Harnessing multivalency and FcγRIIB engagement to augment anti-CD27 immunotherapy

Domain: biology
Published: 2025-12-20
Editorial outcome: minor_revision

Abstract
Despite significant clinical progress, checkpoint blockade remains limited by variable response rates, resistance, and toxicity. Activating costimulatory receptors offers a promising alternative to enhance anti-tumor immunity. However, there is insufficient understanding of how to mimic physiological membrane-anchored costimulatory ligands. Here, we describe a strategy for developing effective agonists of the costimulatory receptor CD27 by increasing both antibody valency and FcγRIIB engagement. Engineered anti-CD27 antibodies capable of tetravalent binding to CD27 and selective FcγRIIB association exhibit potent T cell stimulatory activity and anti-tumor efficacy in pre-clinical models, compared to bivalent counterparts. The anti-tumor effects of the tetravalent antibody are mediated through CD8⁺ T cell activation without evidence of regulatory T cell depletion. Mechanistically, whereas the increase in avidity drives more efficient CD27 clustering, FcγRIIB engagement triggers polarization of receptor clusters to the cell-cell interface and reduces receptor internalization. This work provides a framework for developing more effective agonist-based T cell stimulatory therapies.

Introduction
T cells express unique, somatically rearranged T cell receptors (TCRs) that exhibit a degree of cross-reactivity, thus enabling the recognition of diverse pathogen and tumor-derived antigens. To maintain immunological homeostasis, additional receptor-ligand interactions regulate T cell activation through costimulatory or inhibitory signals1,2. It has now been established that immune evasion is a key mechanism by which tumors grow and metastasize3. Immune suppression within the tumor microenvironment skews the balance of inhibitory and costimulatory signals towards inhibition, leading to T cell ignorance, anergy, and exhaustion4. Antibody-mediated blockade of inhibitory immune checkpoints has shown clinical efficacy across multiple cancer types, with CTLA-4 and PD-1 inhibitors demonstrating particular success5. However, challenges persist, including variable response rates, development of resistance, and immune-related adverse events5.

An alternative approach to enhance anti-tumor immunity involves the direct activation of costimulatory receptors. The premise underpinning this strategy stems from the original work demonstrating that activation of CD28 through ectopic B7 expression on tumor cells leads to their elimination by the immune system4,6. Similar findings with other costimulatory ligands and agonist antibodies targeting TNF receptor superfamily (TNFRSF) members have supported the development of clinical agents4,7. However, the majority of clinical agonists have demonstrated insufficient therapeutic activity, the precise reasons for which remain poorly understood7. Multiple factors have been shown to influence the activity of agonist anti-TNFRSF antibodies, including epitope, isotype, and affinity8–10. Receptor clustering by antibodies is generally required for activation of downstream signaling events, a process that mimics the natural interaction of TNFRSF proteins with their trimeric ligands. Thus, murine IgG1 antibodies targeting members of the TNFRSF exert strong agonism dependent on binding to the inhibitory Fcγ receptor (FcγRII) and introduction of mutations into human IgG1 Fc that enhance affinity towards human FcγRIIB similarly bolster agonism8,11–13. Moreover, various antibody engineering approaches have been proposed that enhance TNFRSF agonism by increasing antibody avidity14–16, or conformational stability17,18.

Antibodies that target the T cell costimulatory receptor CD27 have shown promise in enhancing anti-tumor immunity in preclinical models19–22. The efficacy and mechanism of action have been shown to be influenced by antibody isotype. For example, the activity of an agonistic anti-CD27 antibody, MK-5890, in both primary human tumor cultures and human CD27 knock-in mice, was significantly diminished when the isotype was switched from human IgG1 to IgG2 or a variant that further reduces the interaction with FcγR23. However, targeting CD27 with human IgG1, an isotype associated with strong effector function, resulted in depletion of T cells, especially regulatory T cells, calling into question the choice of using the human IgG1 backbone in CD27 agonists23–25. Given these uncertainties and the limited clinical efficacy of Varlilumab, a human IgG1 anti-CD27 antibody25,26, we sought strategies to generate more effective antibodies with enhanced therapeutic potential.

Here, we explore approaches to optimize the agonistic activity of CD27-targeted antibodies and investigate how valency and FcγR engagements shape their activity in both mouse and human systems. Our data show that these mechanisms act synergistically to enhance agonism and anti-tumor efficacy. Together, these findings outline a strategy for developing therapeutic antibodies that achieve optimal activation of anti-tumor T cells.

Results
Characterization and in vitro activity of a tetravalent anti-mouse CD27 antibody
To assess the impact of increased valency on anti-CD27 antibody potency, we engineered a tetravalent version of the anti-mouse (m) CD27 mAb AT124-1 by incorporating additional Fab arms into its structure (Fig. 1A). Antibodies were produced as mouse IgG1, as this isotype is known to potentiate agonism by anti-TNFRSF antibodies in mice through FcγRII-mediated crosslinking24,27,28. To investigate the requirement for FcγR engagement for bivalent and tetravalent reagents, we also produced anti-mCD27 antibodies incorporating the N297Q (NQ) mutation, which abrogates FcγR binding29. Size-exclusion chromatography (SEC) confirmed that all antibodies existed as monomers in solution, free from aggregates (Supplementary Fig. 1A), and SDS-PAGE analysis established that heavy and light chains of the anticipated size were produced (Supplementary Fig. 1B). The structure of tetravalent anti-mCD27 was also characterized by negative-staining electron microscopy. Class average image analysis revealed the tetra-meric structure of the antibody, displaying four distinct Fab sections (Fig. 1B). The outer Fab sections exhibited a degree of flexibility, consistent with the predicted mobility of the peptide linker connecting them to the inner Fab section (Fig. 1B). Next, we employed surface plasmon resonance (SPR) to investigate the effect of increased valency on the binding strength to mCD27. Our results demonstrated a >5-fold increase in apparent affinity, primarily attributed to a slower dissociation rate of the tetravalent antibody from the receptor (Fig. 1C and Table 1). We also investigated the effects of increased valency on the binding of anti-mCD27 antibodies to mCD27 expressed on the surface of Jurkat cells and observed a significant increase in avidity, as reflected by a ~100-fold decrease in the EC50 value for the tetravalent format (Fig. 1D). Next, we assessed if increased valency and avidity translated into enhanced NF-κB activation, a key downstream signaling pathway of CD2730,31. When applied in solution, neither bivalent nor tetravalent anti-mCD27 antibodies induced GFP expression in mCD27+ Jurkat NF-κB GFP reporter cells (Fig. 2A, left panel). However, both formats induced NF-κB signaling when mCD27+ reporter cells were co-cultured with Chinese hamster ovary (CHO) cells expressing mouse FcγRII (Fig. 2A, right panel and 2B). Importantly, whereas at high antibody concentrations (10–100 nM) the two formats stimulated similar levels of NF-κB activation, tetravalent anti-mCD27 was by far the most active at concentrations ranging from 0.001 to 1 nM (Fig. 2A, right panel). The enhanced potency of tetravalent anti-mCD27 when compared to bivalent anti-mCD27 and the requirement for Fc-mediated crosslinking was similarly observed when OT-I T cell proliferation under conditions of sub-optimal peptide stimulation was used as the readout for CD27 signaling (Fig. 2C). Collectively, our in vitro data suggest that the combination of tetravalency and FcγRII binding produces a level of activation not achievable with bivalent antibodies.

Impact of valency and FcγRII binding on in vivo antigen-specific CD8+ T cell responses and immunotherapy with anti-mCD27 antibodies
To explore the broader implications of our in vitro data, we initially compared the ability of bivalent and tetravalent anti-mCD27 antibodies to boost a CD8+ T cell response in vivo. This was first addressed using adoptively transferred CD45.1+ OT-I T cells and administration of ovalbumin 257–264 peptide (OVA 257–264) combined with either bivalent or tetravalent anti-mCD27 antibodies. Figure 3A–C demonstrates that tetravalent anti-mCD27 treatment led to significantly greater CD8+ T cell expansion compared to bivalent anti-mCD27 or an isotype control. This enhanced T cell proliferation was sustained throughout the contraction phase. Moreover, the “Fc silent” N297A variants of the bivalent and tetravalent anti-mCD27 antibodies did not induce significant CD45.1+ CD8+ T cell expansion, indicating that FcγR crosslinking was required for activity of anti-CD27 antibodies in vivo, thus corroborating our in vitro findings. Of note, this enhanced in vivo activity of the tetravalent anti-CD27 mIgG1 could not be attributed to improved pharmacokinetics, as it exhibited a slightly shorter plasma half-life compared to its bivalent counterpart (Supplementary Fig. 2).

Next, we compared the activity of anti-mCD27 antibodies in an immunotherapy setting wherein mice bearing a B16 melanoma variant that expresses OVA as a surrogate tumor antigen (B16-OVA) were treated with OVA and the antibodies indicated in Fig. 4. Mice were then assessed for endogenous OVA-specific CD8+ T cell expansion and tumor growth. Consistent with the OT-I T cell responses, tetravalent anti-mCD27 elicited the strongest endogenous OVA-specific CD8+ T cell response, surpassing that of the bivalent format, and this response was sustained throughout the sampling period (Fig. 4A–C). This enhanced T cell response correlated with more effective tumor control by tetravalent anti-mCD27 (Fig. 4D). In contrast, but in keeping with their muted OVA-specific CD8+ T cell responses, the Fc-inert mAb did not elicit robust tumor control. These experiments confirm that the tetravalent anti-mCD27 antibody induced a stronger endogenous OVA-specific CD8⁺ T cell response and improved tumor control; although, due to the aggressiveness of B16-OVA tumors and the likely emergence of OVA-loss variants32, tumor growth was ultimately not fully prevented (Fig. 4D and Supplementary Fig. 3).

Next, we conducted experiments to compare bivalent and tetravalent anti-mCD27 antibodies in a second model, the BCL1 lymphoma, which lacks an identified tumor-specific antigen, and in which CD27 agonism had previously been shown to elicit protective T cell responses19,22,24,33. As the Fc-inert anti-CD27 antibodies showed limited activity, we focused on testing antibodies with functional mIgG1 Fc in this model. Bivalent anti-mCD27 provided modest protection from the BCL1 tumor, increasing median survival from 16 days in the isotype control group to 39.5 days (Fig. 5A), which was further increased with the tetravalent anti-mCD27 antibody, with median survival increasing to 71.5 days (Fig. 5A).

Given that BCL1 lymphoma grows primarily in the spleen and FcγRII is abundantly expressed on both malignant and normal B cells34, we next tested the tetravalent anti-mCD27 antibody in the CT26 colon carcinoma model, where FcγRII expression is absent from the tumor and limited to the tumor-infiltrating immune cells. CT26 tumors are known to exhibit heterogeneous responses to immunotherapy with anti-PD-1 antibodies, typically segregating into responders and non-responders35. We observed a similar pattern in mice bearing established CT26 tumors treated with anti-mCD27 antibodies. Strikingly, while the bivalent anti-mCD27 mIgG1 achieved a cure rate of only 50% among responders, the tetravalent anti-mCD27 antibody led to complete tumor regression in all responding mice, highlighting its superior therapeutic potential (Fig. 5B–D). Moreover, administration of a cumulative dose of 660 μg, equivalent to ~30 mg/kg of tetravalent anti-mCD27, did not induce weight loss in mice, suggesting that the enhanced anti-tumor activity was not associated with increased toxicity (Supplementary Fig. 4A). Furthermore, treatment with tetravalent anti-mCD27 mIgG1 did not increase alanine aminotransferase (ALT) or aspartate aminotransferase (AST) enzymatic activity in the blood, both of which are key indicators of liver toxicity (Supplementary Fig. 4B). To understand the cell types involved in the anti-tumor response exerted by tetravalent anti-mCD27, we profiled T cell subsets within the tumor. Data presented in Supplementary Fig. 5 demonstrates that treatment with tetravalent anti-mCD27 induced an increase in both total and activated (4-1BB+ and Granzyme B+) CD8+ T cells without impacting total CD4+ or Foxp3+ CD4+ regulatory T cells. These data support the hypothesis that tetravalent anti-mCD27 exerts its effects through direct co-stimulation of CD8+ T cells.

Tetravalency and FcγRIIB engagement synergize for optimal immune stimulation by anti-human CD27 antibody
To evaluate the clinical relevance of our findings, we explored whether increased valency and FcγRIIB interactions could potentiate the activity of anti-human (h) CD27 antibodies. To this end, we produced the agonistic anti-hCD27 antibody, hCD27.1536, in a standard bivalent human IgG1 format and in a tetravalent configuration similar to the tetravalent anti-mCD27 antibody. Our previous work has shown that this clone recognizes an epitope distinct from that targeted by the clinical antibody Varlilumab and binds to human CD27 with moderate affinity, exhibiting stronger stimulatory activity37. Furthermore, to assess the impact of different FcγR in mediating antibody crosslinking, bivalent and tetravalent anti-hCD27 antibodies were generated using either a wild-type hIgG1 backbone, a hIgG1 variant (V11) with a higher FcγRIIB and a lower FcγRI affinity12, or a variant (NA) that has compromised binding to FcγRs. SEC analysis confirmed that all anti-hCD27 antibodies were monomeric in solution and free from aggregates (Supplementary Fig. 6A). In addition, the overall structure of the tetravalent anti-hCD27 was verified by negative-staining electron microscopy (Supplementary Fig. 6B). We also assessed whether the modified structure of the tetravalent anti-hCD27 antibody affected its thermal stability. Although the melting temperature was slightly reduced compared to the bivalent format (56.7°C vs. 63.5°C; Supplementary Table 1), this is still well above physiological temperatures and the standard accelerated storage temperature of 40°C for biopharmaceuticals, including antibodies38.

SPR analysis revealed that tetravalent anti-hCD27 mAb demonstrated enhanced avidity for hCD27, as evidenced by a ~2-fold increase in apparent affinity and slower dissociation kinetics compared to bivalent anti-hCD27 (Supplementary Fig. 7A and Supplementary Table 2). Furthermore, the increased avidity of tetravalent anti-hCD27 antibodies was also apparent when binding to hCD27+ Jurkat cells was analyzed by flow cytometry, with more tetravalent anti-hCD27 antibody detected at lower concentrations (Supplementary Fig. 7B). The agonistic potential of these anti-hCD27 antibodies was then assessed using hCD27+ Jurkat NF-κB GFP reporter cells. While bivalent anti-hCD27 antibodies (hIgG1, NA, and V11) showed limited activity in the absence of FcγR crosslinking (Fig. 6A, top panels), tetravalent anti-hCD27 antibodies were approximately 1000-fold more potent at inducing NF-κB activation than their bivalent counterparts, even in the absence of FcγRIIB (Fig. 6A, top panels). Although both bivalent and tetravalent antibodies (hIgG1 and V11) showed enhanced activity in the presence of FcγRIIB+ CHO cells, tetravalent anti-hCD27 antibodies demonstrated a significant potency advantage, ranging from 40- to 400-fold, compared to their bivalent counterparts, as evidenced by their lower EC50 values (Fig. 6A, lower panels). We then sought to determine if tetravalency and physiological expression of FcγRIIB in peripheral blood mononuclear cells synergized in promoting T cell proliferation. Interestingly, the addition of bivalent and, to a lesser extent, tetravalent anti-hCD27 hIgG1 had a slightly detrimental effect on cell proliferation, likely due to T cell deletion through antibody engagement of activating FcγRs on blood mononuclear cells (Fig. 6B, left panels). In contrast, both bivalent and tetravalent anti-hCD27 V11 antibodies were able to enhance T cell proliferation, with the tetravalent format demonstrating superior potency, as reflected by lower EC50 values (Fig. 6B, right panels). Moreover, the importance of FcγRIIB engagement was further substantiated by the loss of costimulatory activity of the tetravalent anti-hCD27 NA variant (Fig. 6B, center panels). Thus, the synergistic effect of antibody tetravalency and FcγRIIB engagement is also evident in the human setting.

Tetravalency enhances hCD27 clustering, while FcγRIIB engagement promotes polarization and reduces internalization
The recently resolved structure of the CD27-CD27 ligand (CD70) complex, which comprises 3 receptor molecules bound to homotrimeric CD70, suggests that CD27 signaling is initiated through receptor clustering39. Moreover, ligand-bound CD27 superclusters with a higher receptor density may form through disulfide-bond dimerization of CD27 in the cell membrane39. To explore the effects of antibody valency on the distribution of membrane-anchored CD27, we cultured Jurkat cells expressing hCD27-GFP fusion protein with either bivalent or tetravalent anti-hCD27 hIgG1 V11 and visualized the distribution of receptors by confocal microscopy. Initially, we assessed antibody-induced receptor clustering in the absence of FcγRs and showed that the increased valency afforded by tetravalent anti-hCD27 mAb induced significantly more clusters per cell than bivalent anti-hCD27 at concentrations from 0.1 to 100 nM (Fig. 7A, B). Since the functional activity of tetravalent anti-hCD27 hIgG1 V11 was improved by FcγRIIB binding, we sought to understand how this affects CD27 distribution in the membrane. Therefore, hCD27-GFP+ Jurkat cells were co-cultured with either hFcγRIIB+ CHO cells or control CHO cells together with the tetravalent anti-hCD27 hIgG1 V11 antibody. Whereas CD27 clusters formed in the absence of FcγRIIB were randomly distributed and rapidly internalized within 20–30 min, those formed by tetravalent anti-hCD27 hIgG1 V11 in the presence of hFcγRIIB+ CHO cells were localized to cell-cell contacts (Fig. 7C) and persisted on the cell surface (Fig. 7C, D). Finally, we addressed if hyper-crosslinking of tetravalent anti-hCD27 mAb with a secondary antibody recapitulates the stimulatory effects of FcγRIIB engagement. Using hCD27+ Jurkat NF-κB GFP reporter T cells, we found that hyper-crosslinking increased the stimulatory effects of soluble tetravalent anti-hCD27, as evidenced by the higher GFP signal and increased proportion of GFP+ Jurkat cells (Fig. 7E, F). Importantly, however, engagement of FcγRIIB by tetravalent anti-hCD27 provided even greater levels of GFP (Fig. 7E, F), demonstrating that capture of tetravalent anti-hCD27 by FcγRIIB-expressing cells synergizes with antibody tetravalency to maximize CD27 signaling.

Methods
Mice
C57BL/6J (Charles River UK, Strain code: 027), Balb/c (Strain code: 028, Charles River UK), and OT-I transgenic (Tg) mice (Charles River France, strain code: 642) were purchased from Charles River and stock colonies maintained by the University of Southampton Biomedical Research Facility. For C57BL/6 mice, 54 female 8–12-week-old mice were used. For Balb/c mice, 81 female 8–12-week-old mice were used. For OT-I Tg mice, 20 female 8–12-week-old mice were used. All mice were randomly assigned into experimental groups, with experimental and control animals co-housed. Mice were maintained on a 12-h light and dark cycle, an ambient temperature of 20–24°C, 55% humidity ± 15%, with food and water ad libitum. Mice were kept under specific pathogen-free (SPF) conditions. Daily checks were performed to ensure mice remained healthy, and environmental enrichment was provided. Mice were euthanised by CO2 inhalation or cervical dislocation. All experiments were conducted following University of Southampton ethical approval and in accordance with the Animals (Scientific Procedures) Act 1986 as set out in PPL: P4D9C89EA and PIL: I66C5D543.

CD45.1 OT-I Tg adoptive transfer
To assess antigen-specific responses in vivo, splenocytes from OT-I Tg CD45.1 mice (n=20) containing 1×104 CD8+ T cells were adoptively transferred into C57BL/6J mice. After 24 h, mice were intravenously challenged with a single dose of 30 nmole OVA 257–264 peptide and 200 μg of antibody. Antigen-specific T cell expansion was measured by peripheral blood sampling and identifying CD45.1 CD8+ T cells by flow cytometry, at the time points indicated in Fig. 3.

Tumor models
B16-OVA cells (2×105) were subcutaneously injected into the flank of C57BL/6J (n=48, 8–12-week-old females) mice on day 0. Twenty-four hours later, mice were intravenously injected with a single dose of 5 mg Ovalbumin (Sigma, A5503) and a molar equivalent quantity (1.33 nmol) of bivalent or tetravalent anti-mCD27 antibody. Antigen-specific T cell expansion was measured by peripheral blood sampling and detection of OVA 257–264 -MHC H2-Kb tetramer+ CD8+ T cells by flow cytometry, at the time points indicated in Fig. 4. Tumor growth was monitored every 2–3 days by calliper, and mice euthanised when cross-sectional area reached 225 mm2. This is below the maximum allowed volume of 4000 mm3, equivalent to 400 mm2. For the BCL1 lymphoma model19,57, 5×106 tumor cells obtained from in vivo passage were intravenously injected into Balb/c mice (n=30, 8–12-week-old females) on day 0, and a molar equivalent quantity of bivalent or tetravalent anti-mCD27 antibody (0.5 nmol) was injected intravenously on day 4, as a single dose. Tumor growth was monitored every 2–3 days by splenic palpation, with humane endpoint of gross splenomegaly, where the spleen is enlarged and extended, weighing approximately 500–1000 mg. Survival period to the humane endpoint was plotted using the Kaplan–Meier method with analysis for significance by the log-rank (Mantel–Cox) test. CT26 cells (5×105) were subcutaneously injected into the hind flank of Balb/c mice (n=29, 8–12-week-old females) on day 0, and a molar equivalent of isotype mIgG1, bivalent anti-mCD27 mIgG1, or tetravalent anti-mCD27 mIgG1 (0.66 nmol) was injected on days 10, 13, 16, and 20. Mouse weight was monitored after the first antibody injection. Tumor growth was monitored every 2–3 days by caliper, and mice euthanised when cross-sectional area reached 225 mm2. This is below the maximum allowed volume of 4000 mm3, equivalent to 400 mm2. Survival period to the humane endpoint was plotted using the Kaplan–Meier method with analysis for significance by the log-rank (Mantel–Cox) test. For all tumor experiments, early termination was carried out if mouse welfare was deemed compromised, in accordance with the guidelines proposed by Foltz and Cullere58.

CT26 tumor harvest
CT26 cells (5×105) were subcutaneously injected into the hind flank of Balb/c mice (n=10, 8–12-week-old females) on day 0, and a molar equivalent of isotype mIgG1 or tetravalent anti-mCD27 mIgG1 (0.66 nmol) was injected on days 10 and 13. Mice were euthanised on day 16, and tumors were excised. Tumors were then diced, incubated with 50 μg/ml DNase I (Roche, 4716728001) and 0.5 WU/ml Liberase DL (Merck, LIBDL-RO) for 30 min at 37°C. Digestion was inhibited using cRPMI supplemented with 10 mM EDTA (Fisher Scientific, S311-100) and 20 mM HEPES (Gibco, 15630080). Tumor was then passed through a 70 μm cell strainer (Falcon, 352350), and cells were subsequently stained as specified for flow cytometry.

Antibody bioavailability
200 μg of specified antibody was injected intravenously into C57BL/6J mice (n=6, 8–12-week-old females) on day 0, with blood sampling occurring on days 1, 3, and 7. Serum concentration was determined by ELISA, as previously reported33. Briefly, mCD27-ECD-hFc (R&D Systems) was used as a capture reagent, and bound anti-mCD27 antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse IgG.

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) detection assay
Balb/c mice (n=12, 8–12-week-old females) were intraperitoneally injected with PBS or 200 μg of bivalent anti-mCD27 mIgG1 or tetravalent anti-mCD27 mIgG1 on day 0. Blood sampling was performed on days 3 and 7, after which samples were allowed to clot overnight at 4°C. Serum was then assessed for ALT and AST enzymatic activity using an ALT activity assay (Sigma, MAK052) and AST activity assay (Sigma, MAK055), respectively. The activity assays were performed as per the manufacturer’s instructions.

Cell culture
The Jurkat NF-κB reporter T cell line (Jurkat NF-κB GFP, System Biosciences, TR850A-1), CHO-K1 cells (ATCC, CCL-61) and CT26 colon carcinoma (ATCC, CRL-2638) were grown in Roswell Park Memorial Institute (RPMI) medium 1640 (Gibco, 11875093), supplemented with 10% v/v fetal bovine serum (FBS, Sigma, F9665), 2 mM L-Glutamine (Gibco, 25030081), 1 mM Sodium Pyruvate (Gibco, 11360070), 100 U/ml Penicillin (Sigma, P4333-100ML), 100 μg/ml Streptomycin (Sigma, P4333) (cRPMI). Jurkat cells expressing hCD27-GFP and Jurkat NF-κB GFP mCD27 reporter cells were described previously33,37. CHO hFcγRIIB (provided by Dr Robert Oldham) and CHO mFcγRII cell lines (provided by Dr Hannah Smith) were cultured in cRPMI containing 1 mg/ml geneticin (Gibco, 10131027). Jurkat NF-κB GFP hCD27 cells37 were cultured in cRPMI containing 5 μg/ml puromycin (InVivoGen, ant-pr-1). B16-OVA (provided by Dr Caetano Reis e Sousa) were grown in complete Dulbecco’s Modified Eagle Medium (DMEM, Gibco), supplemented with 10% v/v FBS, 2 mM L-Glutamine, 1 mM Sodium Pyruvate, 100 U/ml Penicillin, 100 μg/ml Streptomycin.

Antibodies
The sequences of the anti-mCD27 and anti-hCD27 (hCD27.15) antibodies were derived from the AT124-1 hybridoma19,33 and US patent 9527916, respectively, and recombinant antibodies were produced as described previously10,33. To produce tetravalent antibodies, additional VH-CH1 domains were encoded in the antibody cDNA, linked to the N-terminus of the heavy chain by a 2(GGGGS) linker. All antibody preparations were endotoxin low (<5 EU endotoxin/mg) as determined using the Endosafe-PTS portable test system (Charles River Laboratories).

Flow cytometry
Fluorescently labeled antibodies were purchased from eBioscience: allophycocyanin (APC)-labeled or PerCP-Cyanine 5.5-labeled anti-mCD8α (53-6.7), APC-eF780-labeled anti-mCD45.2 (104), R-phycoerythrin (PE)-labeled anti-mFoxP3 (FJK-16s), Fluorescein isothiocyanate (FITC)-labeled anti-mCD4 (GK1.5), eFluor450 (eF450)-labeled anti-mCD45.1 (A20), eF450-labeled anti-hCD3 (UCHT1), eF450-labeled anti-m4-1BB (17B5), eF506-labeled anti-hCD4 (RPA-T4); or Biolegend: PE-Cyanine 7-labeled anti-hCD8α (RPA-T8), Alexa Fluor 647-labeled anti-granzyme B (GB11). OVA 257–264 -specific T cells were identified with PE-labeled H2-Kb OVA 257–264 tetramer, produced in-house. Fluorescently labeled secondary F(ab)2 antibodies were purchased from Jackson ImmunoResearch: allophycocyanin (APC)-labeled AffiniPure goat anti-mouse Fcγ fragment, APC-labeled AffiniPure goat anti-human Fcγ fragment. If samples contained erythrocytes, these were firstly lysed using ACK buffer. Viability staining was performed with Fixable Viability Dye eFluor 506 (eBioscience) as per the manufacturer’s instructions. FcγR-blocking was then performed with either 10 μg/ml anti-FcγRII/III (2.4G2, produced in-house) or 10% human AB serum (for human cells), at 4°C for 10 min, prior to a 30 min, 4°C incubation with surface staining antibodies. If secondary detection with F(ab)2 was used, the cells were washed after surface staining and then incubated with the secondary F(ab)2 for 20 min at 4°C. For intracellular staining, cells were fixed and permeabilised using the FoxP3/Transcription Factor Staining Kit, as per the manufacturer’s instructions (eBioscience, 00-5523-00). All antibodies and dilutions used are listed in Supplementary Tables 3 and 4. Cells were then washed before analysis on a BD FACSCanto II or a BD FACSCalibur using the BD FACSDiva software or BD CellQuest software, respectively.

Negative stain electron microscopy (EM)
Glow-discharged carbon-coated copper grids (300 mesh, TAAB Laboratories, C267/050) were floated on a droplet of antibody on parafilm, containing 1–5 μg/ml of tetravalent anti-mCD27 antibody diluted in H2O, for 1 min. The grids were then briefly floated on a drop of H2O and then a drop of 1% w/v uranyl acetate. Finally, the grids were floated on 1% uranyl acetate (Electron Microscopy Sciences, 22400) for 30 s before blotting on filter paper and air-drying overnight. Images were captured using a transmission electron microscope (Hitachi HT7700, Voltage: 100 kV, magnification: 70,000). Class averages were generated using RELION v3.1.3 (Sjors Scheres, MRC Laboratory of Molecular Biology). In brief, micrographs were uploaded to RELION, which auto-selected particles in a template-free manner using the Laplacian of Gaussian method. Particles then underwent 2D classification into class averages, with averages selected that were well resolved and contained real particles. 2D classification was repeated to refine class averages into recognizable tetravalent antibodies, with representative class averages selected.

SPR
The apparent affinity of antibodies for their target proteins was analyzed using a Biacore T200 (Biacore). The target protein was captured using an anti-hFc or anti-his antibody that had been amine-coupled to a CM5 chip (Cytiva, 29104988), as per the manufacturer’s instructions. Target proteins were mCD27-ECD-hFc (R&D Systems, 574-CD-050) or hCD27 ECD-Fc-his (R&D Systems, 382-CD-100), as indicated in Fig. 1 and Supplementary Fig. 7. The amount captured was optimized for each receptor. Antibodies diluted in HBS-EP+ (Cytiva, BR100669) to the concentrations indicated in Fig. 1 and Supplementary Fig. 7 were injected into the flow cell at 30 μl/min for 300 s, to allow the antibodies to associate with the target protein. HBS-EP+ buffer was then injected into the flow cells at 30 μl/min to allow the antibody to dissociate, before the chip was regenerated with 3M MgCl2 at 20 μl/s for 1 min. Kinetics were then assessed in the Biacore Kinetics v3 software.

SDS-PAGE and SEC
Protein samples (1–2 μg) were incubated with reducing Lammeli buffer containing additional SDS (62.5 mM Tris pH 6.5 (Sigma, 20–160), 4% w/v SDS (VWR chemicals, 44215HN), 10% v/v glycerol (Sigma, G9012), 0.04% v/v bromophenol blue (VWR Chemicals, BDH7392-2) and 62.5 mM DTT (R0861)) for 30 min at ambient temperature. Protein samples were boiled for 5 min and then loaded onto a 10% Bolt Bis-Tris Gel (Invitrogen, NW00100BOX), and separation was performed at 100 V for 15 min and then 140 V for 1 h. The gel was fixed using 10% acetic acid (VWR Chemicals) and 25% isopropanol (Fisher Scientific, 10173240) and then stained with 60 mg/L Brilliant Blue (Sigma, B0770) in 10% acetic acid. Excess Coomassie Blue staining was removed by multiple washes with 10% acetic acid on a rocker for 30 min to 1 h. The gel was imaged using the UVP BioSpectrum AC Imaging System (UVP). Antibodies (20–40 μg/sample) were assessed by analytical SEC using a Zorbax GF-250 column (Agilent) and a 0.2 M phosphate running buffer pH 7.0 containing 1M dimethyl formamide, at a flow rate of 0.4 ml/min.

Nano differential scanning fluorimetry
PBS control and antibodies (1 mg/ml in PBS) were loaded into the Prometheus NT.48 (NanoTemper technologies). The temperature was increased from 14°C to 95°C at 1°C per minute, with intrinsic protein fluorescence collected at 330 nm and 350 nm. Unfolding temperature was calculated by PR ThermControl software (NanoTemper technologies) using the first derivative of the 350/330 nm fluorescence.

Jurkat NF-κB GFP activation
The Jurkat NF-κB GFP reporter cell lines (System Biosciences) expressing either mCD2733 or hCD2737 were described previously. To investigate NF-κB activation, cells were incubated with anti-CD27 antibodies for 5 h at 37°C and either untransfected, mFcγRII-transfected (provided by Dr Hannah Smith), or hFcγRIIB-transfected CHO cells (provided by Dr Robert Oldham). GFP production was detected by flow cytometry.

Mouse T cell proliferation
Splenocytes 2×105/well from OT-I Tg C57BL/6 mice were stimulated with 1 pM OVA 257–264 peptide (Peptide Synthetics, 47874) and anti-mCD27 antibodies or an isotype control. Cells were incubated at 37°C, 5% CO2 for 48 h before 1 μCi of 3H-thymidine was added to each well for 16 h. Cells were then lysed, with lysates transferred to filter plates (Opti-plate-96, Perkin Elmer, 101687-172). 40 μl/well Scintillant fluid (PerkinElmer) was then added before incorporation of 3H-thymidine was measured by scintillation counting (TopCount).

Human T cell proliferation
Human PBMCs were isolated from anonymised leukocyte cones from healthy adult donors through the NHS blood and transplant service. The use of human tissue was approved by the East of Scotland Research Ethics Service, Tayside, UK, and via the Faculty of Medicine Research Ethics Committee under submission 19660. PBMCs were isolated from leukocyte cones by density gradient centrifugation using Lymphoprep (Stemcell, 18060). Isolated PBMCs were labeled with 2 μM CFSE (Thermo Fisher Scientific, C34554) for 10 min. CFSE-labeled PBMCs 1×105/well were stimulated with 0.1 ng/ml anti-hCD3 (OKT3) and 0.1–10 nM of anti-hCD27 antibodies or isotype control as indicated in Fig. 6. CFSE dilution in CD4+ and CD8+ T cells was measured after 96 h by flow cytometry. CFSE low cells were identified by comparison to CFSE-labeled cells that were cultured in the absence of any stimulation.

Clustering confocal microscopy
Jurkat cells (1.5×105) expressing hCD27-GFP fusion protein37 were stimulated with either bivalent or tetravalent anti-hCD27 antibodies at the concentrations indicated in Fig. 7 in a final volume of 200 μl in a 96-well U-bottomed plate at 37°C for 10 min. The cells were then fixed with 4% PFA for 20 min before imaging on a poly-D-lysine (Gibco, A3890401) coated μ-slide 18-well microscopy chamber (Ibidi, 81811). Cells were imaged in confocal mode (exposure: 3000 ms; line spacing: 2; 100× objective lens) using an ONI Nanoimager (ONI), before analysis with NimOS v1.18 and ImageJ. Clusters were defined as regions of membrane that had a fluorescence intensity above 15,000, which was determined using the threshold setting on ImageJ (ImageJ).

Cell-cell clustering
Untransfected or hFcγRIIB+ CHO cells (104) were labeled with CellTracer far red (Invitrogen, C34564), as per the manufacturer’s instructions, and the cells were plated onto a poly-D-lysine-coated μ-slide 18-well microscopy chamber and incubated at 37°C in 5% CO2 overnight. Jurkat hCD27-GFP cells (3×104) and antibodies (1 nM) were added to the wells for 10–30 min (as indicated in Fig. 7) before fixing with 4% PFA (Thermo Fisher Scientific, 28908) for 20 min. The cells were imaged in confocal mode (488 laser: exposure: 3000 ms, line spacing: 2; 647 laser: exposure: 150 ms, line spacing: 2; 100× objective lens) using an ONI Nanoimager, before analysis with NimOS v1.18 and ImageJ. Internalization was measured by selecting the intracellular region of the Jurkat hCD27-GFP cells as a region of interest (ROI) on ImageJ and then calculating the mean fluorescence intensity of the ROI.

Statistical analysis
GraphPad Prism 9 was used for statistical analysis. Ordinary one-way analysis of variance (ANOVA) with Tukey’s post-hoc multiple comparison test or a paired-t test was used as indicated in the figure legends throughout. Tumor area was analyzed by calculating the area under the curve (AUC) before performing a one-way ANOVA. Where error bars are shown, they indicate SEM or SD as detailed in the figure legends. Survival analysis was performed using log-rank (Mantel–Cox) test between indicated groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors convincingly demonstrate the importance of FcGR2B engagement for optimal CD27 agonism in both mouse and human systems. They demonstrate that increasing the number of CD27 binding sites per antibody from two (bivalent) to four (tetravalent) allows for equivalent agonism to occur at significantly lower antibody concentrations and that this is the result of an increased avidity effect driven by a lower antibody off-rate.\nIn vivo, tetravalent anti-CD27 antibodies outperformed bivalent ones in promoting antigen-stimulated proliferation of adoptively transferred congenic OT-1 cells and in reducing the growth rate of OVA-expressing B16 melanoma cells in ovalbumin-vaccinated mice. Fc domain silencing abrogated the effect, highlighting the importance of FcGR engagement. Additionally, treatment with tetravalent anti-CD27 antibodies also resulted in a longer survival time in mice challenged with the BCL1 lymphoma cell line compared with bivalent anti-CD27 antibodies.\nFinally, the authors find that modifying human IgG1 Fc domains to increase FcGR2B and decrease FcGR1 binding strongly enhances the capacity of anti-CD27 antibodies to stimulate proliferation of T-cells in human PBMCs cultured ex vivo. These data suggest that CD27 antibodies with wildtype IgG1 Fc domains induce some level of depletion of T-cells, presumably via phagocytosis or ADCC. This finding provide guidance for the design of new clinical TNFR agonist antibodies.\nThe results represent an incremental advance in our understanding of optimal agonists and are not highly novel or unexpected. The manuscript is more appropriate for a journal such as Journal of Immunology or mAbs.\n1. The mechanism of the anti-lymphoma effect (BCL1 lymphoma model) should be further explored to understand whether or not this approach is applicable only to lymphoma. Many lymphomas themselves express FcGR2B on their surface, whereas the vast majority of other tumor types do not. Considering the apparent importance of FcGR2B for the activity of anti-CD27 antibodies, an alternate explanation for the anti-lymphoma activity observed by the authors is that the anti-CD27 antibodies bound FcGR2B on the surface of the BCL1 lymphoma cells and directly provided co-stimulation to T-cells interacting with these lymphoma cells. If this is the mechanism, it would be relevant only for FcGR-expressing tumors, which are primarily lymphomas, and would not be applicable to most other malignancies. Treatment of other tumor types would require that CD27 agonism delivered to T-cells interacting with professional APCs (which do express FcGR) is sufficient to drive an anti-tumor response, since the tumor cells themselves would not express FcGR. If BCL1 lymphoma cells do express FcGR2B, then the authors’ experiment does not test this. Determine whether the BCL1 lymphoma expresses surface FcGR2B by flow cytometry. This line of reasoning should be discussed in the text so that the reader can draw appropriate conclusions regarding the breadth of potential translatability.\n2. Figure 2A shows that Fc receptor expression on the CHO cells is required to induce NFkB agonism in co-cultured anti-murine CD27 mab and murine CD27+ Jurkat cells. However, Figure 5A shows that Fc receptor expression on the CHO cells is not required for agonism of anti-human CD27 mab and human CD27+ Jurkat cells. The reason for this discrepancy is not clear and is not explored. Is it an antibody-specific strength of agonist activity? Discuss.\nMinor.\n3. The results presented in Figures 6A & 6B may perplex some readers. These results indicate that the anti-CD27 antibody with a modified Fc domain that preferentially binds FcGR2B induced more CD27 clustering on Jurkat cells than the anti-CD27 antibody with a modified IgG1 Fc domain. Add an explanation that no FcR is present in the system and so only the valency is being compared.\n4. In Fig1A, highlight the CDR regions.\n5. In Fig 2C, put in legend how many days cells were cultured before analysis.\n6. In Fig 2C, label x-axis as antibody or peptide concentration to avoid confusion.\n7. The NQ mutants have a greater level of activity then control. Saying they are inactive seems an overstatement. Indicate on graphs as NS (not significant) if that is the case.\n8. Starting B16-OVA antibody therapy on day 1 after implantation is very early. One is not sure if the tumor has been established and exhaustion has not been established. Note in text these limitations. Have the authors tested at later times?\n9. If antibody treatments are only on day one, say a single dose, and if repeated, say so.\n10. “lacking a known tumor-specific antigen “ change to an “identified tumor-specific antigen”\n11. “hCD27.15 [34] in a standard bivalent IgG1 format” human or mouse?\n12. If the hCD27.15 mab is the parent of an mab in clinical trials, say the name of the drug.", "reviewer_2": "Al-Shamkhani and colleagues generated tetravalent versions (Fab on Fab) of anti-CD27 antibodies und studied their potency and agonist activity in vitro and in vivo. CD27 is a costimulatory receptor on T cells and agonistic targeting CD27 is a validated clinical concept. However, the anti-CD27 agonist antibody Varlilumab (human IgG1) displayed limited success. In this paper the authors explored how increase of valency from bivalent to tetravalent can improve potency in conjunction with modulated FcγR binding. The authors confirmed previous work showing that agonistic activity of anti-CD27 antibodies depends on FcγR-mediated crosslinking. Importantly, the authors impressively showed by both in vitro and in vivo studies that the potency of agonism was significantly improved for tetravalent mouse and human derived antibodies. A variety of readouts was performed to corroborate improved potency, which could be related to improved clustering and reduced receptor internalization eventually also resulting in improved tumor responses in two different in vivo tumor models. The paper is well written, the data are clearly presented and sound and statistical significance analysis was performed for each experiment. I recommend publication.\nA minor point that might be considered: It is well known that biparatopic antibodies can have beneficial properties regarding receptor clustering (see e.g https://www.tandfonline.com/doi/full/10.1080/19420862.2024.2310890 ). It might be worth briefly discussing this option in the discussion section (do Varlilumab and hCD27.15 bind distinct epitopes? Ref. 34 is not very informative).", "reviewer_3": "Marcus A. Widdess and colleagues developed a strategy to improve the activity of CD27 antibody by increasing both antibody valency and FcγRIIB engagement, presenting a promising avenue for the development of next-generation anti-CD27 therapies. Notably, both multivalency and Fc engineering strategies have been explored for OX40 antibodies and agonist antibodies targeting other TNFRSF receptors. To provide more novel and valuable insights in this field, further mechanistic studies are essential. While the authors focused on the CD27 antibody-mediated costimulation effect, the impact of tetravalent CD27 antibodies on Treg cells should also be investigated, given that the depletion of Treg cells by CD27 antibodies is equally crucial to the costimulation effect in clinical trials. Additionally, evaluating the efficacy of tetravalent CD27 antibodies in CD27 humanized mouse models is warranted.\nMajor points:\n1) Figure 2. There are different types of FcgRII receptors, such as FcgRIIA, FcgRIIB, and FcgRIIC, which type of receptor is involved in this study?\n2)CD27 expression on Tregs plays a critical role in limiting immune responses against tumors. While the authors primarily focus on the agonistic properties of the CD27 antibody, it is important to note that CD27 antibodies used in clinical trials are typically of the human IgG1 subclass, which facilitate CD27 crosslinking and mediate Treg depletion via antibody-dependent cellular cytotoxicity. Therefore, to ensure the clinical relevance and translational value of this work, it is essential to investigate both the agonistic effects and the Treg depletion capabilities of the CD27 antibody. Given their enhanced valency, tetravalent CD27 antibodies may exhibit more potent activity in depleting Tregs, which could significantly impact their therapeutic efficacy in cancer immunotherapy.\n3)Figure 4. To fully evaluate the anti-tumor efficacy of these antibodies, both tumor growth curves and survival curves of the treated mice should be presented.Additionally, a more detailed analysis of the tumor immune microenvironment is warranted. This should include quantifying different T cell populations (at least conventional T cells [Tcon], regulatory T cells [Treg], and cytotoxic T lymphocytes [CTLs]) and assessing the expression of activation and exhaustion markers on these cells. The current data are presented at a single time point, which provides a snapshot of T cell activation. However, a longitudinal analysis over multiple time points could provide more comprehensive understanding of the dynamics of T cell activation and the sustained effects of the treatment.\n4)The toxicity of the tetravalent anti-CD27 antibody has not been assessed. will the tetravalent antibody have a reduced half-life? The safety and PK data will be critical to the translation of this tetravalent agonist antibody into clinical drug development.\n5) There are several clinical candidates, such as varlilumab, all of which have shown limited efficacy in both preclinical models and patients. To ensure that this study can potentially be translated into clinical drug development, the author should evaluate the efficacy and toxicity of the tetravalent version of varlilumab in a humanised CD27 mouse model. Several companies offer humanised CD27 mice.\nMinor points:\n1) Table 1, which model is used to fit the SPR data, 1:2 or 1:1 kinetic model? Is the binding affinity apparent Kd?\n2)The authors should use a consistent name throughout the manuscript, for example, CD8+ T cells and CD8 T cells.\n3) Figure 4C, the tumour volume at the last time point was less than 200mm3. The tumour growth curve should be plotted until some tumours reach 1500 or 2000mm3.\n4) In lines 271-275 of the Methods section, the author should clarify how many doses of antibody were administered. It would be better to inject OVA with an adjuvant to better induce T cell immunity.\n6) In the Figure 5 section, the author should discuss that some human CD27 antibodies are less dependent on FcγRIIb cross-linking, but that FcγRIIb cross-linking can further enhance potency, suggesting that the agonistic activity of CD27 antibodies may be influenced by their epitopes. The bivalent antibody and the tetravalent antibody with the Fc V11 variant did not show a significant difference in Figure 5A.What is the reason?\n7) In Figure 6C, the video should be provided in supplemental information.\n9) For Figure 6E, it would be informative to analyze whether the percentage of GFP+ cells shows significant changes after treatment with either bivalent or tetravalent antibodies. Additionally, to enhance data interpretation, both MFI and the percentage of positive cells could be calculated and presented on the y-axis.\n10) The Discussion section would benefit from a comprehensive analysis of the potential limitations associated with the tetravalent antibody approach, including but not limited to its structural stability, pharmacokinetic properties, and potential immunogenicity.\n11) The term \"Finaly\" in line 175 requires correction to \"Finally.\" Additionally, we suggest performing a comprehensive spell-check of the entire manuscript to identify and rectify any similar typo.\n12) Although Figure S2 shows no aggregation of the tetravalent antibody, this does not mean that it is stable. The thermostability of the tetravalent antibody should be determined.", "reviewer_4": "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": "Point-by-point response\nReviewer #1 (Remarks to the Author):\nThe authors convincingly demonstrate the importance of FcGR2B engagement for optimal CD27\nagonism in both mouse and human systems. They demonstrate that increasing the number of CD27\nbinding sites per antibody from two (bivalent) to four (tetravalent) allows for equivalent agonism to\noccur at significantly lower antibody concentrations and that this is the result of an increased avidity\neffect driven by a lower antibody off-rate.\nIn vivo, tetravalent anti-CD27 antibodies outperformed bivalent ones in promoting antigen-\nstimulated proliferation of adoptively transferred congenic OT-1 cells and in reducing the growth\nrate of OVA-expressing B16 melanoma cells in ovalbumin-vaccinated mice. Fc domain silencing\nabrogated the effect, highlighting the importance of FcGR engagement. Additionally, treatment with\ntetravalent anti-CD27 antibodies also resulted in a longer survival time in mice challenged with the\nBCL1 lymphoma cell line compared with bivalent anti-CD27 antibodies.\nFinally, the authors find that modifying human IgG1 Fc domains to increase FcGR2B and decrease\nFcGR1 binding strongly enhances the capacity of anti-CD27 antibodies to stimulate proliferation of T-\ncells in human PBMCs cultured ex vivo. These data suggest that CD27 antibodies with wildtype IgG1\nFc domains induce some level of depletion of T-cells, presumably via phagocytosis or ADCC. This\nfinding provides guidance for the design of new clinical TNFR agonist antibodies.\nThe results represent an incremental advance in our understanding of optimal agonists and are not\nhighly novel or unexpected. The manuscript is more appropriate for a journal such as Journal of\nImmunology or mAbs.\nWe thank the reviewer for their comments. Despite the success of checkpoint inhibitors such as anti-\nPD-1, anti-CTLA-4, and more recently anti-LAG3 (in combination with anti-PD-1), most patients\nremain refractory to treatment. Therefore, it is critical to explore alternative approaches that could\nbroaden the clinical benefit of immunotherapy.\nCD27 is a key driver of CD8+ T cell responses, and its non-redundant role in human immunity is\nunderscored by genetic deficiencies that lead to susceptibility to EBV-associated malignancies and\nother lymphoproliferative disorders, alongside defects in T cell activation, cytotoxicity, and memory\nformation. However, translating the potent biological effects of CD27 co-stimulation into\ntherapeutics has been challenging. The currently explored anti-CD27 IgG molecules have shown\nlimited efficacy as agonists to date, failing to replicate the effects of the natural, trimeric,\nmembrane-bound CD70 ligand, demonstrating a gap in knowledge as to how to optimally exploit this\ncostimulatory target.\nIn this manuscript, we present new insight into this issue and an approach to overcome these\nlimitations. Unexpectedly, we found that optimal CD27 agonism requires both increased valency,\nspecifically tetravalency compared to bivalency, and stabilisation of the receptor complex at the\nplasma membrane via co-engagement with FcγRIIB. We demonstrate, using three distinct in vivo\npreclinical models (including one added in the revised manuscript), that this strategy represents a\nsignificant advancement in the development of effective CD27 agonists.\n1. The mechanism of the anti-lymphoma effect (BCL1 lymphoma model) should be further explored\nto understand whether or not this approach is applicable only to lymphoma. Many lymphomas\nthemselves express FcGR2B on their surface, whereas the vast majority of other tumor types do not.\nConsidering the apparent importance of FcGR2B for the activity of anti-CD27 antibodies, an\n1\nalternate explanation for the anti-lymphoma activity observed by the authors is that the anti-CD27\nantibodies bound FcGR2B on the surface of the BCL1 lymphoma cells and directly provided co-\nstimulation to T-cells interacting with these lymphoma cells. If this is the mechanism, it would be\nrelevant only for FcGR-expressing tumors, which are primarily lymphomas, and would not be\napplicable to most other malignancies. Treatment of other tumor types would require that CD27\nagonism delivered to T-cells interacting with professional APCs (which do express FcGR) is sufficient\nto drive an anti-tumor response, since the tumor cells themselves would not express FcGR. If BCL1\nlymphoma cells do express FcGR2B, then the authors’ experiment does not test this. Determine\nwhether the BCL1 lymphoma expresses surface FcGR2B by flow cytometry. This line of reasoning\nshould be discussed in the text so that the reader can draw appropriate conclusions regarding the\nbreadth of potential translatability.\nWe would like to thank the reviewer for their insightful suggestion. FcgR2B expression on the BCL\n1\nlymphoma is well documented and we have referenced this in the revised manuscript. To evaluate\nwhether treatment with anti-CD27 agonists is effective beyond FcγRIIB-expressing B cell lymphomas\nor the protein vaccination (OVA)/B16-OVA challenge models, we employed the CT26 colon\ncarcinoma model. These data are presented in new Figure 5B–D. We demonstrate that treatment of\nCT26-bearing mice with the tetravalent anti-mCD27 mIgG1 led to complete tumour regression in all\nresponding mice. In contrast, only half of the responding mice treated with the bivalent anti-hCD27\nmIgG1 survived the tumour challenge. These findings demonstrate that the anti-tumour efficacy of\nan optimised anti-CD27 agonist is not limited to FcγRIIB-expressing B cell lymphomas but also\nextends to tumours lacking FcγR expression. The new findings are fully discussed in the Results\nsection of the revised manuscript\n2. Figure 2A shows that Fc receptor expression on the CHO cells is required to induce NFkB agonism\nin co-cultured anti-murine CD27 mab and murine CD27+ Jurkat cells. However, Figure 5A shows that\nFc receptor expression on the CHO cells is not required for agonism of anti-human CD27 mab and\nhuman CD27+ Jurkat cells. The reason for this discrepancy is not clear and is not explored. Is it an\nantibody-specific strength of agonist activity? Discuss.\nWe agree with the reviewer’s comment that in the human NF-κB reporter assay the requirement for\nFcγRIIB is not absolute, unlike that observed in the reporter assay of murine CD27. However, even in\nthe human assay we observed a substantial enhancement of NFκB activity in the presence of CHO\ncells expressing FcγRIIB for both the tetravalent hIgG1 and tetravalent hIgG1 V11 formats but not\nthe Fc ‘silent’ variants – compare new Fig. 6A lower ‘hFcγRIIB+ CHO’ with upper ‘WT CHO’ panels.\nImportantly, we demonstrated that effective co-stimulation of both human and murine primary T\ncells by the anti-CD27 agonists required FcγR engagement (Fig. 2C for murine anti-CD27 mAb and\nnew Fig. 6B - compare ‘Fc silent’ hIgG1 NA with hIgIG1 V11). Therefore, the overall conclusions that\ntetravalency and FcγRIIB binding combine for optimal agonism is supported by our data. There are\nseveral potential reasons for the variability in dependence on FcγRIIB in the NFκB reporter assay.\nFirst, there is variability in the baseline levels of NFκB activity. Second, we cannot exclude\ndifferences in antibody epitope or downstream cell signalling in the Jurkat reporter cell lines that\ncould affect the sensitivity and therefore the threshold of NFκB activation by mouse and human\nCD27 crosslinking. For these reasons we employed additional assays where possible (in vitro primary\nT cells and in vivo priming) to fully address the dependency on FcγRIIB.\nMinor.\n3. The results presented in Figures 6A & 6B may perplex some readers. These results indicate that\nthe anti-CD27 antibody with a modified Fc domain that preferentially binds FcGR2B induced more\nCD27 clustering on Jurkat cells than the anti-CD27 antibody with a modified IgG1 Fc domain. Add an\nexplanation that no FcR is present in the system and so only the valency is being compared.\nthe effect of valency. We have added a sentence to clarify that no FcγR is present in these\nexperiments.\n4. In Fig1A, highlight the CDR regions.\nWe have re-drawn the Figure as suggested. Based on known structures of Fab fragments we have\nadjusted the position of the linkers and highlighted the relative location of the CDRs.\n5. In Fig 2C, put in legend how many days cells were cultured before analysis.\nThe legend already states that the cells were cultured for 72 hours, with the final 16 hours\nperformed in the presence of tritiated thymidine.\n6. In Fig 2C, label x-axis as antibody or peptide concentration to avoid confusion.\nWe apologise for this omission. The x-axis label has been amended to read ‘antibody concentration\n(Log[nM])’.\n7. The NQ mutants have a greater level of activity then control. Saying they are inactive seems an\noverstatement. Indicate on graphs as NS (not significant) if that is the case.\nWe would like to thank the reviewer for raising this point. The text has been modified to clarify that\nthere was not a statistically significant increase in activity with the NQ variants. This is now also\nstated in the Figure legends where applicable.\n8. Starting B16-OVA antibody therapy on day 1 after implantation is very early. One is not sure if the\ntumor has been established and exhaustion has not been established. Note in text these limitations.\nHave the authors tested at later times?\nB16-OVA tumours are highly aggressive and resistant to long-term T cell immunotherapy due to the\nemergence of OVA loss variants (Kazula et al., Int J Cancer 2011). Therefore, we initiated treatment\nand vaccination early. We have revised the text to acknowledge the limitations of this model and\nhave added the Kazula et al reference. While we have not tested intervention at later time points in\nthis model, we now include new data (Figure 5B–D) demonstrating the activity of the tetravalent\nanti-CD27 antibody in an established CT26 colon carcinoma model. In these new experiments CT26\ntumours were allowed to establish for 10 days to a size of ~100 mm2 prior to treatment. In this\nsetting the tetravalent anti-CD27 produced a cure rate of 100% in responders. We also\ndemonstrated superior activity of the tetravalent antibody in the established BCL1 lymphoma model\n(Figure 5A). Overall, the anti-tumour effects of the tetravalent anti-mCD27 surpassed that of the\nbivalent counterpart in 3 distinct tumour models.\n9. If antibody treatments are only on day one, say a single dose, and if repeated, say so.\nWe have added a ‘a single dose of’ where applicable.\n10. “lacking a known tumor-specific antigen “change to an “identified tumor-specific antigen”\nWe have changed this in the text.\n11. “hCD27.15 [34] in a standard bivalent IgG1 format” human or mouse?\n12. If the hCD27.15 mab is the parent of an mab in clinical trials, say the name of the drug.\nhCD27.15 is one of several anti-human CD27 monoclonal antibodies developed by Aduro Biotech\nand subsequently licensed to Merck. Ultimately, Merck selected hCD27.131A for clinical\ndevelopment as Boserolimab (MK-5980). We selected hCD27.15 for our studies based on its well-\ncharacterized properties. In prior work, we evaluated the binding and functional activity of hCD27.15\nin comparison with other anti-CD27 antibodies, including the clinically relevant Varlilumab. Among\nthese, hCD27.15 exhibited the strongest activity (Heckel et al., Communications Biology, 2022).\nReviewer #2 (Remarks to the Author):\nAl-Shamkhani and colleagues generated tetravalent versions (Fab on Fab) of anti-CD27 antibodies\nund studied their potency and agonist activity in vitro and in vivo. CD27 is a costimulatory receptor\non T cells and agonistic targeting CD27 is a validated clinical concept. However, the anti-CD27\nagonist antibody Varlilumab (human IgG1) displayed limited success. In this paper the authors\nexplored how increase of valency from bivalent to tetravalent can improve potency in conjunction\nwith modulated FcγR binding. The authors confirmed previous work showing that agonistic activity\nof anti-CD27 antibodies depends on FcγR-mediated crosslinking. Importantly, the authors\nimpressively showed by both in vitro and in vivo studies that the potency of agonism was\nsignificantly improved for tetravalent mouse and human derived antibodies. A variety of readouts\nwas performed to corroborate improved potency, which could be related to improved clustering and\nreduced receptor internalization eventually also resulting in improved tumor responses in two\ndifferent in vivo tumor models. The paper is well written, the data are clearly presented and sound\nand statistical significance analysis was performed for each experiment. I recommend publication.\nA minor point that might be considered: It is well known that biparatopic antibodies can have\nbeneficial properties regarding receptor clustering (see e.g\nhttps://www.tandfonline.com/doi/full/10.1080/19420862.2024.2310890 ). It might be worth briefly\ndiscussing this option in the discussion section (do Varlilumab and hCD27.15 bind distinct epitopes?\nRef. 34 is not very informative).\nWe thank the reviewer for their positive comments and valuable suggestions. We agree that\nbiparatopic antibodies represent a promising class of molecules for promoting agonism. In response,\nwe have added a final paragraph to the Discussion to address this comment and have included the\nreview article recommended by the reviewer. The study by Heckel et al. (Communications Biology,\n2022) has now been cited in the Results section to highlight the distinct epitopes recognised by\nVarlilumab and hCD27.15.\nReviewer #3 (Remarks to the Author):\nMarcus A. Widdess and colleagues developed a strategy to improve the activity of CD27 antibody by\nincreasing both antibody valency and FcγRIIB engagement, presenting a promising avenue for the\ndevelopment of next-generation anti-CD27 therapies. Notably, both multivalency and Fc engineering\nstrategies have been explored for OX40 antibodies and agonist antibodies targeting other TNFRSF\nreceptors. To provide more novel and valuable insights in this field, further mechanistic studies are\nessential. While the authors focused on the CD27 antibody-mediated costimulation effect, the\nimpact of tetravalent CD27 antibodies on Treg cells should also be investigated, given that the\ndepletion of Treg cells by CD27 antibodies is equally crucial to the costimulation effect in clinical\ntrials. Additionally, evaluating the efficacy of tetravalent CD27 antibodies in CD27 humanized mouse\nmodels is warranted.\ninsightful suggestions to improve the manuscript. As requested, we have carried out several\nadditional experiments as detailed in the revised manuscript and the point-by-point response below.\nMajor points:\n1) Figure 2. There are different types of FcgRII receptors, such as FcgRIIA, FcgRIIB, and FcgRIIC, which\ntype of receptor is involved in this study?\nTo clarify, Figure 2 refers to anti-mouse CD27 mIgG1 antibodies. Since mice express only a single\nisoform of FcγRII, it is typically referred to as FcγRII rather than FcγRIIB, as is the case in the human\nsystem where multiple FcγRII isoforms exist. Mouse FcγRII is the sole inhibitory FcγR, and the\nhomolog of human FcγRIIB. For these reasons, we believe the use of the nomenclature \"mouse\nFcγRII\" (mFcγRII) in the manuscript is appropriate.\n2) CD27 expression on Tregs plays a critical role in limiting immune responses against tumors. While\nthe authors primarily focus on the agonistic properties of the CD27 antibody, it is important to note\nthat CD27 antibodies used in clinical trials are typically of the human IgG1 subclass, which facilitate\nCD27 crosslinking and mediate Treg depletion via antibody-dependent cellular cytotoxicity.\nTherefore, to ensure the clinical relevance and translational value of this work, it is essential to\ninvestigate both the agonistic effects and the Treg depletion capabilities of the CD27 antibody. Given\ntheir enhanced valency, tetravalent CD27 antibodies may exhibit more potent activity in depleting\nTregs, which could significantly impact their therapeutic efficacy in cancer immunotherapy.\nThe reviewer raises an interesting point, which is addressed in our original manuscript. As shown in\nFigure 6B (originally Figure 5B) there is a dose dependent reduction in activated CD4 and CD8 T cells\nwith anti-human CD27 hIgG1, but this reduction was not potentiated by the tetravalent format of\nthe antibody. Thus, we can conclude that the tetravalent hIgG1 mAb is not more potent at inducing\nT cell deletion than its bivalent counterpart. Importantly, this data also highlights a caveat in clinical\nstudies that used hIgG1, namely that anti-CD27 hIgG1 deletes both effector T cells as well as Tregs.\nHowever, when we converted the antibody to a V11 version, a format with a modified Fc that\nreduces affinity for activating FcγRs and increases affinity for hFcγRIIB, T cell deletion was abolished\nand replaced with enhanced proliferation, consistent with enhanced agonism and CD27 signalling\n(Figure 6B). This highlights the importance of selecting a non-depleting format in order to achieve\neffective agonism.\nIn our revised manuscript, we also further addressed this question in the CT26 mouse model using\nthe tetravalent anti-mCD27 antibody with a mouse IgG1 Fc domain. This isotype preferentially\nengages the inhibitory mFcγRII while showing minimal-to-no binding to activating mFcγRs, mirroring\nthe FcγR binding profile of the human IgG1 V11 variant. In this setting, we observed a marked\nincrease in tumour-infiltrating CD8⁺ T cells, with no corresponding change in Tregs (new\nSupplemental Figure 5). These findings suggest that the mechanism of action of the tetravalent anti-\nmCD27 antibody is not dependent on Treg depletion but rather involves selective enhancement of\nCD8⁺ T cell activation and responses (new Supplemental Figure 5).\n3) Figure 4. To fully evaluate the anti-tumor efficacy of these antibodies, both tumor growth curves\nand survival curves of the treated mice should be presented. Additionally, a more detailed analysis\nof the tumor immune microenvironment is warranted. This should include quantifying different T\ncell populations (at least conventional T cells [Tcon], regulatory T cells [Treg], and cytotoxic T\nlymphocytes [CTLs]) and assessing the expression of activation and exhaustion markers on these\ncells. The current data are presented at a single time point, which provides a snapshot of T cell\nactivation. However, a longitudinal analysis over multiple time points could provide more\n5\ncomprehensive understanding of the dynamics of T cell activation and the sustained effects of the\ntreatment.\nWe thank the reviewer for this suggestion. B16-OVA is known to be resistant to long-term T cell\nimmunotherapy due to the emergence of OVA loss variants (Kaluza et al Int J Cancer 2011). \nAccordingly, in the B16-OVA model shown in Figure 4, the OVA-specific CD8⁺ T cell response induced\nby OVA vaccination and anti-CD27 agonist treatment leads only to a transient delay in tumour\ngrowth, without a significant improvement in long-term survival. We have amended the manuscript\ntext to acknowledge this limitation and added survival data from a B16-OVA experiment in new\nSupplemental Figure 3. To complement this data and to address the reviewer’s point, we have\nadded a longitudinal analysis of antigen specific T cells (Figure 4C). This new data highlights the\nlongevity of the antigen-specific T cell response elicited by the tetravalent anti-mCD27 antibody. The\nnew Figure 4C complements the original graph which showed the antigen-specific response only at\nthe peak of the response.\nThe second well-established tumour model we present, the BCL lymphoma (references 19, 35 and\n1\n58), primarily grows in the spleen, which limits the ability to directly measure tumour burden.\nUnfortunately, it is not possible to use fluorescent or luminescent proteins to monitor tumour load\nbecause the BCL lymphoma does not grow in vitro and is propagated by in vivo passage and\n1\ntherefore manipulation in vitro eg. transfection to express fluorescent or luminescent proteins\ncannot be performed. The BCL is a well-established model and the methods to measure survival of\n1\nprevious publications using this model. For these reasons we present survival data as an indicator of\ntherapeutic efficacy (Figure 5A).\nWe have now included a third model, the CT26 colon carcinoma, in which treatment was\nadministered to mice bearing established subcutaneous tumours. Tumour growth curves and\ncorresponding survival data are shown in Figures 5B–D. In this model, treatment with the tetravalent\nanti-mCD27 resulted in complete tumour regression in responding mice. In contrast, only 50% of the\nresponding mice treated with the bivalent anti-mCD27 survived the tumour challenge.\nMoreover, we performed additional experiments with the CT26 model to understand the cell types\ninvolved in the anti-tumour response. Data presented in Supplemental Figure 5 demonstrates that\ntreatment with tetravalent anti-mCD27 induced an increase in both total and activated (4-1BB+ and\ngranzyme B+) tumour-infiltrating CD8+ T cells without impacting total tumoral CD4+ or CD4+/Foxp3+\nTregs. These data support the hypothesis that tetravalent anti-mCD27 exerts its effects through\ndirect co-stimulation of CD8+ T cells.\nDynamic measurements of the antigen-specific response are shown for the B16-OVA model in Figure\n4C of the revised manuscript. This was carried out by serial blood sampling. Our analysis of the\ntumour microenvironment (TME) in the CT26 model was carried out at a single time point and this\nanalysis showed clear differences in the activation and cytotoxicity markers (4-1BB and granzyme B)\non CD8 T cells. Given that the TME immune phenotyping experiments require mice to be sacrificed\nand that our objective of deciphering the mechanism of action of the tetravalent anti-CD27 mAb was\nachieved; the tetravalent anti-CD27 mAb induced a clear increase in 4-1BB and granzyme B\nexpression on CD8 T cells without Treg depletion, we could not justify to our animal welfare and\nethics board conducting additional time points and sacrificing more animals. The increase in the 4-\n1BB, an activation receptor associated with improved metabolic fitness of CD8 T cells, the\nupregulation of the cytotoxic molecule granzyme B on CD8 T cells and the lack of Treg depletion\nprovides mechanistic insights of how the tetravalent anti-CD27 exerts its potent anti-tumour effects.\nThese new data are presented in Supplemental Figure 5 of the revised manuscript. Thus, our data\nprovides a mechanistic explanation for the effects independent of whether there are additional\n6\neffects on exhaustion markers. It is noteworthy that the relevance of using exhaustion markers in\nmouse models is questionable and confounded by the fact that T cell exhaustion is limited by the\nshort duration time of tumour growth (days) compared to human tumours (months/years).\n4) The toxicity of the tetravalent anti-CD27 antibody has not been assessed. will the tetravalent\nantibody have a reduced half-life? The safety and PK data will be critical to the translation of this\ntetravalent agonist antibody into clinical drug development.\nWe thank the reviewer for raising this important point. Accordingly, we have determined the serum\nconcentration of the bivalent and tetravalent antibody and present the new data in Supplemental\nFigure 2. Fitting the data by nonlinear regression analysis provides t values of 2.1 and 1.8 days for\n1/2\nthe bivalent and tetravalent antibodies, respectively. Furthermore, treatment of CT26-bearing mice\nwith four doses of the tetravalent antibody did not result in a reduction in body weight, which is\ncommonly used as an indicator of toxicity of agonist immunostimulatory mAbs eg. anti-CD40 and\nanti-4-1BB (new Supplemental Figure 4).\n5) There are several clinical candidates, such as Varlilumab, all of which have shown limited efficacy\nin both preclinical models and patients. To ensure that this study can potentially be translated into\nclinical drug development, the author should evaluate the efficacy and toxicity of the tetravalent\nversion of Varlilumab in a humanised CD27 mouse model. Several companies offer humanised CD27\nmice.\nWe appreciate the reviewer’s constructive comments regarding Varlilumab, a first-generation anti-\nhuman CD27 monoclonal antibody that was withdrawn following a lack of efficacy in Phase I/II\nclinical trials.\nIn our study, the tetravalent anti-human CD27 antibody was evaluated using the well-characterised\nhCD27.15 clone. Unlike Varlilumab, which exhibits weak agonist activity, hCD27.15 has been shown\nto co-stimulate T cell proliferation in vitro in its soluble form (Heckel et al., Communications Biology,\n2022). This functional difference is likely due to its distinct epitope, which differs from that targeted\nby Varlilumab. Notably, the same epitope has been used to select other potent anti-CD27 agonists,\nsuch as MK-5890 (Guelen et al., Journal for ImmunoTherapy of Cancer, 2022), suggesting that\nbinding to this specific region of CD27 imparts agonism.\nFurthermore, hCD27.15 exhibits lower affinity and faster dissociation kinetics compared to\nVarlilumab (Heckel et al., Communications Biology, 2022), properties that have recently been linked\nto enhanced receptor clustering and agonistic activity in other anti-TNFRSF antibodies (Yu et al.,\nNature, 2023). Interestingly, the affinity of hCD27.15 is similar to that of the anti-mouse CD27\nantibody used in our study.\nIn summary, both the epitope specificity and binding kinetics of anti-TNFRSF antibodies are critical\ndeterminants of their agonistic function. Our selection of hCD27.15 for engineering into the new\ntetravalent format was therefore driven by a deliberate effort to incorporate these favourable\nfeatures. We have revised the manuscript text to better highlight these key features and the\nselection of hCD27.15 as our mAb of choice.\nThe use of a humanised CD27 mouse model to evaluate the bivalent and tetravalent anti-human\nCD27 hIgG1 V11 formats is not straightforward because the V11 Fc mutations, which are designed to\nenhance binding to human FcγRIIB and reduce binding to the activating human FcγRs, do not exert\nthe same effects on mouse FcγRs.\nD\nmouse FcγRII, the K values are as follows:\nD\n• hIgG1: 2.2 × 10⁻6 M\n• hIgG1 V11: 1.3 × 10⁻6 M\n• mIgG1: 3.9 × 10⁻7 M\nIn contrast, for human FcγRIIB, hIgG1 and hIgG1 V11 show K values of 9.4 × 10⁻7 M and 7.8 × 10⁻8\nD\nM, respectively. These data highlight that a humanised CD27 mouse model cannot faithfully\nreplicate the physiological interaction between the tetravalent anti-CD27 V11 antibody and human\nFcγRIIB.\nTherefore, to test in a humanised mouse model one would need to generate mice with humanised\nCD27 and human FcγRs. Generating these double transgenic mice would take between 18-24\nmonths providing they can be sourced from suppliers and collaborators. Moreover, the human FcγR\ntransgenic mice have variable levels of FcγRs expression that do not always reflect physiological\nlevels seen on human leukocytes and human IgG1 has a much shorter half-life in mice.\nGiven these caveats, we felt it was more appropriate to investigate anti-human CD27 antibodies\nusing our human PBMC assay. This assay employs whole human PBMCs, thereby preserving not only\nphysiological levels of human CD27 expression on T cells but also the native expression profile of\nhuman FcγRs on B cells, myeloid cells, and NK cells such as would be present in human blood. This\nsystem proved invaluable for distinguishing the functional profiles of the various anti-human CD27\nantibody formats, including tetravalent vs. bivalent, and hIgG1 vs. Fc-silent (hIgG1 NA) vs. hIgG1 V11,\noffering key mechanistic insights that would be obscured in murine models lacking human FcγR\nbiology.\nMinor points:\n1) Table 1, which model is used to fit the SPR data, 1:2 or 1:1 kinetic model? Is the binding affinity\napparent Kd?\nAs detailed in the table legend, the bivalent (2:1) fit was used to model affinity. (2:1) has now been\nincluded for clarity. Affinity is assumed to be apparent affinity due to multivalent binding, with table\n1 and supplementary table 2 (previously supplementary table 1) updated to reflect this.\n2)The authors should use a consistent name throughout the manuscript, for example, CD8+ T cells\nand CD8 T cells.\nNoted and changed for consistency.\n3) Figure 4C, the tumor volume at the last time point was less than 200mm3. The tumor growth\ncurve should be plotted until some tumors reach 1500 or 2000mm3.\nTo clarify, tumour size is determined by cross-sectional area and is the product of measurable\nperpendicular tumour dimensions (length × width). The units on the y-axis of the tumour growth\ngraphs are mm2. A cross-sectional area of 200 mm2 is approximately equivalent to a volume of 1500\nmm3.\n4) In lines 271-275 of the Methods section, the author should clarify how many doses of antibody\nwere administered. It would be better to inject OVA with an adjuvant to better induce T cell\nimmunity.\nagonist was used as an adjuvant. CD27 agonists exhibit potent adjuvant-like properties for enhancing\nCD8⁺ T cell responses, surpassing those of convenYonal adjuvants such as TLR4 agonists (Rowley and\nAl-Shamkhani, J Immunol, 2004).\n6) In the Figure 5 section, the author should discuss that some human CD27 antibodies are less\ndependent on FcγRIIb cross-linking, but that FcγRIIb cross-linking can further enhance potency,\nsuggesting that the agonistic activity of CD27 antibodies may be influenced by their epitopes. The\nbivalent antibody and the tetravalent antibody with the Fc V11 variant did not show a significant\ndifference in Figure 5A. What is the reason?\nWe agree with the reviewer that some antibodies can elicit agonism in their soluble form and are\nless dependent on FcγR crosslinking, and that this ability is influenced by the specific epitope\nrecognized by the antibody. The anti-human CD27 clone hCD27.15 used in our study is known to\ntarget an epitope associated with stronger agonistic activity (please refer to our response to\nReviewer 3, Major Point 5, for a detailed discussion on the roles of epitope specificity and affinity).\nWe have updated the manuscript text accordingly in light of the reviewer’s comment.\nThe reviewer also asked, “The bivalent antibody and the tetravalent antibody with the Fc V11 variant\ndid not show a significant difference in Figure 5A. What is the reason?” The tetravalent anti-hCD27\nhIgG1 V11 clearly demonstrated greater activity at lower concentrations compared to the bivalent\nanti-hCD27 hIgG1 V11, as indicated by the highly statistically significant difference shown in Figure\n5A (lower right panel). Specifically, the EC₅₀ values for the tetravalent and bivalent V11 antibodies in\nthe presence of FcγRIIB were 0.0002 and 0.008 nM, respectively, a 40-fold difference.\n7) In Figure 6C, the video should be provided in supplemental information.\nAs detailed in the Figure and Methods section, this experiment was conducted as a time course\nrather than a live video recording, with cells fixed at the indicated time points and subsequently\nimaged. Representative images are shown in the Figure.\n9) For Figure 6E, it would be informative to analyze whether the percentage of GFP+ cells shows\nsignificant changes after treatment with either bivalent or tetravalent antibodies. Additionally, to\nenhance data interpretation, both MFI and the percentage of positive cells could be calculated and\npresented on the y-axis.\nWe thank the reviewer for their constructive suggestion, and we have now included these graphs as\nFigure 6F.\n10) The Discussion section would benefit from a comprehensive analysis of the potential limitations\nassociated with the tetravalent antibody approach, including but not limited to its structural\nstability, pharmacokinetic properties, and potential immunogenicity.\nWe thank the reviewer for this important comment. We have now added new data on the half-life\nand thermostability of the tetravalent antibody, which show only a modest reduction compared to\nconventional IgG. These results are included and discussed in the revised manuscript.\nRegarding immunogenicity, the tetravalent antibody sequence is composed almost entirely of native\nhuman IgG components. As such, the likelihood of eliciting a strong anti-drug immune response is\nexpected to be low. It is worth noting that clinically approved chimeric antibodies such as infliximab\nand cetuximab, despite containing a significantly higher proportion of non-human (murine)\nsequence, have been successfully used in patients, further supporting the tolerability of antibody\nformats with structural modifications. In response, we have added a final paragraph to the\nincreased immunogenicity.\n11) The term \"Finaly\" in line 175 requires correction to \"Finally.\" Additionally, we suggest performing\na comprehensive spell-check of the entire manuscript to identify and rectify any similar typo.\nNoted and corrected.\n12) Although Figure S2 shows no aggregation of the tetravalent antibody, this does not mean that it\nis stable. The thermostability of the tetravalent antibody should be determined.\nThermostability was assessed using nano differential scanning fluorimetry (nanoDSF), which\ndemonstrated that the tetravalent antibody has a melting temperature of 56.7 °C. Although this is\nslightly lower that of the IgG molecule (63.5 °C), it is well above physiological temperatures and the\nstandard accelerated storage temperature of 40°C for monoclonal antibodies. These results are\nsummarised in Supplemental Table 1 and discussed in the revised manuscript in the Results and\nDiscussion.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe would like to thank the reviewer for their constructive comments and suggestions.\n10"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done additional experimental work and addressed my concerns.", "reviewer_2": "In their revision, the authors addressed my critical point adequately. I recommend publication.", "reviewer_3": "The authors have addressed most of the comments raised during revision. However, several minor points still require clarification before acceptance to Nature Communications.\n1. The layout of the main figures requires overall optimization to improve clarity and visual appeal.\n2. Although treatment of CT26-bearing mice with the tetravalent antibody did not result in a reduction in body weight, additional safety parameters, such as hepatotoxicity (ALT, AST et al.), should also be assessed to further substantiate the safety profile of the tetravalent antibody.\n3. Regarding the response to minor point 9), the figure referred to by the authors as Figure 6F does not appear to correspond; it may in fact be Figure 7F?", "reviewer_4": "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