 10/29 (34.5%) participants were identified in the 2-week lead-in cohort and 4/11 (36.4%) participants were in the 3-week lead-in cohort, but no objective responses (PR or CR) were observed (online supplemental figure S2). The study was terminated early before the planned enrollment was completed and prior to identification of the MTD for reasons other than safety or lack of efficacy; therefore, no formal conclusions regarding efficacy can be drawn.
Pharmacokinetics
PK parameters following intravenous administration were primarily derived from dosing at C1D15, when dense PK sampling was planned. At each DL, three to four participants were enrolled (except DL3, n=9). Overall, plasma exposure increased with increasing dose (online supplemental figure S3a), and the overall PK profiles following intravenous administration were consistent across DLs. Total PK variability between participants was moderate to high, with a coefficient of variation (CV) of 40–127%. The maximum concentration measured at the end of 4-hour infusion (Cmax) and area under the plasma concentration versus time curve was slightly less than proportional to the dose increase across the full explored dose range (18–720 µg, 2-week lead-in cohort) (online supplemental table S11). The geometric mean half-life ranged from 25 to 71 hours across DLs. The Cmax increased from 2 ng/mL for the 18 µg dose to 37 ng/mL for the 720 µg dose.
Across multiple cycles, the end-of-infusion level was stable over time, with few exceptions showing a steady reduction across first cycles followed by stabilization. Initial PK profiles were also obtained for three participants dosed by SC route (online supplemental figure S3b).
Immunogenicity
Treatment-emergent ADAs were observed in 8 of 29 (27.6%) participants in the 2-week lead-in cohort and in 1 of 11 (9.1%) participants in the 3-week lead-in cohort (online supplemental table S12). Reduced plasma exposure to SAR443216 was observed in four of the participants who were confirmed as ADA-positive. One participant was ADA-positive at baseline, which could be attributed to prior therapy with trastuzumab; however, the pre-existing ADA did not appear to significantly impact plasma exposure to SAR443216. This participant completed 10 cycles and experienced clinical benefit, achieving SD followed by progressive disease at EOT. The median time to ADA onset was 60.0 days (Q1–Q3: 34–106 days) in the 2-week lead-in cohort, and 108 days in the 3-week lead-in cohort.
Cytokine analysis and immunophenotyping
SAR443216 induced increases in serum interleukin (IL)-2, IL-6, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) following each administration/infusion starting at C1D4 (second infusion), indicating T-cell activation and target engagement (figure 3). IL-2, IL-6, TNF-α, and IFN-γ levels peaked at 10 hours post-infusion, then returned to baseline within 24 hours of each infusion, with median fold-changes of approximately 10–100; this pattern was observed across all participants. The highest concentrations of IL-2 were observed at C1D8, whereas those of IL-6, IFN-γ, and TNF-α were observed at C1D15 (first target dose) in the 2-week lead-in cohort and at C1D22 (first target dose) in the 3-week-lead-in cohort. These levels decreased after reaching the target dose, indicating immune tolerance.
Following each infusion of SAR443216, a rapid decrease in peripheral lymphocyte count was observed. Lymphocyte redistribution was accompanied by transient expression of the T-cell activation markers such as CD57, PD-1, and HLA-DR, cytotoxicity marker granzyme B, and proliferation marker Ki67. A decrease in regulatory T cells was also observed (figure 4a). These data suggest a robust peripheral pharmacodynamics response including proinflammatory cytokine secretion, CD4 and CD8 T-cell activation and proliferation, and regulatory T-cell reduction, on SAR443216 administration.
Figure 4. Characterization of immune cells based on immunophenotyping in periphery and immune response modulation in the tumor microenvironment. a. T cell heatmap based on flow cytometry panel by treatment and analysis visit - exposed population, b. Immune response modulation in the tumor microenvironment.
Slight increases have been observed for other CRS/inflammatory markers, including C-reactive protein (CRP), ferritin, white blood cell (WBC) and neutrophils during the first cycle, with a similar pattern observed for the 2-week and 3-week lead-in groups (online supplemental figure S4). This observation is consistent with the pharmacological effect of the drug; however, only pre-dose values are available for these parameters.
TME analysis
Baseline HER2-expression measured by IHC in local laboratories and corresponding treatment duration are shown in online supplemental figure S5a. HER2-expression was also measured in a subset of participants (n=26) via multiplex immunofluorescence (online supplemental figure S5b). Among participants with below-median HER2 expression levels, 4 of 13 participants achieved SD (31%), whereas among participants with above-median HER2 expression level, 7 of 13 participants (54%) achieved SD. Changes in immune cells in response to SAR443216 within the TME were measured via multiplex IHC using pretreatment and post-treatment (C2D15 or EOT) paired tumor biopsies in a subset of participants (n=12). At C2D15 or EOT, increased levels of proliferation biomarker Ki67 in CD3 T cells were observed in 11 of 12 participants. A similar increase in Ki67 levels was observed in CD8 and CD4 T cells (10 of 12 and 11 of 12 participants, respectively) (figure 4b). However, no clear trend was observed for changes in T-cell abundance, other immune cell subsets, or functional biomarkers.
Discussion
The results of this Phase 1 study demonstrate that treatment with SAR443216, a trispecific TCE targeting HER2, CD3, and CD28, is feasible and well tolerated in participants with R/R solid tumors. The modulation of immune cells through co-stimulatory approaches holds significant promise as an antitumor therapy.24 A multitarget approach, such as bispecific or trispecific antibodies can be beneficial in the treatment of solid tumors, wherein tumor heterogeneity renders single-target therapies less effective.25–27 Blinatumomab, a first-in-class bispecific TCE (CD19/CD3), based on a similar mechanism, was approved by the Food and Drug Administration in 2014 and the European Medicines Agency in 2015 for the treatment of B-cell acute lymphoblastic leukemia.28 Following blinatumomab approval in 2014, seven T cell-engaging compounds have reached the market, two of which are approved for treatment of solid tumors: tebentafusp29 and tarlatamab.30 This highlights the growing interest in TCE as a promising therapeutic approach for solid tumors. Recent studies suggest that trispecific antibodies offer a promising approach for tumor treatment by enhancing immune cell engagement.31–33 These antibodies have been engineered with two antigen-binding sites on T cells (CD3 and CD28) and one antigen-binding site on cancer cells. The presence of the CD28-targeting domain significantly enhances T-cell activation compared with that of antibodies lacking this domain.17 21 22 Wu et al developed a trispecific antibody targeting CD38, CD3, and CD28 that exhibited potent T-cell-mediated killing of CD38+myeloma cell lines, with 3-log to 4-log greater in vitro cytotoxic potency than daratumumab.22
SAR443216 is a human IgG4-based trispecific antibody engineered to activate T cells through co-engagement of CD3 and CD28 on T cells and HER2 on tumor cells. This first-in-human, Phase 1 dose-escalation study of SAR443216 in participants with advanced HER2+solid tumors demonstrated a manageable safety profile. Pulmonary toxicity (eg, pneumonitis or interstitial lung disease) and decrease in LVEF were not reported in any participant, and the only case of cardiac failure was confounded by pre-existing cardiac conditions. Given that cardiotoxicity is a known class effect of HER2-directed therapies, a potential contribution of SAR443216 to this case cannot be excluded; however, the pre-existing troponin and significant pro-BNP elevation with specific ECG changes already present at the time of enrollment suggested an alternative cause. In this study, to reduce the risk of severe CRS, an intraparticipant dose escalation (lead-in dose) was used in C1, and low-grade (grade 1 or 2) AEs were most commonly reported, including CRS, asymptomatic increase in TA levels, fever, and IRRs, which were consistent with the mechanism of action of the engaging T cells and cytokine release. Dual T-cell stimulation via CD3 and CD28 did not increase the severity of AEs related to cytokine release as compared with AEs resulting from T-cell stimulation via CD3 alone. Similarly, in a previously reported Phase 1 study of NJH395, an immune-stimulator antibody conjugate, with a TLR7 agonist payload targeting HER2+tumor cells, CRS was observed as a common but manageable AE. However, toxicities and ADAs at high doses represented significant clinical challenges for use in humans.34 A comparable safety profile was observed with runimotamab, an HER2×CD3 bispecific antibody, which demonstrated dose-dependent CRS, ALT/AST elevations, and other immune-mediated toxicities. Antitumor activity with runimotamab monotherapy was limited; however, the addition of trastuzumab improved tolerability, reduced CRS frequency, and enabled higher runimotamab dosing, leading to clinically meaningful responses (30.4%) in heavily pretreated participants. These findings suggest reduced on-target/off-tumor toxicities with the combination of HER2-TCE and trastuzumab.35 In the current study with SAR443216, no fatal TEAEs were observed throughout the treatment period. Deaths were attributed to disease progression across the cohorts and were not related to SAR443216. Compared with other HER2-targeting treatments, no serious pulmonary and cardiac toxicities were observed in this study. The study was terminated before the planned enrollment was completed for reasons unrelated to safety or efficacy; the termination was related to the sponsor’s portfolio reprioritization decision. DCRs were 34.5% in the 2-week lead-in cohort and 36.4% in the 3-week lead-in cohort, though no objective responses were achieved in this heterogeneous and heavily pretreated population. Enrolled participants showed high heterogeneity in terms of tumor types, HER2 status, HER2 expression, and HER2 mutations/aberrations; >50% had received different prior HER2 targeted treatments. More than half of participants enrolled had HER2 low expression (online supplemental figure S5). HER2 expression may have an impact on clinical efficacy, as the percentage of stable disease was lower in participants with HER2-low tumors compared with those with tumors expressing HER2 above median. For PK analysis, only three to four participants were enrolled at each DL (except DL3 with n=9), warranting cautious data interpretation due to the small sample size. The number of participants was limited to understand the source of the total variability observed for each DL. Nevertheless, the Cmax was generally stable over time across multiple cycles, with few exceptions showing a steady minor reduction across the initial cycles followed by stabilization. When ADA were negative, these particular profiles suggested at least two possible mechanisms:1 increased availability of the cellular target (HER2) once it becomes free following prior therapy with an anti-HER2 agent (as suggested by the therapy received prior to study entry vs first dosing with SAR443216 in the context of SD), or2 neutralization of soluble HER2 allowing increased binding to cellular targets after a few weeks. Overall, the relatively stable Cmax (end-of-infusion levels) and pre-DLs suggested no remarkable change in the PK characteristics of SAR443216 over time. In addition, the elimination half-life of SAR443216 was consistent across DLs, suggesting that target-mediated elimination of SAR443216 did not reach saturation. Approximately 25% of participants receiving SAR443216 developed ADAs with only about half showing potential impact on plasma exposure to SAR443216, demonstrating that immunogenicity was not a critical concern with SAR443216 treatment. One participant was ADA-positive at baseline, which could be attributed to prior therapy with trastuzumab; however, the majority of participants who had received prior trastuzumab-based therapy did not present with ADAs.
Biomarker analysis demonstrated that SAR443216 induced an increase in the levels of multiple serum proinflammatory cytokines, such as IFN-γ and IL-2. T-cell activation markers such as CD57, PD-1, and HLA-DR, cytotoxicity marker granzyme B, and proliferation marker Ki67 in CD4 and CD8 T cells were also induced on SAR443216 administration. These data support the mechanism of action by demonstrating increased T-cell activation and target engagement. The serum cytokine increases also correlated with the safety profile. Among the seven participants with grade 2 CRS, two (who also presented with concurrent grade ≥3 TA elevation) had the highest elevations of IFN-γ and IL-6 (>2,000-fold changes compared with baseline) among all participants; increases in cytokine levels in other participants with grade 1 or 2 CRS were comparable with those in participants without CRS. IFN-γ and IL-6 also peaked in four of eight participants with grade ≥3 TA elevation, regardless of the presence and severity of concurrent CRS. These data suggest that the transient asymptomatic TA elevations observed in a high proportion of participants treated with SAR443216 could be linked to cytokine release even in the absence of classic CRS symptoms.
Although Ki67, the proliferation biomarker increased in tumor-infiltrating T cells, including CD8 and CD4 T cells, this did not translate into increased T-cell abundance in the TME or enhanced clinical activity. Investigation of other immune cell subtypes and functional biomarkers, including tumor-associated macrophages; IDO1, HLA-DR, and PD-L1; granzyme B (CD8 cytotoxicity); PD-1, PD-L1, and T-cell immunoglobulin and mucin-domain containing-3 (TIM3, activation/inhibition biomarkers); and forkhead box P3 (FoxP3) (CD4 regulatory cells), did not reveal a clear trend in functional changes in T cells or tumor-associated macrophages.
This study has limitations due to its early termination before the completion of planned enrollment, resulting in limited safety and efficacy data to draw any definitive conclusions. Nevertheless, this study provides initial results for SAR443216 use in humans, demonstrating that treatment with this trispecific TCE SAR443216 is feasible in advanced HER2+solid tumors with manageable toxicities. To achieve improved responses and survival rates, combination strategies can be evaluated in future studies. Further evaluations are essential to fully characterize the efficacy and safety profile of SAR443216 in participants with advanced HER2+solid tumors.
Supplementary material
Acknowledgements
The authors and Sanofi would like to thank the trial participants and their families. The authors thank Dimitri Carene (biostatistics and data analysis), Eric Boitier (biomarker analysis), Magali Carcenac (pharmacokinetics), and the bioanalysis and immunology teams at Sanofi France for their contributions to data generation. Medical writing support for this manuscript was provided by Ujara Shaikh MPharm and Sarabjeet Kaur PhD of Sanofi.
Footnotes
Funding: This study was funded by Sanofi.
Provenance and peer review: Not commissioned; externally peer reviewed.
Patient consent for publication: Not applicable.
Ethics approval: The study received ethical approval from the institutional review boards or ethics committees of all participating centers, including the WCG IRB and The University of Texas MD Anderson Cancer Center IRB for USA (Approval No. Not available), the Research Ethic Committee I, China Medical University Hospital (Approval No. CMUH110-REC1-120), the Institutional Review Board II, National Cheng Kung University Hospital (Approval No. B-BR-110-048), the Institutional Review Board I, Kaohsiung Medical University Chung-Ho Memorial Hospital (Approval No. KMUHIRB-F(I)-20230192), the Seoul National University Hospital Institutional Review Board (Approval No. H-2104-202-1216), Asan Medical Center Institutional Review Board (Approval No. 2021-0744), COMITE DE PROTECTION DES PERSONNES ILE DE FRANCE V Hôpital Saint-Antoine (Approval No. NA), and the Comité de Ética de Investigación con medicamentos (CEIM) Parc Taulí, Plaça Torre de l’Aigua, s/n, 08202 Sabadell (The study was managed by old regulation and EudraCT number was used. EudraCT: 2021-000086-32). Written informed consent was obtained from each participant, and all procedures were conducted in accordance with the Declaration of Helsinki.
Data availability free text: Qualified researchers may request access to patient-level data and related documents, including clinical study report, study protocol with any amendments, blank case report form, statistical analysis plan, and dataset specifications. Patient-level data will be anonymized, and study documents will be redacted to protect the privacy of trial participants. Further details on Sanofi’s data sharing criteria, eligible studies, and process for requesting access can be found at: https://www.vivli.org.
Data availability statement
Data are available upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Data are available upon reasonable request.