Safety and biologic activity of a bispecific T cell receptor targeting HIV Gag in males living with HIV: a first-in-human trial

Domain: neuroscience
Published: 2026-01-31
Editorial outcome: minor_revision

Abstract
HIV persistence in reservoirs despite antiretroviral therapy (ART) is a barrier to a permanent cure. We present the affinity-enhanced TCR bispecific IMC-M113V as a potential therapeutic for targeted HIV reservoir elimination. Preclinical studies demonstrate that IMC-M113V redirects T cells towards cells expressing the variable viral peptide, Gag 77-85, presented by HLA-A*02:01 at low copy number, without binding to HIV-negative cells. Here, we conduct a first-in-human, open-label single ascending dose study of IMC-M113V (1.6-15 µg) in twelve HLA-A*02:01-positive males living with HIV on suppressive ART (EudraCT number 2021-002008-11). Participants receive one intravenous infusion of IMC-M113V on Day 1 and are monitored through Day 29 to evaluate safety, tolerability (primary endpoints) and pharmacodynamic (PD) activity (secondary endpoint). IMC-M113V is well tolerated and not associated with any serious adverse event. PD activity is dose-dependent and strongest in participants with highly IMC-M113V-sensitive Gag 77-85 variant sequences. Thus, we provide a promising foundation to evaluate multiple and higher doses of IMC-M113V as a strategy for achieving ART-free virological control.

Introduction
Durable antiretroviral (ART)-mediated suppression of HIV replication has led to dramatic reductions in mortality and normalisation of lifespan for people living with HIV (PLWH). ART is not curative, however, and must be taken continuously for life due to the persistence of a reservoir of replication-competent HIV proviruses, which cause rebound viremia if treatment is interrupted1. Reservoirs are composed primarily of CD4+ T cells, but HIV DNA is also found in other cell types, including hematopoietic progenitors, macrophages, dendritic cells, microglial cells and astrocytes2. The complete eradication of reservoirs is an extremely ambitious goal that has to date only been achieved by hematopoietic stem cell transplantation (HSCT) with CCR5Δ32/Δ32-deleted donor cells3–7. The high morbidity and mortality associated with HSCT restrict its application to people with life-threatening malignancies for which there are no other treatment options. Consequently, the intent of investigational therapies is to safely reduce reservoirs to levels that would lead to sustained virological control after ART withdrawal, i.e., a ‘functional cure’8.

Because the host immune response curtails HIV replication following acute infection and may, in rare cases, lead to a state of prolonged virological suppression in the absence of ART, novel modalities that can recapitulate immune-mediated viral control and/or directly target reservoirs for elimination are under evaluation9–12. However, diverse immune evasion strategies used by HIV, including the acquisition of escape mutations that do not impede replication, could limit their effectiveness. For example, broadly neutralising antibodies (bNAb) in PLWH led to a delay in viral rebound for up to 6 months after ART interruption, and for even longer durations in PLWH who initiated ART very early, but typically in individuals who had not acquired detectable bNAb resistance mutations prior to treatment13–16. Furthermore, this post-treatment control was largely dependent on maintaining plasma bNAb levels above a predefined threshold, although in some cases modest reductions in viral reservoirs were observed13,15,16. Other technologies, such as adoptive cell therapies, bispecific antibodies and gene editing, have shown promise in animal models, and clinical proof of concept is beginning to emerge17–20. Still, a broadly applicable and scalable therapy that fulfils the target product profile of a functional cure remains elusive21.

Immune mobilising monoclonal T cell receptors against cancer (ImmTAC) and viruses (ImmTAV) are soluble bispecific proteins comprised of an affinity-enhanced T cell receptor fused to a CD3-specific single chain variable fragment (scFv) (Fig. 1a). The TCR binds to a specific peptide when presented by HLA molecules on the target cell surface; the scFv binds to CD3 on any T cell in the vicinity, leading to the formation of an immune synapse and elimination of the target cell22,23. The ImmTAC platform has been clinically validated by tebentafusp, which binds to an HLA-A*02:01-presented peptide derived from the melanocyte antigen, gp100, and which conferred a significant survival benefit to HLA-A*02:01-positive patients with metastatic uveal melanoma in a randomised controlled trial24. ImmTAV molecules are an adaptation of this technology and aim to provide targeted elimination of viral reservoirs in people with chronic hepatitis B25, HIV and other persistent infections26. We previously reported that an ImmTAV molecule that specifically recognises an immunodominant HLA-A*02:01-restricted HIV peptide, Gag 77–85, along with its common escape variants, could redirect T cells to eliminate HIV-infected cells from ART-suppressed PLWH after viral reactivation in vitro27. This ImmTAV molecule was active at picomolar concentrations, indicating exquisite sensitivity to the Gag peptide, which may be crucial to its efficacy in vivo, given that HIV proteins are expressed at very low levels in reservoirs28–30. Further engineering was undertaken to produce the clinical candidate drug IMC-M113V, which was advanced to a first-in-human (FIH) study in HLA-A*02:01-positive PLWH on a suppressive ART regimen.

Given the excellent overall safety profile of current ART and the benefit/risk considerations for a therapy with a new mechanism of action against HIV, IMC-M113V was initially evaluated in a single ascending dose (SAD) study to identify tolerable and biologically active doses. To ensure safety, principal eligibility criteria were: documented virological suppression on ART for at least one year, current CD4+ T cell count >500 cells/µL and CD4+ T cell nadir >200 cells/µL. In addition, the duration of ART at study entry was restricted to ≤7 years to maximise the potential to detect pharmacodynamic (PD) activity against Gag-expressing cells, which are indicative of a transcriptionally and translationally active HIV reservoir and which decline in frequency with long-term viral suppression31,32. Here, we present both the preclinical data supporting the clinical study rationale, and the results of the SAD study, which demonstrate the safety, tolerability and initial PD activity of IMC-M113V. These data inform the evaluation of IMC-M113V in multiple dose schedules, where it has the potential to confer post-treatment viral control through targeted reservoir reduction.

Results
Binding kinetics and activity of IMC-M113V against prevalent Gag 77–85 variants
IMC-M113V was derived from a previously described ImmTAV molecule (m121) that was modified to meet manufacturing specifications27. The TCR domain was engineered to bind HLA-A*02:01-Gag 77–85 (cognate sequence, SLYNTVATL) complexes with an enhanced affinity of ~100 pM (KD at 25°C) (Fig. 1a, b). Some of the preclinical assays described here were performed with the closely related precursor molecule, IMC-M113VRES, which differs from IMC-M113V by a single amino acid and demonstrates a similarly enhanced binding affinity to the cognate peptide HLA (pHLA). This residue is located within the TCR domain and was mutated to support the manufacturability of IMC-M113V by reducing the risk of a non-enzymatic post-translational modification.

Gag 77–85 variants have arisen over the course of the HIV pandemic because of immune selective pressure and/or recombination events, with Y79F representing the most prevalent variant globally (Supplementary Fig. 1a)33. To address anticipated inter- and intra-individual viral diversity, the TCR domain of IMC-M113V was engineered to achieve >1000-fold enhancement in affinity for the 8 most prevalent (global frequency ≥2%) Gag 77–85 variants identified using the Los Alamos National Laboratory HIV sequence database (Supplementary Fig. 1a), all of which are stably presented by HLA-A*02:01 (Fig. 1b). TCR binding affinities for the variants ranged from 0.16 to 1.62 nM (Fig. 1b). The potency of IMC-M113V against T2 cells presenting the Gag 77–85 cognate and variant peptides was assessed in a T cell redirection assay with titrated peptide concentrations. Peptide half-maximal effective concentration (EC50) values correlated with the measured affinities and ranged from 0.31 nM for the V82I variant to 5.29 nM for the Y79F/T84V double mutant across the two effector donors tested (Supplementary Fig. 1b). To confirm binding of the TCR to Gag-positive cells, the TCR domain (Fc-tagged and lacking the anti-CD3 scFv) of IMC-M113VRES was used to stain an HLA-A*02:01-transduced HIV-infected CD4+ T cell line (C8166) (Fig. 1c), which was then analysed using total internal reflection microscopy. This enabled quantification of TCR-bound pHLA complexes (epitopes) on the cell surface23,34. HLA-A*02:01-Gag 77–85 complexes were detected at a median copy number of 3/cell (range 0–14) on infected cells. Despite some background signal (range 0–8) on uninfected cells due to non-specific binding of the secondary antibody, pHLA density was significantly higher on infected cells (p < 0.0001 (Fig. 1d). In a parallel experiment to determine the HLA-A*02:01-Gag 77–85 density on T2 cells pulsed with titrated peptide concentrations, 1 nM peptide yielded a median epitope count of 2 (range 0–13) pHLA/cell (Supplementary Fig. 1c). Taken together, these results indicate that IMC-M113V is sensitive to very low levels of viral pHLA complexes expressed on the cell surface.

IMC-M113V potency against HIV-infected cells in vitro
The potency of IMC-M113V against HIV-infected cells was determined using an infected cell elimination assay, in which HLA-A*02:01-transduced HIV-infected C8166 cells were co-cultured with purified CD8+ T cells from healthy donors (n=6) for 7 days, together with titrated concentrations of IMC-M113V. Dose-dependent elimination (calculated using the equation described in “Methods”) was observed across all effector donors, with a mean EC50 value of 6.5 pM (range 1.2–13.1 pM, Fig. 2a). IMC-M113V also eliminated targets infected with the most prevalent global variant, Y79F, in a dose-dependent manner (n=4 effector donors, mean EC50 value <100 pM), albeit less efficiently than the cognate virus, consistent with the weaker binding affinity of IMC-M113V for the Y79F variant (Figs. 1b and 2b and Supplementary Fig. 1b).

In vitro assessment of IMC-M113V off-target reactivity
To assess the potential for off-target reactivity, IMC-M113V was tested in clinically validated screening assays incorporating whole blood and a panel of normal cells associated with vital organs obtained from HLA-A*02:01-positive healthy donors35. In these assays, interferon-γ (IFN-γ) release, alone or together with other cytokines, was used as a sensitive readout for T cell activation in vitro (Fig. 2c). No statistically significant cytokine production above background levels was observed in whole blood, or in co-cultures of immune cells and cardiomyocytes, astrocytes or lung epithelial cells, when incubated with IMC-M113V at concentrations below 10 nM (Fig. 2c). As the biologically active dosing range was anticipated to be 0.1–1 nM, the reactivity at 10 nM was not considered to be a concern.

FIH safety of IMC-M113V in people living with HIV
Given the HLA restriction of IMC-M113V, the study began with pre-screening of potentially eligible participants (n=89) for the HLA-A*02:01 allele. We estimated the prevalence of HLA-A*02:01 to be in the range of 23–45% among PLWH populations in the countries where the study was conducted, and this was borne out in the pre-screening data (Fig. 3)36,37. Of the 33 HLA-A*02:01-positive participants who were eligible for screening, 16 completed screening, of whom four were excluded due to a CD4+ T cell count <500 cells/µL. The first and last participants were enrolled on 6 July 2022 and 12 December 2022, respectively, at 7 hospital sites in the United Kingdom, Belgium and Spain. Altogether, 12 participants were enrolled in three single ascending dose cohorts (Fig. 3). Although females were eligible and invited to participate, all study participants were male, with a median age of 38 years (range 27–60 years). The median duration of ART on Day 1 was 3.5 years (range 2–7 years), the median CD4+ T cell count was 882 cells/µL (range 466–1460 cells/µL), and the median CD4+ T cell nadir was 550 cells/µL (range 330–846 cells/µL) (Table 1).

Participants were enrolled in accordance with the planned dose escalation scheme (described in “Methods”), with a single individual being enroled at each of the first two dose levels (1.6 µg and 5 µg), to minimise exposure to potentially sub-therapeutic doses38. At the 15 µg dose level, 4 participants were enroled initially, of whom 2 met the pre-specified criteria for PD activity. This cohort was subsequently expanded to a total of 10 participants to enable further evaluation of this PD activity (Fig. 3). As the primary endpoint, safety and tolerability of single doses of IMC-M113V were assessed by incidence and severity of treatment-emergent adverse events (TEAE). Fifteen TEAEs were recorded in 6/12 (50%) participants: 7 were deemed unrelated to the study drug, and 8 were considered possibly related (Supplementary Table 1). Of the six participants who experienced TEAEs, 5 (83%) experienced the following possibly related events: fatigue (1 Grade 1, 1 Grade 2) lasting 2–4 days, phlebitis, herpes simplex, itch, skin redness (all Grade 1) and two episodes of eczema (1 Grade 1, 1 Grade 2). Five of the 6 aforementioned skin AEs were observed in the same participant. Apart from one procedure-related event, the onset of all these AEs was 5–20 days after administration of IMC-M113V. No participant developed fever, cytokine release syndrome or neurotoxicity of any grade, nor were there any serious adverse events. There were no laboratory AEs of Grade ≥3 severity. CD4+ T cell counts were >500 cells/µL post-dosing in all participants, including those who had a pre-dose (Day 1) CD4+ T cell count of <500 cells/µL (n=2) despite meeting all eligibility criteria at screening. All plasma HIV RNA (viral load, pVL) values were below the limit of detection or detected at ≤40 copies/mL post-dosing and through follow-up.

Pharmacokinetics of IMC-M113V
Serum IMC-M113V concentrations measured pre-dose and up to 24 h after IV administration were used to derive pharmacokinetic profiles for each dose level using non-compartmental analysis. IMC-M113V was detectable at all dose levels administered, and peak concentrations were observed immediately after infusion (Fig. 4). Overall, exposure increased in a dose-proportional manner, with Cmax values ranging from 171 to 3,650 pg/mL and area under the curve (AUClast) values from 1220 to 45,400 h*pg/mL. Serum IMC-M113V levels decreased below the lower limit of quantification (LLOQ) by 12 h post-administration of the 1.6 µg dose but remained detectable at all time-points up to 24 h for the 5 and 15 µg doses. Based on the 15 µg dose (n=10), clearance (CL) ranged from 2.03 to 12.8 L/day; the steady-state volume of distribution (Vss) was 1.93–11.2 L, and the terminal half-life was estimated to be 14.5–21.6 h.

Pharmacodynamic activity of IMC-M113V
A biomarker-guided dose escalation strategy was implemented in this SAD study in order to identify a tolerable dose range on which to base a future multiple dose schedule. Target-specific and dose-proportional increases in proinflammatory cytokines and chemokines in the serum have been observed following treatment with tebentafusp, consistent with its mechanism of action as a T cell engager39,40. As shown in the preclinical studies described earlier, IFN-γ release is a direct and sensitive measure of T cell activation. However, interleukin-6 (IL-6)-producing cells, which are largely of myeloid and stromal origin41,42, can amplify the initial T cell activation signal, potentially offering higher sensitivity to detect target engagement39. To determine whether a robust IL-6 signal could be detected in the context of low Gag 77–85 peptide presentation, which is anticipated in ART-treated PLWH, we first assessed IMC-M113VRES-mediated IL-6 induction in a T cell activation assay with titrated peptide concentrations. We confirmed that IL-6 production by peripheral blood mononuclear cells (PBMC) occurred only in the context of TCR engagement with HLA-A*02:01-Gag 77–85, at peptide concentrations as low as 100 pM (Supplementary Fig. 2 and Fig. 2C).

In the clinical study, serum IL-6 was quantified pre-dose, 8 and 24 h post-dose, and on Day 8, together with IFN-γ, TNF, IL-2, IL-8, IP-10, and IL-10. Baseline IL-6 values were below the LLOQ in 9/12 participants, while the remaining three participants all had values within the normal range. A small increase of 2-3-fold from baseline in IL-6 was noted in each of the single participant cohorts, with a peak occurring at 8 h for the 1.6 µg dose and 24 h for the 5 µg dose. Five of the 10 participants in the 15 µg dose cohort showed a >4-fold increase in IL-6 from baseline, of whom two had a >10-fold rise (Fig. 5a). The highest recorded value in any participant was 62.8 pg/mL (Fig. 5 and Supplementary Fig. 3). By Day 8, IL-6 levels had declined to baseline or below 10 pg/mL. Modest changes were observed in other cytokines in the 15 µg dose cohort, with 1/10 and 3/10 participants showing a >2-fold rise in IFN-γ and IL-8, respectively, at 8–24 h post-dose, and 1/10 showing an early 4-fold rise in TNF on Day 8 (Supplementary Fig. 3). We also assessed T cell activation ex vivo in a subset of participants in the 15 µg dose cohort with available PBMC samples, pre- and post-dose, by flow cytometry (Supplementary Figs. 4 and 5). This showed a post-dose increase in the frequency of CD4+ and CD8+ T cells expressing the early activation marker, CD69, in 3/6 participants, together with a significant increase in CD69 mean fluorescence in both cell subsets overall (n=6, Fig. 5d). In addition, after ex vivo stimulation with phorbol myristate acetate (PMA)/ionomycin, IFN-γ, granzyme B (GzB), and perforin expression were increased post-dose in several participants, with significant changes being detected in CD8+ T cells for GzB and perforin (n=5, Fig. 5d).

To explore the relationship between the IL-6 response and baseline HLA-A*02:01-Gag 77–85 expression, we quantified HIV cell-associated RNA (HIV CA-RNA) as a measure of the active reservoir. We also sequenced gag transcripts to determine the presence of Gag 77–85 cognate or variant sequences, given that IMC-M113V binding affinity for pHLA is affected by known mutations in this epitope (Fig. 1b and Supplementary Fig. 1b)43. Median HIV CA-RNA copy number was 639 (range 112–2777) copies/106 TBP (TATA-box binding protein) for the 15 µg cohort. Gag 77–85 sequences detected in HIV CA-RNA included previously characterised variants (Fig. 1b and Supplementary Fig. 1) and some less prevalent variants for which the IMC-M113VRES binding affinity was determined reactively (Fig. 5b and Supplementary Fig. 6).

The relationship between baseline HIV CA-RNA copy number, dominant Gag 77–85 sequence, and peak IL-6 level post-dose is shown in Fig. 5c. IL-6 elevations of >10-fold were observed in 2/3 of the participants with picomolar affinity Gag 77–85 variants; the measured affinities to these variants ranged from 76 to 178 pM. One participant with a 178 pM affinity variant (cognate sequence) but no IL-6 response had very low HIV CA-RNA levels (112 copies/106 TBP). In contrast, IL-6 elevations of >10-fold were observed in 0/7 participants with nanomolar affinity Gag 77–85 variants, for which the measured affinities ranged from ~1.3 to 261 nM. Although the study was not powered to test the correlation between the IL-6 response and HIV CA-RNA expression, a trend was observed for the three participants with a picomolar Gag 77–85 variant (Pearson r2=0.87) but not for the seven participants with nanomolar variants (Pearson r2=0.02). These observations are consistent with in vitro data showing that the magnitude of IMC-M113V-mediated cytokine response was dependent on both the peptide concentration and the affinity of IMC-M113V for the peptide variant (Supplementary Figs. 1b and 2).

Effect of a single 15 µg dose of IMC-M113V on the HIV reservoir
HIV CA-RNA and intact proviral DNA were quantified pre (Day 1) and post dose (Days 8 and 29) in an exploratory analysis to determine any impact on the size of the reservoir. Values did not change significantly throughout follow-up (Fig. 6).

Methods
ImmTAV molecules, pHLA complex stability and TCR binding affinity measurements
IMC-M113VRES was derived from the closely related ImmTAV molecule m12127 that was modified to meet manufacturing specifications. Late in preclinical development, a single amino acid change in the TCR domain was introduced to reduce the risk of non-enzymatic post translational modification, yielding the clinical lead candidate drug, IMC-M113V. IMC-M113V and IMC-M113VRES show comparable target binding affinity and specificity. In vitro studies of IMC M113VRES described here were performed to complement data from preclinical experiments supporting regulatory submissions, all of which were performed using IMC-M113V.

To determine the stability of Gag 77–85 pHLA complexes, their response to ILT2—which recognises the α3 domain and β2-microglobulin of HLA-A—was evaluated through biolayer interferometry (Octet® RED384—Sartorius). Soluble biotinylated Gag 77–85 pHLA complexes were immobilised on a streptavidin-coated biosensor as a ligand, and binding to soluble ILT2 was then assessed at 30 min intervals over 4 h at 37°C. The complex t1/2 (h) was then determined by plotting the ILT2 response against time and fitting the resulting curve using non-linear regression (GraphPad Prism v8.3 or later). Binding affinities of IMC-M113V or IMC-M113VRES to Gag 77–85 pHLA complexes were determined by single-cycle kinetic analysis using surface plasmon resonance (SPR) (Biacore 8K) at the specified temperatures. Dissociation rate constant (kd) values lower than 9.63E-06 s−1 were constrained when determining the binding affinities (KD).

Analysis of Gag 77–85 variant prevalence
Nucleotide sequences (and metadata) were downloaded from the Los Alamos National Laboratory website (https://www.hiv.lanl.gov/ accessed 17th September 2019) and trimmed to the region containing Gag 77–85 with some additional sites on either side. The trimmed sequences were then aligned to the HXB2 reference sequence and translated to the amino acid sequence; any indels or sequences that did not provide full coverage of Gag 77–85 were excluded. Only one sample of each patient identifier was included in the analyses; sequences with no patient ID were assumed to be from unique individuals. Gag 77–85 inter-subtype variation was determined from this dataset and then combined with the published regional estimates of subtype prevalence to extrapolate per-region diversity33.

Cell lines and primary cells
T2 cells (#CRL-1992) were obtained from American Type Culture Collection (ATCC; Manassas, VA). C8166 cells (#88051601) were obtained from European Collection of Authenticated Cell Cultures (ECACC, UK) and genetically modified to express HLA-A*02:01 and beta-2-microglobulin using lentiviral transduction. Both cell lines were cultured in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific 42401018) supplemented with 10% foetal calf serum, 1% (v/v) penicillin/streptomycin, and 2 mM of L-glutamine. Primary Human Bronchial Epithelial Cells (HBEpC) were obtained from Promocell (#C-12640) and cultured in Promocell airway epithelial cell growth medium. IPSC derived astrocytes (Astro1g) were obtained from Cellular Dynamics (#ASC-100-020-001-PT) and cultured in iPSC Astrocyte maintenance medium. Induced Pluripotent Stem Cells (iPSC) derived cardiomyocytes were obtained from Cellular Dynamics (#C1006), NCardia (#PCMI-1031-1) and Takara Bio (Cellartis) #Y10076 and cultured according to the supplier’s instructions. PBMC were obtained from Cellular Technologies Ltd (Cleveland, OH, USA), Discovery Life Sciences (Huntsville, AL, USA – previously Conversant Bio) or Stemcell Technologies GmbH (Cologne, Germany). CD8+ T cells were enriched from PBMC by positive selection using magnetic beads in accordance with the manufacturer’s instructions (CD8 microbeads, 130-045-201, Miltenyi Biotech, Surrey, UK). Healthy donors gave written informed consent to provide blood samples in compliance with an approved study protocol. Approval was obtained from the National Research Ethics Committee South Central - Oxford A Research Ethics Committee, UK A) (reference: 13/SC/0226) in accordance with the principles of the Declaration of Helsinki.

T cell redirection assays
T2 cells were incubated with Gag 77–85 peptides for 2 h before co-culture overnight with HIV-negative donor PBMC at a previously optimised effector/target ratio, together with IMC-M113V or IMC-M113VRES at specified concentrations. IFN-γ enzyme-linked immunospot (ELISpot) assays were performed according to the manufacturer’s recommendations (BD Biosciences #551849); spots were quantified using the BD ELISpot reader (Immunospot Series 5 Analyzer, Cellular Technology Ltd, Shaker Heights, OH, USA). IL-6, IFN-γ, and TNF were quantified in the supernatants using an MSD immunoassay (V-PLEX proinflammatory kit; Meso Scale Technologies) following the manufacturer’s recommendations.

Quantification of pHLA complexes on Gag 77–85 positive cells
Epitope quantification on the cell surface was performed as follows23,34: HLA-A*02:01-transduced C8166 cells were infected with HIV-1 IIIB (Centre for AIDS Reagents, National Institute for Biological Standards and Control) by spinoculation. After 48 h, cells were stained with 50 nM rabbit Fc-tagged IMC-M113VRES (TCR domain), goat anti-rabbit-CF640R (Biotium #BT20176-1), and Alexa Fluor 488 Annexin V (Thermo Fisher Scientific #A13201), then fixed with 4% paraformaldehyde and transferred to poly-L-lysine-coated glass-bottomed chambers. Tile images (phase-contrast and annexin V) were acquired using a Nikon ECLIPSE Ti2 microscope with an oil 100× objective to identify alive cells before Z-stack (0.8 µm apart) fluorescent images were captured covering the entire 3D cell surface. Fluorescent spots (TCR bound to pHLA = 1 epitope) on each Z-stack were counted (minimum 40 cells/condition) using the Nikon NIS-Elements software. For peptide-pulsed targets, T2 cells were incubated with Gag 77–85 peptide for 2 h, following which staining and imaging were performed as described above.

Infected cell elimination assay
pNL4-3 (Centre for AIDS Reagents, National Institute for Biological Standards and Control, #ARP2006; donated by Dr Malcolm Martin courtesy of the NIH AIDS Research and Reference Reagent Program) plasmid was mutated to express either Gag 77–85 cognate (SLYNTVATL) or 79F variant (SLFNTVATL) sequence. HLA-A*02:01-transduced C8166 cells were infected with HIV-1 IIIB or mutated pNL4-3 by spinoculation. After 48 h, infected cells were co-cultured with CD8+ T cells purified from HIV-negative donor PBMC at a 1:2 ratio, together with IMC-M113V at the specified concentrations for 7 days. Remaining infected cells were quantified by intracellular Gag p24 staining, which was performed along with CD3 and CD8 antibodies following live/dead staining, fixation, and permeabilisation (Supplementary Table 2)67. Flow cytometry was performed using the MACSQuant X (Miltenyi), and results were analysed using FlowJo (v10) using the gating strategy shown in Supplementary Fig. 7. Per cent elimination of HIV-infected cells was calculated using the following equation: [(% p24+ cells without IMC-M113V − % p24+ cells with IMC-M113V)/(% p24+ cells without IMC-M113V)] × 100. Apparent negative elimination values observed at the lower concentrations of IMC-M113V tested were normalised to zero.

Specificity assessment against whole blood and normal cells
For whole blood cytokine release assays, fresh whole blood from healthy donors (n=9) was incubated with IMC-M113V alone, IMC-M113V and exogenous Gag 77–85 peptide, or stimulatory anti-CD3 antibody (positive controls) for 4 h. Cytokine release was measured in serum supernatants and compared to negative controls (blood incubated for 4 h in the absence of IMC-M113V) using an MSD immunoassay (V-PLEX proinflammatory kit; Meso Scale Technologies, Rockville, MD). For assays involving iPSC-derived cardiomyocytes (n=3 lots) or astrocytes, cells were incubated with IMC-M113V and HIV-negative donor (n=3) PBMC (effector/target ratio of 10:1) for 48 h68. Cytokine release was measured in the supernatants using an MSD immunoassay. To assess IMC-M113V reactivity to bronchial epithelial cells (n=3 lots), these cells were incubated with IMC-M113V and HIV-negative donor (n=3) PBMC for 24 h, following which IFN-γ ELISpot assays were performed as described above. The assays were performed in triplicate using 96-well plates. IMC-M113V, together with the Gag 77–85 peptide, was included as a positive control.

Study design and participants
IMC-M113V-103 (also known as Soluble T cell Receptors In Viral Eradication, STRIVE) is a phase 1/2a, open-label dose escalation study (EudraCT 2021-002008-11, 29 Oct 2021). Given the requirement for the HLA-A*02:01 allele and the restriction of prior ART experience to a maximum of 7 years for eligibility, a multi-centre design was used. The primary objective was to determine the safety and tolerability of IMC-M113V when administered as a single dose during ART, which would guide the selection of doses for evaluation in multiple dose schedules. A starting dose of 1.6 µg was selected based on the minimum anticipated biological effect level (MABEL), in accordance with European Medicines Agency guidance69,70. Dose escalation increments were maximally 3-fold, provided there were no emerging safety signals, and escalation was permitted by the modified toxicity probability interval (mTPI-2) algorithm71. Dose escalation could continue until either of the following occurred: (1) at least 50% participants (minimum n=4) met pre-specified criteria for pharmacodynamic activity, namely, a ≥4-fold increase in serum IL-6 ≤24 h or a ≥33% reduction in absolute lymphocyte count at 24 h, both relative to baseline; (2) the dose was associated with unacceptable toxicity as defined by the mTPI-2 algorithm. The rationale for stopping escalation in the SAD study once the pre-specified pharmacodynamic activity threshold had been reached was to expedite transition to the multiple ascending dose portion of the study, in which antiviral activity was to be assessed. A minimum 4-fold increase in serum IL-6 was based on prior studies that showed a positive correlation with overall survival in patients treated with tebentafusp, which uses the same anti-CD3 mechanism of action as IMC-M113V39.

Participants were people with HIV aged 18–65 years, with a positive test for HLA-A*02:01 as assessed by a central laboratory high-resolution (4-digit) assay, treatment with ART for a minimum of 1 year and maximum 7 years at the time of planned first dose, with pVL < 50 copies/mL, CD4+ T cell count >500 cells/µL and CD4+ T cell nadir >200 cells/µL. Participants’ sex was determined based on self-report. Males and females were eligible to participate, but as this was an FIH study, sex and gender were not considered in the design. Participants were excluded if they were a known HIV controller (pVL < 2000 copies/mL in the absence of ART for at least 12 months) or had initiated ART within 12 weeks of a confirmed diagnosis of primary HIV infection, or had active co-infection with hepatitis B or C virus.

The study was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines. The protocol was approved by the Medicines and Healthcare Products Regulatory Agency, UK (CTA 36781/0011/001-0001), the Federal Agency for Medicines and Health Products, Belgium (1296758) and the Agencia Española de Medicamentos y Productos Sanitarios, Spain (RD 1090/2015). Ethical approval was obtained from East of England—Cambridge East Research Ethics Committee, UK (21/EE/0242), University of Gent Medical Ethics Committee (BC-11136), Belgium and the Drug Research Ethics Committee of the Hospital Universitario Germans Trias y Pujol, Spain. The study was registered with the European Union Drug Regulating Authorities Clinical Trials Database (EudraCT number 2021-002008-11). Participating sites were: Imperial College Healthcare NHS Trust, London, UK; Guy’s & St Thomas’ NHS Foundation Trust, London, UK; Manchester University NHS Foundation Trust, UK; Chelsea and Westminster Hospital NHS Foundation Trust, UK; Kings College Hospital NHS Foundation Trust, UK; Universitair Ziekenhuis Gent, Belgium; Universitair Ziekenhuis Brussel, Belgium; Hospital Universitari Vall d’Hebron, Barcelona, Spain; Hospital Universitario Germans Trias y Pujol, Badalona, Spain; Hospital Universitario Ramon y Cajal, Madrid, Spain; Hospital Clinico San Carlos, Madrid, Spain. Participants gave informed written consent before being screened for enrolment.

Study oversight was provided by a study safety team (SST), comprising the Sponsor and Investigators. The SST was responsible for dose decision-making, including escalations and modifications for safety reasons. For each cohort, dose escalation decisions were based on the SST’s review of all relevant available cumulative safety and PD data. An independent Data Monitoring Committee (DMC) comprising three clinicians with relevant expertise was also convened. The DMC provided oversight of safety considerations and recommendations regarding steps to ensure participant safety and the ethical integrity of the study.

Procedures
IMC-M113V was given intravenously over 2 h on Day 1. Premedication with antipyretic (paracetamol 1 g orally or ibuprofen 400 mg orally, or their equivalents) and an oral non-sedating antihistamine was given to all participants within 30 min prior to the start of infusion, to minimise the incidence and/or severity of CRS and infusion-related reactions, which were anticipated based on experience with T cell engagers in oncology settings72. IMC-M113V infusions were administered at clinical research units at the sites. Participants were observed for a minimum of 16 h for monitoring of vital signs and, if necessary, provision of supportive care, then discharged the following day. Follow-up visits were conducted at outpatient clinics at the participating sites on Days 8, 15, 22 and 29. Clinical safety assessments included complete (Screening visit) and directed physical examinations (all other visits), vital signs measurement, 12-lead electrocardiogram in triplicate, haematology, clinical chemistry [Days 1 (pre-dose), 2, 8, 15, 22, 29], CD4+ T cell count [Days 1 (pre-dose), 2, 8 and 29, using a quantitative flow cytometry panel comprising antibodies to CD3, CD16, CD56, CD45, CD4, CD8, CD14, CD19] and pVL [Days 1 (pre-dose and 8 h post-end of infusion), 2, 8 and 29; Abbott RealTime HIV-1 assay, Abbott m2000 system, detection limit—40 copies/mL]. All analyses were performed at a central laboratory (Q2 LabSolutions) according to the manufacturer’s instructions.

Pharmacokinetic assay
Plasma concentrations of IMC-M113V were measured using a validated electrochemiluminescence immunoassay (ECLIA). This assay format was based on step-wise capture of free IMC-M113V in patient serum samples via immobilised pHLA, followed by detection with reporter-labelled recombinant human CD3.

Pharmacodynamic activity
Serum cytokines and chemokine measurements were performed at a central laboratory (Q2 LabSolutions) in accordance with their standard operating procedures. Serum IL-6 was measured using ECLIA from Cobas C6000 (Roche Diagnostics). Serum levels of IFN-γ, TNF, IL-2, IL-8 and IL-10 were measured using the 5-plex Meso Scale Discovery electrochemiluminescence assay; the chemokine IP-10 (CXCL10) was also measured by electrochemiluminescence. LLOQ for IL-2, IL-6, IL-8, IL-10, IFN-γ, TNF, and IP-10 were 1.78 pg/mL, 2.5 pg/mL, 1.18 pg/mL, 0.60 pg/mL, 3.52 pg/mL, 1.38 pg/mL, and 2.32 pg/mL respectively. Values below LLOQ were set at the LLOQ. For analysis of T cell activation and effector function, cryopreserved PBMC were thawed, rested for 1 h at 37°C, followed by surface marker staining or, for intracellular cytokine staining, stimulation with PMA (Abcam #ab147465) and ionomycin (Merck #I3909) for 4 h. Brefeldin A (BioLegend #420601) was added after 30 mins. Cells were then stained with Live/Dead UV Blue (Thermo Fisher Scientific #L23105), followed by surface markers before permeabilisation and intracellular staining for 20 min at RT. Cells were fixed in 4% PFA, washed, and acquired on the ID7000 spectral analyser (Sony). Antibodies used in this panel are described in Supplementary Table 2. Flow cytometry analysis was performed on Omiq (OMIQ.ai) with the gating strategy shown in Supplementary Figs. 4 and 5. Data was exported from Omiq and plotted using GraphPad Prism 10.5.0.

Sequencing of gag from HIV CA-RNA
CD4+ cells were isolated from PBMC (CD4 Microbeads, Miltenyi Biotech #130-045-101), following which RNA was extracted using the RNeasy Mini Kit (Qiagen #74104). cDNA was synthesised and amplified (RT-PCR1) using SuperScript III One-Step RT-PCR System (Thermo Fisher Scientific #11732020) with Platinum Taq DNA Polymerase (Thermo Fisher Scientific #11304011) and SUPERase·In RNase Inhibitor (Thermo Fisher Scientific #AM2694) and the following primers14:
gag_F_RTPCR1: 5′-AAATCTCTAGCAGTGGCGCCCGAACAG-3′
gag_R_RTPCR1: 5′-TAACCCTGCGGGATGTGGTATTCC-3′.
The following touchdown thermal cycling conditions were used73: 50°C for 30 min followed by 94°C for 2 min and then 94°C for 30 s, 64°C for 30 s, and 68°C for 3 min (3 cycles); 94°C for 30 s, 61°C for 30 s, and 68°C for 3 min (3 cycles); 94°C for 30 s, 58°C for 30 s, and 68°C for 3 min (3 cycles); 94°C for 30 s, 55°C for 30 s, and 68°C for 3 min (41 cycles); and then 68°C for 3 min. The total reaction volume was 13.5 μL containing a maximum of 2000 ng RNA. The amplified product from RT-PCR1 was diluted with 15 μL of nuclease-free water. Two microliters of the diluted RT-PCR1 product was next amplified (PCR2) in a 25 μL reaction volume using Platinum Taq DNA polymerase High Fidelity (Invitrogen), the same thermocycling conditions as RT-PCR1 (excluding the RT 50°C for 30 min step) and the following nested primers14:
gag_F_PCR2: 5′-GCGCCCGAACAGGGACYTGAAARCGAAAG-3′
gag_R_PCR2: 5′-TATCATCTGCTCCTGTATC-3′
TapeStation 2200 (Agilent) and Qubit 3.0 (Invitrogen) were used to confirm PCR2 amplification before proceeding to library preparation using Nextera XT Library Preparation Kit (Illumina #FC-131-1096) and Nextera XT Index Kit v2 Set A (Illumina #FC-131-2001) as per the manufacturer’s protocol. Amplified indexed libraries were purified using Illumina purification beads and analysed on a TapeStation 2200 and Qubit 3.0 prior to producing a 4 nM library pool. The pooled library was paired-end sequenced on a MiSeq Sequencer using a 300-cycle MiSeq Reagent Kit v2 (Illumina).

For sequence analyses, Fastq files were processed as follows: Quality and adaptor trimming were performed using Trim Galore (v0.6.2) with default parameters, specifying the amplification primers and Nextera adaptors. FastQC was used to assess read quality. Mapping was carried out against the HIV-1 HXB2 reference annotation (obtained from NCBI, November 2022; ASM310297v1 / GCA_003102975.1) using BWA INDEX and BWA MEM (v0.7.17) with PCR duplicates removed. MultiQC was used to assess mapping statistics. iVar variants (v1.3.1) were used to call variants from the aligned BAM files, and iVar consensus (v1.3.1) was used to generate consensus sequences. SAMtools Depth (v1.3) was used to compute the alignment depth along the HIV-1 genome. Sequence plotting and visualisation were performed in R using the packages muscle (v3.38.0), seqinr (v4.2.16), ggplot2 (3.3.6) and ggmsa (v1.2.3). Variants obtained in the region of interest (HIV Gag 77–85) at a frequency of >3% were reported.

Quantification of HIV gag CA-RNA
Quantification of HIV gag CA-RNA was performed using ddPCR based on the previously published method with some modifications74. CD4+ cells were isolated from PBMC (CD4 Microbeads, Miltenyi Biotech #130-045-101), following which RNA was extracted using the RNeasy Mini Kit (Qiagen #74104) and quantified using the Qubit 3.0 and a Broad Range RNA Kit (Thermo Fisher Scientific #Q10211). A minimum of two replicates each containing up to 350 ng RNA were analysed using the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad #1864021) and the QX200 ddPCR System (Bio-Rad), following the manufacturer’s protocol. The following primers and probe were used for the quantification75:
CS-gag forward primers: 5′- GACTAGCGGAGGCTAGAAGGA GAGA’-3′
CS-gag reverse primers: 5′- CTAATTTTCCSCCDCTTAA TAYTGACG’-3′
CS-gag FAM/BHQ Probe: 5′-AT+G+GGT+GC+GAGA-3′
For some participants, primers and/or probes were customised based on the gag sequence determined in CA-RNA (Supplementary Table 3). Droplets were prepared using an Automated Droplet Generator (Bio-Rad) and cycled at 95°C for 10 min; 40 cycles of (94°C for 30 s, 59°C for 1 min) and 98°C for 10 min, using a ramp rate of 2°C to improve droplet separation. Droplets were analysed on a QX200 Droplet Reader (Bio-Rad) using QX Manager software (Bio-Rad, version 1.2). RNA of the human TBP gene was also quantified using the TaqMan Gene Expression VIC Assay (20×) from Invitrogen (Hs00427620_m1). For this purpose, RNA was diluted 20-fold, and 2 μL was used per reaction. HIV gag CA-RNA values were normalised to TBP expression and are presented as HIV CA-RNA copies/106 TBP76,77.

Quantification of intact proviral DNA
The cross subtype intact proviral DNA assay (CS-IPDA) was performed using the QIAcuity Four 5-plex system (Qiagen) as previously described with some modifications55. Briefly, CD4+ T cells were isolated from cryopreserved PBMC using the EasySep CD4 T cell isolation kit (Stemcell Technologies #17952), followed by direct lysis (DLR, Viagen #301-C) of cell pellets (~1 million cells) and XhoI restriction digestion (end volume 75 µL). Samples were analysed in triplicate with 20 µL (undiluted) input per reaction for the CS-IPDA assay that was performed without the restriction enzyme XbaI. Samples were diluted 1/100 for the RPP30 normalisation assay and analysed in triplicate (5 µL input). The same primer probe sets were used except for LTR-gag (Supplementary Table 3). Analysis was performed using the QIAcuity Software Suite 2.5.0.1 (Qiagen).

Ethical approvals for the use of human material
All uses of human material have been approved for this study. The use of PBMC from healthy donor volunteers for the in vitro investigations was approved by the Oxford A Research Ethics Committee under study protocol reference 13/SC/0226. All participants provided written informed consent.

Ethical approval for the use of samples obtained from PLWH treated with IMC-M113V was obtained through the IMC-M113V-103 study (EudraCT 2021-002008-11, 29 Oct 2021), which was approved by the relevant ethics bodies at each participating site as indicated in the Study design and participants section. All participants provided written informed consent.

Statistical analysis
In vitro cytokine release assays performed for IMC-M113V specificity assessment against whole blood and normal cells were analysed using Wilcoxon matched-pairs signed rank and Holm-Šídák multiple comparison tests (p value threshold for significance (alpha) 0.05). P values for changes from baseline (pre-dose) in the HIV CA-RNA and intact proviral DNA copies were calculated using a mixed-effects model and Šídák multiple comparison test. All analyses were performed using GraphPad Prism Version 10.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This well written article investigates the affinity of a novel bispecific TCR targeting HIV Gag and CD3 in people living with HIV. The investigators first investigate the affinity of the TCR to a range of Gag variants at position 77-85 and then assess the binding of IMC-M113V to cells transduced with HLA-2 and infected with HIV. They show high affinity to a range of variants and TCR-bound pHLA complexes on the cell surface of infected cells with a median copy number of 3/cell (range 0-14). Although significantly higher than uninfected cells, there was some background binding of 1/cell (range 0-8). There was minimal off target reactivity of IMC-M113V when cytokine production was assessed in cardiomyocytes, astrocytes or lung epithelial cells. Then in a phase 1 study in PWH with HLA02.01 alleles, they showed that IMC-M113V was safe, had minimal adverse events and there was a dose dependent increase in serum IL6, an expected pharmacodynamic effect of TCR binding. Interestingly, in participants with high affinity binding of the TCR, they showed a relationship between baseline HIV CA RNA and IL6 (but this was only 3 participants). This correlation was weaker for the 7 participants with nM variants. The manuscript is clear and well written. I only had a few minor questions and comments\n1. The in vitro model looking at binding of TCR (Supp Fig S1) in productively infected cells. Have any experiments been done with latently infected cells that undergo latency reversal? This might be a more realistic model to estimate sensitivity of binding\n2. The sequencing of gag in cell associated HIV RNA is a very interesting angle to the study. Given it was bulk sequencing, an assessment of quasispecies was probably not possible. Could limiting dilution sequencing of CA HIV also be performed?\n3. Did the investigators look at the frequency of p24+ cells in participants? The HIV flow assay could also be helpful and may be a more direct read out of the potential effects of IMC-M113V on the reservoir.\n4. In the adverse events, skin rash was quite common. What are the potential reasons for this. This should be discussed further in the discussion\n5. In figure 2a, there is very wide variation in percentage elimination of HIV infected clels. Close to one log variation in the EC50. What are the potential reasons for this?\n6. The HIV subtype of the participants should also be included in the patient demographics\n7. The modest elevation of IL6 with the 15ug dose was encouraging. What would be a safe level of IL6 from a therapeutic such as this bispecific antibody? This should be discussed further in the discussion.\n8. The potential negatives of this approach ie HLAA2 specific, gag variant escape, low presentation of gag in latency and potentially toxicities, need to be better explained for a non expert audience.", "reviewer_2": "The submitted manuscript reports on preclinical studies and results of a first in human, single ascending dose study of a novel affinity-enhanced bispecific T cell receptor (TCR) (IMC-M113V) targeting Gag-positive cells in 12 participants, as a potential strategy for HIV elimination or functional cure. The TCR is fused to a CD3-specific chain, a variable HIV peptide Gag77-85, which incorporates the most prevalent Gag 77-85 variants. As described by the authors, the concept is adapted from TCR technology applied to cancer treatment.\nThe manuscript is very well written, leading to few comments or suggestions. The study design, analysis, methods and assays used are clearly and thoroughly delineated. The authors describe the design and functionality of the novel bispecific TCR targeting cells expressing Gag well.\nResults presented indicate IMC-M113V was safe and well-tolerated as single doses, without cytokine release syndrome or ICANS. Notably and as described by the authors, this strategy appears to have measurable impact at low levels of HIV gag protein in vitro, which is relevant to strategies targeting the latent reservoir. Impact was suggested by levels of serum IL-6 which correlated with drug-sensitive virus in participants, assessed by target binding affinity. Results are to inform a subsequent study of multiple doses of IMC-M113V at higher doses.\nSpecific suggestions are listed below:\nAbstract:\n- No recommended edits\nIntroduction\n- Line 65-68. Would revise to clarify that the HIV reservoir also resides in other cells in addition to CD4+ cells.\n- Lines 80-82 – consider providing a reference to support the statement; two are provided in the next sentence but confirm both apply to both sentences.\n- Line 88-90 – consider referring to Figure 1a here to help describe/visualize the TCR receptor.\n- Line 112 – would define “PD” with first mention.\n- Consider in the Intro or discussion providing information on the prevalence of individuals, or PWH if known, who are HLA-A*02:01 positive, to help frame the impact this strategy could have, or how it could be expanded/adapted to allow it to be effective for PWH without that HLA allele.\nResults\n- Lines 143 – 146. Consider discussing as a limitation the overlap in the median copy of complexes/cell between infected and uninfected.\n- Lines 175 – 177. The numbers presented on participants prescreened in the text is n=89, but in Figure 3 it is 74. If 47 were excluded due to negative HLA-A*02:01, that leaves 27 participants (not 26). Please reconcile.\nMethods\n- Well-delineated\nDiscussion\n- Authors address the limitation of using HIV cell-associated RNA as to measure/estimate the active HIV reservoir.\nFigures\n- Would make the font size for some legends larger as they are difficult to read, i.e. Figure 5\n- Consider making the figure of the bispecific TCR more detailed and reference Figure 1a (as suggested above) to help explain the novel design.\nFigure S2 – would either describe what “index” refers to or label more specifically.", "reviewer_3": "Vandekerckhove and colleagues describe the evaluation of an experimental bispecific soluble TCR targeting a HIV-1 Gag77-85 peptide restricted by the prevalent HLA-A*02:01 epitope (IMC-M113V) in a first-in-human single administration, dose ranging study in 12 virally suppressed volunteers with chronic HIV-1 infection to assess the safety/tolerability and pharmacodynamics (PD) of the product as a novel approach to reduce the HIV-1 viral reservoir.\nThe biological plausibility of this approach was described in the Introduction as these biospecifics can target “undruggable” epitopes on T-cells as peptide and not protein recognition is sufficient; high levels of expression are not needed; intracellular peptides can be detected by cell-surface TCR detection; the product carries an scFv with an immune synapse elimination effector mechanism of action. The first in drug class biospecific ImmTAC tebentafusp is FDA approved for uveal melanoma and is being studied for reservoir reduction in chronic HBV infection as well as other pathogens. Previous in vitro studies with IMC-M113V showed promise in re-directing T-cells to eliminate HIV-1 infected cells derived from ART suppressed individuals after viral reactivation. Thus, the next step in evaluating this product as a possible drug for HIV-1 sustained viral remission was a first-in-human safety/tolerability and PD study.\nVolunteers were chosen with high circulating CD4+ T-cell levels and high historical nadirs (relatively intact immune systems); viral suppression greater than 1 year; cART duration less than 7 years (maximize target availability). Study product was directly assessed for PD. Plasma IL-6 cytokine levels were assessed as a primary marker for PD activity (immune impact) as this correlation of surrogacy was established for tebentafusp. The product was well tolerated. No cytokine release syndrome episodes were noted nor were any SAEs. Dose dependent IL-6 levels were noted and highest in volunteers with the highest IMC-M113V sensitive Gag77-85 sequences in baseline cell associated RNA. The authors conclude that IMC-M113V should advance to more extensive clinical testing as an approach to achieving a functional HIV-1 cure (sustained cART-free viremic remission).\nThe pre-clinical data demonstrate high sensitivity of the TCR with Gag binding at picomolar concentration, as well as potency against HIV-infected cells. The clinical data from a first-in-human dose escalation study, most importantly, showed few adverse events. However, there was little evidence for effector function with only an increase in IL-6 activity, but no changes in other cytokines. There was no change in cell-associated HIV RNA or HIV proviral DNA, although this would have been a high bar for a first-in-human study. The strength of the study lies in the platform (immune mobilizing monoclonal T cell receptor) which was previously used in cancer, to now target a virus. Decreasing the HIV reservoir is critical to achieving a functional cure of HIV, and new mechanisms will help achieve this goal.\nThe weaknesses of the study lie in the lack of sufficient data to demonstrate effector function. The authors use IL-6 as the marker of T-cell activity but it’s unclear why other cytokines such as IFN-y aren’t elevated. Assessing other markers of T-cell activation such as CD69 positivity would have also been helpful. Overall, the authors present a novel protein for HIV elimination using a platform that has been proven in cancer treatment, but with limited exploration of T-cell activation in humans.\n.\nAttention to the points below would strengthen an already strong manuscript.\nMajor points:\n1. A brief comparison to other CD3-HIV co-binding proteins would be helpful here (e.g., the HIV env/CD3/CD28 tri-specific antibody in Promsote et al., Nat Comm 2023).\n2. The authors include a brief discussion of bNAbs for HIV treatment, particularly for the viral reservoir. It should be noted that some studies have shown reductions in viral reservoir, particularly when started in acute HIV (e.g., Gunst et al, Nat Med 2022).\n3. Figure 2c: The choice of cytokines presented here and throughout the paper should be better justified. Here the authors use IFN-y as the marker of activity, but throughout the rest of the paper, present IL-6 as the primary marker. This is critical because of the varying responses between IFN-y and IL-6 (both elevated in vitro in Figure 2c, but variable response in humans in Figure S3). Please provide justification for using IFN-y in the off-target reactivity study.\n4. Figure 2c: The figure suggests that IMC-M113V induces less IL-6 than the anti-CD3 alone but induces similar IFN-y to anti-CD3 alone. This raises the question if this due to greater CD4+ T-cell activation in the anti-CD3 alone group. Please clarify if these are statistically significant differences, and if so, why that might be the case.\n5. Figure 2c and lines 168-172: The authors state that “No cytokine production above background levels was observed in whole blood…” This statement should be supported by a statistical test. Visually, Figure 2c shows increasing IL-6 and IFN-y production at increasing concentrations of IMC-M113V even in the absence of Gag peptide, at least in whole blood.\n6. Figure S3 and lines 280-283: Why were there few changes in other cytokines? The prior study by Middleton et al. on tebentafusp is used to justify IL-6 as a primary correlate of activation, but that study also found increased IL-10 which was not increased here. Perhaps this is due to the biomass of target in a solid tumor model vs the HIV-1 viral reservoir overall, but this should be noted by the authors here. Furthermore, the preclinical data (Figure 2c) showed dramatic increases in IFN-y which we again don’t see here, which begs the question of the degree of T-cell activation. Did the authors look at markers of T-cell activation such as CD69?\n7. Lines 322-324: It would be helpful to address the limitation of the HLA restriction. The HLA-A*02:01 restriction in uveal melanoma does not present a large barrier to utility; for HIV, it severely limits the impact. This study is certainly an excellent proof-of-concept, but there is little overlap globally between the specific HLA subtype and HIV prevalence (Olivier, JAMA Net Open 2023).\n8. Better define the strengths and weakness of measuring impact on the viral reservoir. While the authors are appropriately circumspect about making much of a safety and PD study, they do employ an intact proviral DNA assay (IPDA) as an indication of reservoir impact. I would strongly encourage this team to collaborate with labs that are skilled in reservoir measurements (Siliciano, Trautmann, Deeks, Douek, Chaumont, etc.). For the next phase of clinical evaluation, the team would probably want to use culture-based assays such as quantitative viral outgrown assays (QVOA) which are as close to the gold standard now in 2025. There are others such as TILDA, etc.\n9. Strongly consider using scRNASeq for next studies to better define surrogates of activity.\n10. Taken together, I would urge the authors to note that these are future directions as the clinical development plan matures.\nMinor points:\n1. Figure 1d: it is surprising that half of the uninfected cells had >1 pHLA-TCR complex. This is only slightly lower than a median of 3 in infected cells. but I wonder how prevalent the Gag peptide used here is in uninfected cells.\n2. Table 1 and Figure 5: It is worth noting that one patient was previously a long-term non-progressor, and that same patient also had an actively decreasing CD4 count (lower at enrollment than prior nadir by 100 cells/mL). Furthermore, this patient also had the greatest fold increase in IL-6. Is this a coincidence or is there something unique in this patient’s T-cell response?\n3. Discussion: The authors state, “We anticipate that a multiple dose schedule incorporating higher doses with be needed.” This certainly appears to be true, and as such, but it is best to not overstate the lack of off-target binding. First, it is not certain that there is no off-target binding without statistical tests in Figure 2c, and even so, higher doses may result in more off-target binding.\n4. Methods: The authors should justify the use of pre-treatment with an antipyretic and antihistamine for all volunteers and note this limitation when assessing the adverse events data.\n5. Figure 5c and S3: The authors can consider presenting these figures with absolute values rather than fold-change, which can be difficult to interpret given the LLOQ.\n6. Define the acronym PD as pharmacodynamics at first mention.", "reviewer_4": "The authors present preclinical results and an open-label single dose escalation study of a first in-human TCR bispecific molecule targeting HIV Gag epitope 77-85 in HLA-A*02+ individuals on ART. The treatment was administered to 12 participants and follow-up was conducted at various timepoints for 29 days. The data suggests the molecule does not have off-target effects and clears cells that present the epitope by HLA-A*02. The treatment is well-tolerated, and the authors evaluated the pharmacodynamics of the agent in vivo. The findings show there was no impact on the reservoir/proviral load using the CS-IPDA assay, even transiently, in any patient. Overall, the manuscript is clear, concise and well written. Major and minor comments for the authors are highlighted below:\nMAJOR:\n1. It is unclear whether, in the authors' opinion, there is any signal of efficacy to justify an ongoing study testing this agent at higher dose levels. While it is appreciated that the safety signal is high, without any signal of efficacy it is unclear how dose escalating with the increased toxicity expected, is justified. This should be discussed along with the potential reasons (other than dose) why no signal of efficacy was observed?\n2. While this approach has been used with success in melanoma, the approach in HIV is yet to be realised. Further, it is telling that the majority of participants enrolled on this study are white which is the population where HLA A0201 is the most prevalent. Can the authors speculate on the broader impact of this HLA A0201-restricted therapy to the PLWH population given the prevalence of the virus in the African continent? This also warrents discussion.\nMINOR:\n- Please confirm that all participants were high resolution typed at A0201. And if not, please jusitfy why.\nIn the abstract, the authors should specify primary and secondary endpoints assessed.\n• Lines 125-128: The IMC-M113VRES molecule should be more clearly introduced. Authors should note Y79F is the most prevalent variant when it is first mentioned (which is stated later in line 158).\n• Lines 129-135: Authors should specify how many variants were identified and how many they tested.\n• Lines 153-156: These lines should be rewritten for clarity as it was hard to follow.\n• Line 154: C8166 is introduced here as an HIV-infected CD4+ T cell line, but this should be mentioned sooner in line 142.\n• Line 176: The participant numbers in this line differ from the CONSORT diagram in Fig. 3. This line states eligible participants is n=89, however Fig. 3 shows n=74. This line states the HLA-A*02+ participants is n=33, however Fig. 3 shows n=26.\n• Line 177: The authors should specify the screening criteria that resulted in 4/16 failing the screen. Additionally, these criteria should be added to Fig. 3, as exclusion criteria is listed in the figure too.\n• Line 244-246: These lines should be rewritten to improve flow and clarity, and specify the pM and nM affinity ranges.\n• Line 246: The authors should specify the pM affinity variant.\n• The IMC-M113VRES molecule was assessed often in the Results section but was not discussed in the Discussion.\n• Line 433-435: Equations should be referenced in the main text.\n• Line 466: This states the criteria threshold is 50% participants, but states the minimum is n=4; should it be n=6?\n• Line 519-520: Amend Supplementary Table 2 to add all fluorophores listed in these lines.\n• Some other limitations should be addressed in the discussion: n=1 in Cohorts 1 and 2, and the short window of follow-up post-infusion.\n• The journal expects that the title and/or abstract must indicate when findings apply to only one sex or gender.\n• The methods section should include whether sex and/or gender were considered in the study design.\n• Add n values (# participants or replicates) to all figure legends where applicable.\n• For the present study, the ART regimens reported in Table 1 do not seem necessary to reveal.\n• CONSORT checklist not coinciding with correct page numbers in manuscript.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Referees\nWe would like to thank the reviewers for their constructive comments and suggestions.\nReviewer 1\nAlthough significantly higher than uninfected cells, there was some background binding of 1/cell (range\n0-8).\nThe background binding seen with the uninfected cells (HIV pHLA-negative) in Figure 1d can be\nattributed to the non-specific binding of the goat anti-rabbit*CF640R secondary antibody. The infected\nC8166 cells stained with only the secondary antibody (no TCR) show the same 0-8 pHLA/cell range seen\non the uninfected cells stained with TCR + secondary antibody.\n• For clarity, we have amended the figure 1d legend to explain the ‘no TCR’ control.\nThe manuscript is clear and well written. I only had a few minor questions and comments.\n1. The in vitro model looking at binding of TCR (Supp Fig S1) in productively infected cells. Have any\nexperiments been done with latently infected cells that undergo latency reversal? This might be a more\nrealistic model to estimate sensitivity of binding\nWe agree that this is a more realistic model. Work is ongoing to improve sensitivity of the imaging\nmethod sufficiently to detect HIV peptides on ex vivo infected cells.\n• We also respectfully refer the reviewer to our prior publication: Elimination of Latently HIV-\ninfected Cells from Antiretroviral Therapy-suppressed Subjects by Engineered Immune-\nmobilizing T-cell Receptors. Yang H et al. Molecular Therapy 2016, doi: 10.1038/mt.2016.114.\nThis publication contains comprehensive data indicating that the TCR binds to latently infected cells\nthat have undergone latency reversal. We showed specific elimination of ex vivo CD4+ T cells from\nART-treated PLWH, after activation in vitro to reverse latency. CD4+ T cells were co-cultured with\nautologous or allogenic CD8+ T cells together with a precursor molecule that is almost identical to\nIMC-M113V (same specificity and affinity). Dose-dependent elimination was observed, indicating\nsensitivity of TCR binding to infected cells after latency reversal.\n2. The sequencing of gag in cell associated HIV RNA is a very interesting angle to the study. Given it was\nbulk sequencing, an assessment of quasispecies was probably not possible. Could limiting dilution\nsequencing of CA HIV also be performed?\nBulk sequencing of the active reservoir was performed to identify the dominant target sequence in\nparticipants in an attempt to explore potential associations with pharmacodynamic responses. This\nmethod successfully identified low frequency target variants in some of the participants (Supplementary\nFig 5) but has limitations, which we acknowledge.\nLimiting dilution sequencing of HIV CA-RNA is an excellent suggestion, however, it was unfortunately\nnot possible to do this for this single ascending dose (SAD) study due to sample limitations.\nIn the longer term, the impact of quasispecies on the efficacy of IMC-M113V will be an important area of\ninvestigation. Limiting dilution methods, along with other approaches such as near full length sequencing,\nwill certainly be powerful approaches to support this.\n• We have amended the Discussion to address the reviewer’s point (lines 351-363).\n3. Did the investigators look at the frequency of p24+ cells in participants? The HIV flow assay could\nalso be helpful and may be a more direct read out of the potential effects of IMC-M113V on the reservoir.\n1. We agree that a protein-based assay would provide a more direct read out of IMC-M113V effects on\nthe reservoir and is therefore highly desirable. We appreciate the suggestion to do the HIV flow\nassay; however, we would like to highlight that Pardons et al. employed leukapheresis, which we\ninfer was to ensure that the cell input in the assay was sufficient to yield a reproducible signal because\nthe frequency of p24+ cells was so low (doi: 10.1371/journal.ppat.1007619). Specifically, a signal\nwas detected in ART-treated individuals only after activation of CD4+ T cells with PMA/ionomycin\nand the median (IQR) frequency was 4.3 (0.7-8) per million cells.\nSimilarly, a FISH-Flow assay was successfully used by Gunst J et al. to demonstrate small but\nstatistically significant reductions in the proportion of p24+ cells in participants in the eCLEAR study\n(https://doi.org/10.1038/s41591-022-02023-7). However, these participants were ART-naïve at baseline\nand thus had a measurable baseline signal (10-100-fold higher than in ART-treated individuals by HIV\nflow, Pardons et al.) that enabled detection of a post-intervention reduction.\nWe have not implemented the HIV flow or FISH-flow assay yet, as the majority of the current trial sites\ndid not have access to leukapheresis, but these assays are under consideration for future studies.\n• We have amended the Discussion to provide an explanation for why a p24+ assay was not used in this\nstudy (lines 372-376)\n4. In the adverse events, skin rash was quite common. What are the potential reasons for this. This should\nbe discussed further in the discussion\nThe majority of adverse events affecting skin (n = 5/6) were observed in one participant. The skin redness\nwas procedure-related (a reaction to adhesive on the ECG electrodes); the herpes simplex episode\noccurred 5 days after study drug administration in a participant who had a prior history of HSV infection\nand was therefore deemed a recurrent episode. Three events (eczema and itch) occurred 20 days after\nadministration of IMC-M113V and were therefore deemed unlikely to be related, based on the lack of\ntemporal association, the molecule half-life of <18 hours and the lack of biological plausibility.\nAn episode of phlebitis occurred in another participant 16 days after IMC-M113V administration and was\nrecorded as possibly related to IMC-M113V by the investigator. However, the Sponsor’s determination\nwas that this was also unlikely to be related, for the same reasons given for the events described above.\n• We have clarified this in the Results section (lines 200-204) and have added a statement to the\nDiscussion summarising these details (lines 400-405).\n5. In figure 2a, there is very wide variation in percentage elimination of HIV infected cells. Close to one\nlog variation in the EC50. What are the potential reasons for this?\nThe percent elimination in this assay indicates that IMC-M113V is highly potent in vitro (EC values\n50\n1.2-13.1 pM). We would like to highlight that despite a ~1 log variation in EC , the values reflect\n50\nextremely low drug concentrations (equivalent to ~0.1-1.0 ng/ml), which can accentuate any expected\ninter-donor variability. The inter-donor variability likely relates to variation in the proportions in CD8+ T\ncell subsets across the donors (particularly effector memory cells, as reported in Yang H et al. 2016).\n6. The HIV subtype of the participants should also be included in the patient demographics\n• We have added this information to Table 1.\nPlease note that this was obtained from viral resistance tests performed at clinical sites as part of standard\nof care and a viral subtype was not available for all participants.\n7. The modest elevation of IL6 with the 15ug dose was encouraging. What would be a safe level of IL6\nfrom a therapeutic such as this bispecific antibody? This should be discussed further in the discussion.\nThank you for the question. In our opinion, a safe threshold cannot be defined because: 1) while IL-6 is a\nkey mediator of toxicity in CRS, the pathology is driven by a cascade of immune activation events, with\nother cytokines and various immune cell populations contributing to organ dysfunction; 2) patient-\nspecific factors can increase the risk for CRS of Grade ≥ 2 (hypotension or hypoxia), eg. lung disease,\nadrenal insufficiency; 3) our oncology studies show that there is overlap in the range of IL-6 levels across\nseverity grades.\nIn case of interest, the mean (SEM) level of serum IL-6 at 8 hours post-dose in patients receiving\ntebentafusp for metastatic cutaneous (mCM) or uveal (mUM) melanoma is shown below (Hamid, O. et al.\nTumor microenvironment (TME) features and serum cytokines in patients with metastatic uveal and\ncutaneous melanoma treated with tebentafusp. EJC Skin Cancer, Volume 2, 100140).\nSerum IL-6 (pg/ml) - mean (SEM)\nmCM mUM\nNo CRS 24.1 (7.7) 20.3 (12.8)\nGrade 1 30.7 (9.2) 433.7 (159.5\nGrade 2 126.8 (55.2) 1267 (377.5)\nGrade ≥ 3 N/A 34071.6 (32543.5)\nThese data provide some insight into the relationship between serum levels and CRS severity grade.\nHowever, for the reasons outlined above, avoidance of CRS depends on careful monitoring for clinical\nsymptoms and signs, particularly at the first dose, and serum cytokine levels are confirmatory rather than\npredictive.\nThe duration of the cytokine response is also pertinent. The IL-6 elevations in this study were transient,\n(consistent with the serum half-life of IMC-M113V), therefore, it is expected any cytokine-related\nadverse events – even at doses >15 µg - would be transient, as has been noted with tebentafusp in patients\nwith uveal melanoma (https://doi.org/10.1158/1078-0432.CCR-25-1513).\n• We have included a statement in the Results on the highest IL-6 value recorded in this study (lines\n244-245) and modified the text in the Discussion to link the lack of CRS to the low levels of\ncytokines observed (lines 315-316). As CRS could also be the result of off-target toxicity, we have\nadded text to highlight the importance of monitoring for this when evaluating higher doses and\nmultiple dose schedules (lines 405-406).\n8. The potential negatives of this approach ie HLA-A2 specific, gag variant escape, low presentation of\ngag in latency and potentially toxicities, need to be better explained for a non expert audience.\nWe acknowledge these potential limitations of the approach and have expanded the discussion to explain\nhow these have been or will be addressed through clinical development.\n• Considerations for expanding to other HLA alleles beyond HLA-A2 are addressed in lines 422-430.\n• Overcoming and / or mitigating Gag variant escape and low presentation of Gag in latency are\naddressed in lines 288-306 and lines 413-422.\n• Toxicities and mitigation strategies are discussed in lines 319-324 and lines 388-406.\nReviewer 2\nComments on the Introduction:\n- Line 65-68. Would revise to clarify that the HIV reservoir also resides in other cells in addition to\nCD4+ cells.\n• We have amended this sentence as suggested (now lines 64-66).\n- Lines 80-82 – consider providing a reference to support the statement; two are provided in the next\nsentence but confirm both apply to both sentences.\n• We have added references to this sentence (now lines 78-84).\n- Line 88-90 – consider referring to Figure 1a here to help describe/visualize the TCR receptor.\n• Thank you for the suggestion – now included.\n- Line 112 – would define “PD” with first mention\n• This has been corrected (now line 114).\nConsider in the Intro or discussion providing information on the prevalence of individuals, or PWH if\nknown, who are HLA-A*02:01 positive, to help frame the impact this strategy could have, or how it could\nbe expanded/adapted to allow it to be effective for PWH without that HLA allele.\nThank you for the suggestion.\n• We have clarified the prevalence of HLA-A*02:01 in the Results section (lines 179-181) and\nexpanded the Discussion to explain the estimated prevalence of HLA-A*02:01 in PLWH, including\nestimates based on recent high-resolution HLA typing in selected populations in East and Southern\nAfrica (lines 424-425).\n• We have added a statement to explain that the ImmTAX platform can be deployed to produce TCR\nbispecific molecules that target other HLA alleles, with references to support this (lines 428-430).\nResults: Lines 143 – 146. Consider discussing as a limitation the overlap in the median copy of\ncomplexes/cell between infected and uninfected.\nPer our response to Reviewer 1, there is some background binding with the uninfected cells (HIV pHLA-\nnegative), which can be attributed to the non-specific binding of the goat anti-rabbit*CF640R secondary\nantibody, as indicated by the same pHLA/cell range seen in the ‘no TCR’ control.\n• We have amended the text in the Results (lines 149-151) to reflect this point.\nIn addition, some overlap in the signal from infected and uninfected experimental conditions could also\nreflect the presence of p24-low cells in the former (Fig. 1c shows that the MFI of p24 expression in\ninfected cells spans a 1-2 log range), together with a range in the level of HLA class I on the cell surface.\nThe reported range in pHLA copies/cell cannot be further refined, which is a limitation of this method.\nResults: Lines 175 – 177. The numbers presented on participants prescreened in the text is n=89, but in\nFigure 3 it is 74. If 47 were excluded due to negative HLA-A*02:01, that leaves 27 participants (not 26).\nPlease reconcile.\nThank you for pointing out this error. The text is correct and Figure 3 has been corrected.\nDiscussion\n- Authors address the limitation of using HIV cell-associated RNA as to measure/estimate the active HIV\nreservoir.\n• This is discussed in lines 380-382.\nFigures\n- Would make the font size for some legends larger as they are difficult to read, i.e. Figure 5\n• We have increased the font size in the graphs in all figures.\n- Consider making the figure of the bispecific TCR more detailed and reference Figure 1a (as suggested\nabove) to help explain the novel design.\n• We have amended Figure 1a and included more details to explain the design and mechanism. We\nhope these changes have helped to make it clearer.\nFigure S2 – would either describe what “index” refers to or label more specifically.\n• We have changed ‘index’ to ‘cognate’ and amended the figure legend to explain what this means.\nReviewer 3\nGeneral: The weaknesses of the study lie in the lack of sufficient data to demonstrate effector function.\nThe authors use IL-6 as the marker of T-cell activity but it’s unclear why other cytokines such as IFN-y\naren’t elevated. Assessing other markers of T-cell activation such as CD69 positivity would have also\nbeen helpful. Overall, the authors present a novel protein for HIV elimination using a platform that has\nbeen proven in cancer treatment, but with limited exploration of T-cell activation in humans.\nThis SAD study achieved its primary objective by identifying the 15 µg dose as a suitable initial step dose\nto test in a subsequent multiple ascending dose (MAD) study. Demonstration of T cell effector function\nwas an exploratory objective that will be assessed in depth in the ongoing MAD study. Nevertheless, we\nappreciate the concern regarding the lack of impact on cytokines other than IL-6 and the lack of analysis\nof effector function beyond IL-6. We have addressed this in our response to comments #3 and #6 below.\n1. A brief comparison to other CD3-HIV co-binding proteins would be helpful here (e.g., the HIV\nenv/CD3/CD28 tri-specific antibody in Promsote et al., Nat Comm 2023).\nWe appreciate the suggestion. The HIV trispecific antibody, N6/αCD3-αCD28, binds to HIV Env.\nPromsote et al. reported that this antibody elicited high levels of CD4+ and CD8+ T cell activation in the\npresence of HIV-infected cells in vitro, with evidence of some activation in the absence of HIV. The\nmolecule also elicited HIV antigen expression and T cell activation in ex vivo assays with PBMC from\nART-treated individuals. In an in vivo study, naïve Rhesus macaques developed high levels of T cell\nactivation after IV or SC administration of the antibody, together with broad induction of\nproinflammatory chemokines and cytokines to substantial levels that were, surprisingly, not associated\nwith adverse events. However, a similar trispecific antibody, αCD38/αCD3-αCD28 (SAR442257), was\ntested in a Ph1 trial in refractory / relapsed multiple myeloma and non-Hodgkin’s lymphoma. The study\nwas terminated during dose escalation due to safety events (CRS in over half the participants, recurrence\nof CRS, and high rates of CMV and EBV reactivation). https://doi.org/10.1182/blood-2024-204614\nThis study highlights the risk of combined CD3/CD28 agonism, even with monovalent antibodies, and in\nour opinion, confirms that the high levels of T cell activation in the NHP study were concerning, despite\nthe apparent lack of adverse effects.\nTwo other T cell engager approaches are briefly summarized below, in case of interest to the reviewer. As\nthere are no clinical publications on these we have not discussed them in our manuscript.\n- HIV dual-affinity retargeting agents (DARTs) comprise Env-CD3 co-binding proteins and were\nshown to specifically engage HIV-infected target cells in vitro and re-direct T cell killing of these\ntargets in vitro and ex vivo (reviewed by Nordstrom et al. https://doi.org/10.1016/j.jve.2022.100083).\nIn SHIV-infected macaques on ART, the DART molecules bound to circulating T cells without\ncausing T cell activation or cytokine induction. No impact on viral parameters was observed. A first-\nin-human study evaluating single and multiple dose schedules of one of the HIV DARTs (MGD014)\nin PLWH reported that the molecule did not induce activation markers or serum cytokines despite\nbinding to the majority of CD4+ and CD8+ T cells in the blood (Norstrom J et al., IAS 2022, abstract\n# OAA0403).\n- Sengupta et al (doi: 10.1073/pnas.2123406119) reported on the in vitro evaluation of a CD3\nbispecific incorporating a TCR mimic that binds an HIV Pol peptide in complex with HLA-A*02:01\nas the targeting arm. The molecule was potent against infected primary CD4+ T cells in vitro, but to\nthe best of our knowledge no clinical trials have been initiated.\n2. The authors include a brief discussion of bNAbs for HIV treatment, particularly for the viral reservoir.\nIt should be noted that some studies have shown reductions in viral reservoir, particularly when started in\nacute HIV (e.g., Gunst et al, Nat Med 2022).\n• We have noted this and have amended the text in the introduction to include reference to Gunst et al.\n2022 (lines 78-84). The study by Gaebler et al., which showed a statistically significant reduction in\nthe intact intact reservoir, is also cited (lines 82-84 and reference #16).\nWe note that Gunst et al reported a reduction in intact proviruses in the eCLEAR study, which\nassessed bNAbs +/- HDAC inhibitor in PLWH at ART initiation versus ART alone. In this well-\ndesigned trial, they also reported a significant decrease in intact proviruses from baseline through one\nyear in the ART-only arm. Of interest, there was also a reduction in the active reservoir (proportion of\np24+ cells) in two of the intervention arms. Because eCLEAR enrolled a mix of people with recent\n(<6 months) and chronic infection it is difficult to ascertain the relative contributions that infection\nrecency and administration of bNAbs at the time of ART initiation – a unique aspect of this study -\nmade to the reported outcomes.\nWe are also aware of the data from the RIO and FRESH studies that showed a positive effect of bNAbs\non post-treatment control in PLWH who initiated ART during acute infection. To our knowledge these\nstudies have not yet been published.\n3. Figure 2c: The choice of cytokines presented here and throughout the paper should be better justified.\nHere the authors use IFN-y as the marker of activity, but throughout the rest of the paper, present IL-6 as\nthe primary marker. This is critical because of the varying responses between IFN-y and IL-6 (both\nelevated in vitro in Figure 2c, but variable response in humans in Figure S3). Please provide justification\nfor using IFN-y in the off-target reactivity study.\n• We have amended the Results (lines 170-172 and 224-232) to provide a clearer explanation of the\nrationale for the choice of read-outs for the preclinical and clinical data that are presented in the\nmanuscript.\nIn brief, in this manuscript a variety of read-outs were used to evaluate IMC-M113V preclinically and\nclinically. IFN-γ is a sensitive and direct measure of T cell activation in vitro and was therefore used for\ninitial screening for potency (on-target activity) of IMC-M113V (Figure 1, Supp Figure S1b) and off-\ntarget reactivity in assays with a panel of normal cells (Figure 2c). A granzyme B Elispot assay was also\nperformed in parallel; this was not included in this manuscript because the results were similar to those\nobtained with the IFN-γ assay. For the whole blood assay, we used a panel of cytokines - IFN-γ, TNF-α,\nIL-6, IL-2 and IL-1β - but included only the IFN-γ and IL-6 data in Figure 2c, as similar results were\nobtained with the other cytokines.\n• We can include the data for the other cytokines evaluated in the preclinical assays if the reviewer\nconsiders this to be essential.\nFor the clinical study we evaluated changes in multiple serum cytokines as a secondary endpoint. (A copy\nof the study protocol is provided with this submission). A 4-fold increase in serum IL-6 was selected as a\ncriterion of biological (pharmacodynamic) activity to inform dose escalation decisions. This choice of\ncytokine was based on clinical data with tebentafusp, which showed substantially greater fold changes in\nserum IL-6 than IFN-γ in patients with metastatic uveal melanoma (mean ~35-fold increase for IL-6 vs.\n7-fold for IFN-γ (Middleton M et al. 2020, doi: 10.1158/1078-0432.CCR-20-1247 and Carvajal R et al.\n2022, doi: 10.1200/JCO.21.01805). The same phenomenon was observed in this SAD study.\n• This is explained in lines 224-228 and discussed in lines 325-330.\n4. Figure 2c: The figure suggests that IMC-M113V induces less IL-6 than the anti-CD3 alone but induces\nsimilar IFN-y to anti-CD3 alone. This raises the question if this due to greater CD4+ T-cell activation in\nthe anti-CD3 alone group. Please clarify if these are statistically significant differences, and if so, why\nthat might be the case.\nThis is an intriguing observation. We have not investigated this because the experimental condition in\nquestion is the Gag peptide (‘pep.’) positive control, in which target cells are saturated with peptide\n77-85\n(10 µM) and cultured with PBMC effectors plus a very high concentration (5 nM) of IMC-M113V.\nWe wish to emphasise that the anti-CD3 antibody condition is included only as a second positive control and\nthere was no intent to make comparisons between these two controls.\n5. Figure 2c and lines 168-172: The authors state that “No cytokine production above background levels\nwas observed in whole blood…” This statement should be supported by a statistical test. Visually, Figure\n2c shows increasing IL-6 and IFN-y production at increasing concentrations of IMC-M113V even in the\nabsence of Gag peptide, at least in whole blood.\n• Thank you for the recommendation. We have included a statistical test and amended the Results text\n(lines 172-175) and figure legend.\nWe would like to highlight that there was no significant increase in IL-6 in the whole blood assay, nor\nIFN-γ in the normal cell screen in the presence of IMC-M113V. There was a significant increase in\nIFN-γ in the whole blood assay at the 10 nM concentration only. As this concentration is outside the\nanticipated clinical dose range (based on the range of drug concentrations at which there is no broad\nloss of specificity against a panel of normal cell types), this result is not considered clinically\nrelevant. We have included a statement to clarify this in the Results (lines 175-176).\n6. Figure S3 and lines 280-283: Why were there few changes in other cytokines? The prior study by\nMiddleton et al. on tebentafusp is used to justify IL-6 as a primary correlate of activation, but that study\nalso found increased IL-10 which was not increased here. Perhaps this is due to the biomass of target in\na solid tumor model vs the HIV-1 viral reservoir overall, but this should be noted by the authors here.\nFurthermore, the preclinical data (Figure 2c) showed dramatic increases in IFN-y which we again don’t\nsee here, which begs the question of the degree of T-cell activation.\nWe agree with the comment that the size of the active HIV-1 reservoir (i.e. the fraction that expresses\nviral antigens) is likely to represent a small biomass of target relative to that of a solid tumour, and is\ntherefore a key factor determining the magnitude of all cytokine responses in the study participants. This\nalso explains the apparent disconnect between the IFN-γ signal in vitro and in the clinical study.\n• We have amended the Discussion to make the point that the target burden (reservoir size) and low\ndose is one of the likely explanations for the minimal changes in IFN-γ and other cytokines including\nIL-10 (lines 316-319).\nIn Figure 2c, the strong IFN-γ signal from the positive control reflects both high target expression (cells\npulsed with a high concentration of peptide) and high IMC-M113V concentration (5 nM), both of which\ndo not reflect conditions in PLWH (intentionally, given that this is a positive control for the cross-\nreactivity assay).\nThe other key determinant is dose. This is borne out by emerging data from our ongoing MAD study\n• We refer the reviewer to data showing dose-dependent cytokine elevations (presented by Mothe B et\nal. at CROI 2025, abstract #104; https://www.croiconference.org/abstract/1861-2025/) which are\nappended to this document, together with additional confidential data.\nThese data show that a consistent increase in IFN-γ is observed only at doses ≥ 300 µg, and typically\nis only ~2-fold relative to baseline, whereas at lower target doses (60 and 120 µg), only 1/5\nparticipants showed an increase of similar magnitude. In this SAD study, only 1/10 participants in the\n15 µg dose cohort showed a >2-fold increase above pre-dose levels (Supplementary Fig. 3), which is\nconsistent with the trend observed in the ongoing MAD study.\nThe lack of IL-10 induction in this SAD study (vs. Middleton 2020) is consistent with the lack of\ninduction of IFN-γ in the majority of participants, since IL-10 typically follows IFN-γ in acute\ninflammatory responses (as shown by Stacey A et al, 2009; https://doi.org/10.1128/jvi.01844-08).\nThe known sensitivity of IL-6 as an indirect biomarker of T cell activation was the justification for its use\nin guiding dose escalation decisions (as explained in our response to Comment #3 above). The observed\nresponse rate of a >4-fold increase in 5/10 participants in the 15 µg cohort in this SAD study bears this\nout.\n• We address the apparent disconnect between clinical IL-6 and IFN-γ responses (and other cytokines)\nin the Discussion. We include a reference to a study of blinatumomab (CD3xCD19 bispecific) in\nminimal residual B-lineage acute lymphoblastic leukemia (MRD+ B-ALL), a low tumour burden\nsetting, where elevations in IL-6 without a concomitant IFN-γ were observed in several patients (lines\n342-350).\nDid the authors look at markers of T-cell activation such as CD69?\n• We appreciate the question. To address the reviewer’s concern, we have included new data in the\nmanuscript (shown below and now included in Figure 5) providing evidence for CD4+ and CD8+ T\ncell activation and upregulation of cytolytic molecules post-dosing.\nThis was performed in a subset of participants from the 15 µg dose cohort, based on sample availability.\nThe Results (lines 249-257), Discussion (lines 339-344) and Methods (lines 625-635) have been updated\nand the gating strategy is included as a new supplementary figure (Figure S4). In the Discussion we make\nthe point that the changes in CD69 expression are commensurate with the low dose and anticipated low\ntarget expression. We believe this is a reasonable conclusion given that no change in T cell activation\nmarkers was observed in PLWH in the Phase 1 study of MGD014, the Env-specific DART, discussed in\npoint #1 above.\n7. Lines 322-324: It would be helpful to address the limitation of the HLA restriction. The HLA-A*02:01\nrestriction in uveal melanoma does not present a large barrier to utility; for HIV, it severely limits the\nimpact. This study is certainly an excellent proof-of-concept, but there is little overlap globally between\nthe specific HLA subtype and HIV prevalence (Olivier, JAMA Net Open 2023).\nThank you for this comment and for mentioning the Olivier et al. study, which we agree makes a critical\npoint regarding the risk of racial and ethnic inequities in trial eligibility for HLA-based therapeutics and\nthe need to find solutions. Specifically, the development of HLA-based therapeutics for HIV must address\ndifferences in HLA allele frequencies in the African continent vs. EU/US.\nRegarding the comment that the HLA restriction severely limits impact, this was also raised by Reviewers\n1 and 2. However, allele frequencies alone do not provide a complete picture and consideration of the\nabsolute numbers of potentially eligible patients is important. We outline these below, basing our\nestimation on recently published high resolution HLA typing in Eastern and Southern African\npopulations (Banjoko et al, 2025. https://doi.org/10.1038/s41598-025-06704-4).\n- The HLA-A*02:01 allele frequency in this study was 5% in S. Africans, vs. 11% in African\nAmericans and 22% in European Americans (broadly similar to Olivier et al).\n- Considering that there are an estimated 7.7 million adults living with HIV in S. Africa, a potential\n~0.75 million PLWH could be eligible for treatment with IMC-M113V based on HLA-A*02:01\nprevalence. This is greater than the estimated number of HLA-A*02:01-positive PLWH in EU5 and\nUSA combined (0.65 million, adjusted for demographic mix in these countries).\nBanjoko et al. also show that HLA-A*02:01 is among the top 5% most frequent alleles in Eastern and\nSouthern African populations, therefore, adequate population coverage will likely require targeting HLA-\nA*02:01 alongside other prevalent alleles in these regions.\n• We have expanded the Discussion to discuss how the restriction of the current drug to HLA-A*02:01\nalone can be overcome (lines 422-430).\n8. Better define the strengths and weakness of measuring impact on the viral reservoir. While the authors\nare appropriately circumspect about making much of a safety and PD study, they do employ an intact\nproviral DNA assay (IPDA) as an indication of reservoir impact. I would strongly encourage this team to\ncollaborate with labs that are skilled in reservoir measurements (Siliciano, Trautmann, Deeks, Douek,\nChaumont, etc.). For the next phase of clinical evaluation, the team would probably want to use culture-\nbased assays such as quantitative viral outgrown assays (QVOA) which are as close to the gold standard\nnow in 2025. There are others such as TILDA, etc.\n• We have expanded the Discussion to better address the strengths and weaknesses of measuring\nimpact on the viral reservoir (lines 364-384).\nWe greatly value the experience and expertise of labs skilled in reservoir measurements and will continue\nto liaise with experts in this area. We appreciate the advice on the alternative reservoir assays (measuring\nthe replication competent reservoir and the frequency of cells with msRNA) and will aspire to expand on\nthe analyses described here in future studies.\n9. Strongly consider using scRNASeq for next studies to better define surrogates of activity.\nWe appreciate the suggestion. We are intending to incorporate this in the ongoing MAD study.\n10. Taken together, I would urge the authors to note that these are future directions as the clinical\ndevelopment plan matures.\nThank you for your constructive comments and suggestions.\nMinor points\n1. Figure 1d: it is surprising that half of the uninfected cells had >1 pHLA-TCR complex. This is only\nslightly lower than a median of 3 in infected cells. but I wonder how prevalent the Gag peptide used here\nis in uninfected cells.\nThe Gag peptide is not present in uninfected cells. The background binding seen with the uninfected cells\n(HIV pHLA-negative) can be attributed to the non-specific binding of the goat anti-rabbit*CF640R\nsecondary antibody. The infected C8166 cells stained with only the secondary antibody (no TCR) show\nthe same 0-8 pHLA/cell range as the uninfected cells stained with TCR + secondary antibody.\n• We have amended the text in the Results (lines 149-151) to clarify this point.\nAs clarified in our response to Reviewer 2, the low median pHLA copy/cell in the infected cell condition\nand the overlap in this signal with uninfected cells could also reflect the presence of p24-low cells in the\nformer (Fig. 1c shows that the MFI of p24 expression in infected cells spans a 1-2 log range), together\nwith a range in the level of HLA class I expression on the cell surface. The reported range in pHLA\ncopies/cell cannot be further refined, which is a limitation of this method.\n2. Table 1 and Figure 5: It is worth noting that one patient was previously a long-term non-progressor,\nand that same patient also had an actively decreasing CD4 count (lower at enrollment than prior nadir\nby 100 cells/mL). Furthermore, this patient also had the greatest fold increase in IL-6. Is this a\ncoincidence or is there something unique in this patient’s T-cell response?\nThe CD4 nadir in this participant was >4 years prior, before he started ART. Values for Screening,\nWeek 1 and Week 2 were 760, 474 and 613 cells/µL (all within normal range). This degree of fluctuation is very\ncommon, irrespective of HIV status, and does not indicate an actively decreasing CD4 count. It likely\nreflects fluctuations in absolute lymphocyte count since the CD4% values for the same time points were\n40.2, 42.7, 39.6% (also all within normal range). We state in the Results that two participants (including\nthis one) had a CD4 count <500 cells/µL on Day 1 despite normal values at Screening (lines 207-209).\n• We have amended the footnote in Table 1 to clarify that the count was normal at Screening.\nIt is intriguing that this individual had the greatest fold increase in IL-6. Given that they had lost their\nlong-term non-progressor status several years prior to enrolling in the study, we have avoided speculating\non the relevance of any distinct immunological features of this phenotype. However, identifying baseline\ncovariates that may predict response to treatment is an important goal for ongoing and future studies.\n3. Discussion: The authors state, “We anticipate that a multiple dose schedule incorporating higher\ndoses with be needed.” This certainly appears to be true, and as such, but it is best to not overstate the\nlack of off-target binding. First, it is not certain that there is no off-target binding without statistical tests\nin Figure 2c, and even so, higher doses may result in more off-target binding.\nThank you for this comment. As stated in our response to your major comment #5, we have included a\nstatistical test in the text and figure legend and amended the Results text.\n• The text now reads: No statistically significant cytokine production above background levels was\nobserved in whole blood, or in co-cultures of immune cells and cardiomyocytes, astrocytes or lung\nepithelial cells, when incubated with IMC-M113V at concentrations below 10 nM. As the\nbiologically active dosing range was anticipated to be 0.1-1 nM, the reactivity at 10 nM was not\nconsidered to be a concern.\nWe agree that the risk of off-target binding may be increased at higher doses, however, the extensive\nnormal cell screening that was performed as part of the regulatory submission demonstrated a lack of off-\ntarget binding at a wide range of clinically relevant drug concentrations. Further details can be provided if\nneeded.\n• To address the reviewer’s concern, we have amended the Discussion to emphasise the importance of\nmonitoring for safety signals that may be caused by off-target binding, irrespective of preclinical\nsafety data, throughout clinical development (lines 388-406).\n4. Methods: The authors should justify the use of pre-treatment with an antipyretic and antihistamine for\nall volunteers and note this limitation when assessing the adverse events data.\n• We have modified the text in the Methods (lines 594-598) and added a reference to a review by\nGeraud A et al., 2024, (https://doi.org/10.1016/j.ejca.2024.114075) which covers the toxicities\nassociated with T cell engagers and their management and prevention, including use of\npremedication.\nRegarding the suggestion that non-steroidal premedication could limit the assessment of adverse events\ndata, while they are often used with T cell engagers to reduce the incidence of CRS or infusion-related\nreactions or the severity of these events once triggered, they are unlikely to mask them completely. This\nwould be a legitimate concern with corticosteroid premedication.\n5. Figure 5c and S3: The authors can consider presenting these figures with absolute values rather than\nfold-change, which can be difficult to interpret given the LLOQ.\nWe have replaced Figure 5c and Supplementary Figure S3 with new graphs showing the absolute values\nof IL-6 and the other cytokines analysed.\n6. Define the acronym PD as pharmacodynamics at first mention.\n• This has been corrected.\nReviewer 4\n1. It is unclear whether, in the authors' opinion, there is any signal of efficacy to justify an ongoing study\ntesting this agent at higher dose levels. While it is appreciated that the safety signal is high, without any\nsignal of efficacy it is unclear how dose escalating with the increased toxicity expected, is justified. This\nshould be discussed along with the potential reasons (other than dose) why no signal of efficacy was\nobserved?\nWe appreciate the comment. We wish to clarify here - and we state in the manuscript - that the aim of this\nSAD study was to identify tolerable, biologically active doses for further evaluation in multiple dose\nschedules. The SAD design is not intended to assess efficacy but is an accepted approach to identifying a\nsafe dose range that can be evaluated for efficacy in subsequent multiple dose schedules. This approach is\nconsistent with EMA/CHMP guidance on risk mitigation in first-in-human trials.\nhttps://www.ema.europa.eu/en/documents/scientific-guideline/guideline-strategies-identify-and-mitigate-\nrisks-first-human-and-early-clinical-trials-investigational-medicinal-products-revision-1_en.pdf\nAs the reviewer points out, dose escalation for any new investigational agent poses a risk of increased\ntoxicity but is necessary to identify a safe and effective dose. The study protocol (included in this\nsubmission) incorporates multiple risk mitigations for both the SAD and MAD studies to ensure that the\nbenefit / risk ratio is favourable.\nWhile a signal of efficacy is not a pre-requisite for exploring higher doses, we did observe a dose-\ndependent serum IL-6 signal (Figure 5b) which is a positive finding that provides evidence of\npharmacodynamic activity. This, together with the tolerability of the doses tested to date, justified\nproceeding to the MAD study.\nPer our response to Reviewer 3, we believe that the principal reasons for the minimal increase in serum\ncytokines (a possible surrogate for efficacy) other than IL-6 are the low dose range tested in this SAD\nstudy and the low biomass of target (active HIV reservoir). This is borne out by emerging data from our\nongoing MAD study.\n• Data showing dose-dependent cytokine elevations (Mothe B et al. CROI 2025, abstract #104 and\nconfidential data) are appended to this document to illustrate this point.\n• We have included new data to provide evidence of T cell activation and enhanced effector function,\nas requested by Reviewer 3. The enclosed figure provided for Reviewer 3 above (now Figure 5d in\nthe manuscript) shows CD4+ and CD8+ T cell activation and upregulation of cytolytic molecules\npost-dosing.\n• Please refer to our response to Reviewer 3’s major comment #6 for full details.\n2. While this approach has been used with success in melanoma, the approach in HIV is yet to be\nrealised. Further, it is telling that the majority of participants enrolled on this study are white which is\nthe population where HLA A0201 is the most prevalent. Can the authors speculate on the broader impact\nof this HLA A0201-restricted therapy to the PLWH population given the prevalence of the virus in the\nAfrican continent? This also warrents discussion.\nThank you. This point was made by the other reviewers and we agree that further drug development\ntargeting other HLA alleles, in addition to HLA-A*02:01, will be needed to have maximum impact in\nhigh HIV prevalence regions.\n• Please refer to our response to Reviewer 3’s major comment #7 and amendments to the Discussion to\naddress this point (lines 422-430).\nRegarding the majority of white participants in this SAD study, this is not wholly attributable to the HLA-\nA*02:01 restriction but reflects the demographics of the PLWH at the study sites, together with multiple\nfactors that influence enrolment in a first-in-human study. For example, women from African countries\nwho are of child-bearing potential account for the majority of WLWH at the participating site; protocol\nrestrictions, including contraception requirements, are known barriers to participation for this group.\nNevertheless, development of TCR bispecific molecules for other HLA alleles would certainly be\nwarranted if a reproducible signal of post-treatment control is observed in the MAD study.\nMinor comments Please confirm that all participants were high resolution typed at A0201. And if not,\nplease jusitfy why.\n• High resolution typing was performed in all participants. This is now clarified in Methods (line 559)\nIn the abstract, the authors should specify primary and secondary endpoints assessed.\n• We have amended the abstract accordingly.\n• Lines 125-128: The IMC-M113VRES molecule should be more clearly introduced.\n• Thank you for this comment. We have edited this section to provide more clarity (lines 125-130 and\n434-441).\nAuthors should note Y79F is the most prevalent variant when it is first mentioned (which is stated later in\nline 158).\n• We have now introduced Y79F as the most prevalent variant at first mention earlier in this section\n(lines 132-133).\n• Lines 129-135: Authors should specify how many variants were identified and how many they tested.\n• 8 variants were identified with an estimated global frequency of >2% and all 8 were tested. We have\namended the text to reflect this (line 135).\n• Lines 153-156: These lines should be rewritten for clarity as it was hard to follow.\n• Thank you. We have amended this text and hope it is clearer (now lines 157-160).\n• Line 154: C8166 is introduced here as an HIV-infected CD4+ T cell line, but this should be mentioned\nsooner in line 142.\n• Thank you. This has been corrected (now lines 145-146).\n• Line 176: The participant numbers in this line differ from the CONSORT diagram in Fig. 3. This line\nstates eligible participants is n=89, however Fig. 3 shows n=74. This line states the HLA-A*02+\nparticipants is n=33, however Fig. 3 shows n=26.\n• Thank you for pointing out this error. We have corrected Figure 3.\n• Line 177: The authors should specify the screening criteria that resulted in 4/16 failing the screen.\nAdditionally, these criteria should be added to Fig. 3, as exclusion criteria is listed in the figure too.\n• We have updated the text and Figure 3 to clarify the reasons for screen failure.\n• Line 244-246: These lines should be rewritten to improve flow and clarity, and specify the pM and nM\naffinity ranges.\n• Thank you for the suggestion. We have modified the text to address this and included the affinity\nranges (now lines 270-274).\n• Line 246: The authors should specify the pM affinity variant.\n• We have included the details.\n• The IMC-M113VRES molecule was assessed often in the Results section but was not discussed in the\nDiscussion.\n• We appreciate that this may have caused confusion. We have added text to further clarify the use of\nIMC-M113VRES in the Results and Methods, as pointed out earlier. Because it has the same\nspecificity as the clinical molecule, IMC-M113V (1 amino acid difference in one of the CDRs) and it\nwas used to complement preclinical assays that supported the regulatory submission, which were\nperformed only with nominated clinical candidate drug IMC-M113V, we think it is more appropriate\nto explain this in the Methods rather than the Discussion (lines 434-441).\n• Line 433-435: Equations should be referenced in the main text.\n• We have included a reference in the Results section where potency against HIV-infected cells in vitro\nis presented (lines 160-161).\n• Line 466: This states the criteria threshold is 50% participants, but states the minimum is n=4; should it\nbe n=6?\nThe criteria allowed dose escalation until pre-specified criteria for pharmacodynamic activity were met in\na minimum of 2/4 participants. This criterion was met in the 15 µg cohort after the first 4 participants had\nreceived IMC-M113V, so dose escalation was halted, and we expanded the cohort to a total of 10\nparticipants to provide a more robust evaluation of this dose (as permitted by the protocol).\n• This point is explained in the Results (lines 193-196).\n• Line 519-520: Amend Supplementary Table 2 to add all fluorophores listed in these lines.\n• We have updated Supplementary Table S2 to include this information.\n• Some other limitations should be addressed in the discussion: n=1 in Cohorts 1 and 2, and the short\nwindow of follow-up post-infusion.\nWe appreciate that the rationale for single patient cohorts for Cohorts 1 and 2 was not made explicit in the\ntext. This strategy is not a limitation but an important aspect of the study design, which contributes to a\nfavourable benefit / risk ratio by minimizing exposure of participants to sub-therapeutic doses.\n• We have added explanatory text in the Results (line 193) and included a reference to a publication\nexplaining the utility of accelerated titration designs for Phase 1 studies.\nWe do not consider the window of 28 days’ follow-up to be a limitation, given that:\n1) this was a single dose study and the observed PK profile of IMC-M113V indicates that it has a very\nshort serum half-life;\n2) toxicity associated with ImmTAX molecules is primarily within the first hours-days of first exposure to\na given dose; cumulative toxicity is not observed. This is highlighted in a recent publication on\ntebentafusp that showed that most treatment-related adverse events occurred within the first 4 weeks,\nduring administration of step-up doses (treatment in this study was given once weekly until disease\nprogression occurred. DOI: 10.1056/NEJMoa2304753).\n• The journal expects that the title and/or abstract must indicate when findings apply to only one sex or\ngender.\n• We have amended the title accordingly.\n• The methods section should include whether sex and/or gender were considered in the study design.\n• The relevant section in the Methods has been updated accordingly (lines 562-564).\nAdd n values (# participants or replicates) to all figure legends where applicable.\n• We have included n values in all figures and / or figure legends.\n• For the present study, the ART regimens reported in Table 1 do not seem necessary to reveal.\n• We appreciate this thoughtful comment and have removed this information.\n• CONSORT checklist not coinciding with correct page numbers in manuscript.\n• We have updated the CONSORT checklist to ensure reference to correct page numbers.\nChanges in serum cytokines in the IMC-M113V multiple ascending dose study (ongoing)\nThe above data from Cohorts 1-3 (15/30/60, n = 5; 20/40/120, n = 5; 20/40/300 mcg, n = 6) were\npresented at CROI 2025 (Mothe B et al., abstract #104). Confidential data from Cohort 4 (20/40/300/600\nmcg) from the ongoing MAD study is included on the right. Enrolment to this cohort is ongoing. S1, S2,\nS3 refer to step doses. T1 refers to the target dose for each cohort (60, 120, 300 and 600 mcg\nrespectively).\nTo aid interpretation of the above data, we show the maximum fold change (FC) for each participant and\neach cytokine below."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed all my questions and concerns", "reviewer_2": "The authors have effectively addressed my comments in the rebuttal and revised manuscript. The authors have also effectively addressed the comments and concerns of the other reviewers. I do not have any additional comments or concerns and feel the revisions have improved the manuscript significantly. I support publication of the manuscript.", "reviewer_3": "The authors have provided highly detailed and effective responses to all of the myriad of questions that were raised to the critique. The process was a lovely scientific dialogue, as well, and not merely a punch list of answers to get the paper across the finish line. Kudos.", "reviewer_4": "The authors have addressed all my questions.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter