Supramolecular coiled-coil peptide platform for site-specific antibody drug conjugate engineering

Domain: chemistry
Published: 2026-03-02
Editorial outcome: minor_revision

Abstract
Antibody conjugates play a central role across multiple healthcare sectors with a prime example being antibody-drug conjugates (ADCs). Although widely used lysine and hinge cysteine conjugation methods yield products, the lack of site-specificity and spatial control along with the highly heterogeneous composition are significant limitations. Herein, we describe a facile supramolecular assembly method based on heterodimer coiled-coil formation for site-specific antibody conjugation that perseveres antibody antigen binding sites for target engagement. The platform method affords uniform loading of diverse payloads including anti-cancer agents, polymers, enzymes, fluorophores, etc. under mild aqueous conditions. Further, the facile convergent approach capitalizes on the independent strengths and flexibility of protein expression and peptide chemistry culminating in a final self-assembly step. An ADC loaded with monomethyl auristatin E targeting ErbB2/Her2 positive tumors significantly reduces tumor volume in a human ovarian cancer xenograft model outperforming the antibody alone with validated performance against a best-in-class therapeutic.

Introduction
Antibody-drug conjugates (ADCs) are transformative treatments against cancer 1–5. Classical and still widely used bioconjugation methods for ADCs include reacting protein primary amines with an entity containing an activated ester or converting primary amines to thiols, via Traut’s reagent, or using hinge cysteines for subsequent coupling to a maleimide functionalized moiety 6–8. Although they are useful reactions, their selectivity and specificity are poor with ill-defined, highly heterogeneous products being obtained from a single reaction 9,10. Alternative methods use unique reactive sites such as a C-terminal thioester or N-terminal amine, however yields remain low 11,12. Newer techniques rely on biorthogonal chemistry, the selective chemical transformation within a complex biological milieu, 13,14 and include: 1) incorporation of a non-natural amino acid into the protein such as azide-modified phenylalanine 15–18 or glycan remodeling 19–21 for a subsequent specific click-chemistry reaction (e.g., azide fragment with an alkyne functionalized molecule); 2) disulfide rebridging 22,23; and, 3) enzymatic ligation reactions (e.g., AviTag, SorTag) 24–27 and split proteins tags (e.g. SpyTag, Intein Tag) 28–31. Each of these methods possess a number of advantages for the preparation of ADCs, but none have advanced to preparation of clinically approved products 32,33.

A supramolecular assembly synthesis strategy offers benefits such as ease of use, selectivity, specificity, bio-orthogonality, and complementarity to other methods. Supramolecular assembly, the ability of smaller entities to spontaneously organize into well-defined larger structures through non-covalent interactions, is widely used in materials science, chemistry, and biological chemistry 34–38. Alpha-helical coil peptides are protein structural motifs capable of orthogonal and selective self-assembly into coiled-coil structures (Figure 1A). Although they rely on non-covalent interactions, the presence of multiple contact points within the assembly significantly contributes to stability with dissociation constants in the sub-nanomolar range 39. Due to modular design and easily controlled molecular features, by varying the peptide sequences, coiled-coils are used in many biomedical applications 40–44 including hydrogel fabrication 45,46, nanoparticle modification 47,48, fusion protein preparation 49,50, and cell therapy engineering 51,52.

Here, we report a biorthogonal platform based on supramolecular assembly of coiled-coil peptides for preparing antibody-conjugates, including antibody-drug conjugates (ADCs). The Fc region of an IgG with two heavy chains is poised for a heterodimer coiled-coil supramolecular interaction: two C-terminal receiving peptides on the antibody assemble with two separately synthesized docking peptides containing the conjugated payload of interest (Figure 1B). This facile synthetic strategy enables uniform stoichiometric site-specific antibody conjugation while maintaining antibody functionality. It is a convergent bioconjugation method where the final self-assembly step occurs under mild aqueous conditions facilitating an easy, modular pairing of antibodies to payload(s) simplifying final production and purification. We site-specifically pair antibodies with fluorophores, cytotoxic drugs, peptides, polymers, nucleic acids, and enzymes. The resulting antibody-conjugates retain function and bioactivity, and a prepared ADC excels in a murine xenograft human ovarian cancer model.

Results
Antibody-receiving peptide production and characterization. We identified two sets of peptides which form dimeric coiled-coil structures without self-interaction to create the bioconjugate platform 53. Each of the four peptide sequences (P1, P2, P3 and P4) comprise four amino acid heptad repeats containing different patterns of glutamic acid or lysine for promoting electrostatic interaction between the coils and leucine or isoleucine for hydrophobic interaction (Supplementary Table 1). We introduced a single unnatural azide-bearing amino acid into the C-terminus of these peptides to enable subsequent conjugation, generating a novel set labeled as P1N3, P2N3, P3N3, P4N3. Small peptides bearing unnatural amino acids are readily synthesized via solid-phase peptide synthesis at milligram scale 54,55 unlike large antibodies that usually require elaborate optimization of genetic encoding and protein expression 16,56,57. Individually, the peptides do not form an organized structure but spontaneously form a heterodimeric coiled-coil structure when mixed at 1:1 molar ratio with its pairing peptide (P1N3 when mixed with P2N3, and P3N3 when mixed with P4N3). The assembled coiled-coil structure is stable over time and under endosomal-lysosomal pH conditions (Supplementary Figure 1).

Next, we designed a set of plasmids encoding the trastuzumab heavy chain fused at the C-terminal end with one of the receiving peptide sequences (P1, P2, P3, or P4; Supplementary Figure 2). We expressed the antibodies in Expi293F cells and collected the protein via Protein A column chromatography. Incorporation of the peptide at the C terminal end of the antibody does not impair antibody production, as titers are similar between peptide modified and native antibodies and ranged from 40 to 80 mg/L (Supplementary Table 2, 3). A fully folded antibody possesses two identical peptide tags linked to the two heavy chains at the Fc region. The far-UV CD spectrum displays a broad minimum at 218 nm for the unmodified αHER2 antibodies and for the peptide-tagged antibodies (αHER2-P1, αHER2-P2, αHER2-P3, and αHER2-P4), characteristic of a significant presence of β-sheets in IgG (Supplementary Figure 3) indicating that coil tag does not interfere with the protein folding. The peptide-modified antibodies, except αHER2-P2, elute slightly earlier (αHER2-P1 t=6.95 min; αHER2-P2 t=7.15 min; αHER2-P3 t=7.03 min; αHER2-P4 t=7.00 min) than the native αHER2 antibodies (t=7.15 min) during SEC chromatography, consistent with an increase in mass due to peptide insertion (Figure 1C). The slower elution of αHER2-P2 is likely attributed to the lysine-rich P2 sequence, which also contributes to peak tailing. Antibodies modified with the P3 peptide display a uniform peak and were used for further experiments. SDS-PAGE analysis additionally confirms the modification of the heavy chain with the coil peptide (Figure 1D, Supplementary Figure 4A). Non-reduced antibodies with the inserted peptide exhibit a shift toward a higher molecular weight region compared to unmodified antibodies. Upon reduction, the light chain remains intact, while the heavy chain-coil peptide conjugate shifts to a higher molecular weight region, as anticipated based on the conjugate design. Finally, the MALDI-TOF MS spectrum reveals a major peak at 156 kDa for αHER2-P3, corresponding to the expected molecular weight of the full antibody (trastuzumab Mw ≈ 148 kDa) with two receiving peptides (P3 Mw ≈ 4 kDa each) (Supplementary Figure 4B,C). To confirm the recombinant antibody binds the antigen (ErbB2/Her2 protein), we performed an ELISA assay and compared it to the commercial αHER2 without the peptide tag and αHER2 with a P3 receiving peptide tag. The EC50 values for all tested samples are: 0.110 ± 0.024 nM and 0.133 ± 0.003 nM respectively, indicating that the peptide tag does not compromise target binding of the antibody (Figure 1E). We then measured the affinity of coiled-coil formation when one of the peptides is fused to the antibody. We immobilized the biotin-labeled docking peptide (P4N3-biotin) on a BLI sensor and exposed it to αHER2-P3 receiving peptide antibody at various concentrations. A shift in the interference pattern occurs only between the complementary pair of a docking peptide (P4N3) and the corresponding P3 receiving peptide on αHER2 antibodies (αHER2-P3). The calculated dissociation constant (Kd) is 6 x 10-10 M, indicating a highly stable structure and similar in magnitude as that reported by Plaper et al 58 (Figure 1F). Repeating the affinity experiment using microscale thermophoresis (MST), an immobilization-free assay format, affords similar Kd values in nanomolar range (Supplementary Figure 5). The interaction between the docking and receiving peptides is specific as antibodies without a receiving peptide and those with a non-complementary receiving peptide tail, do not elicit a BLI response when mixed with the docking peptide (Supplementary Figure 6). Next, we evaluated if conjugation at the Fc region interferes with its interaction with the neonatal Fc receptor (FcRn), which regulates IgG recycling. We plated native trastuzumab antibody or αHER2-P3 on an ELISA plate and incubated with biotin-labeled human FcRn receptor, followed by detection with streptavidin-HRP conjugates. The αHER2-P3 antibodies maintain their affinity to FcRn after introducing the coil, however the binding is slightly lower than for native trastuzumab (Figure 1G). Overall, these findings indicate that the introduction of a coiled-coil peptide tag preserves the key properties of the mAb and its antigen-binding capabilities.

Conjugation principle. With introduction of a coil peptide tag preserving the properties of the mAb and its antigen-binding capabilities, we next investigated payload loading onto the antibody which occurs in two steps: 1) attachment of the payload to the docking peptide via a strain-promoted azide-alkyne cycloaddition reaction; and 2) self-assembly of the payload-conjugated docking peptide with the receiving peptide on the antibody. This convergent approach enables expanded payload chemistry on the docking peptide independent of antibody modification, thereby separating the steps, minimizing the reactions with the antibody, and avoiding denaturation or aggregation of monoclonal antibody in organic solvents and/or harsh pH conditions. Thus, this facile plug-and-play supramolecular assembly opens opportunities for novel chemistries and payloads to be conjugated to the docking peptide.

Docking peptide conjugation. To demonstrate the utility of the methodology, we prepared docking peptides linked with different entity classes to include a fluorophore (e.g., DBCO BP Fluor 647), antimitotic agents mertansine (DM1) and monomethyl auristatin E (MMAE), a DNA oligo (e.g., polyA15 CCC), a polymer (e.g., PEG10k), an enzyme (e.g., HRP), a biotin molecule, and a phospholipid (e.g., DSPE). DBCO modified payloads, except HRP, were mixed is a small excess (1.2 eq) with azide bearing docking peptide (P4N3) (Figure 2A). The coupling reactions occur in minutes as monitored through depletion of DBCO absorbance peak at 310 nm (Figure 2B). The estimated reactions half-life with P4N3 is 26 ± 2 min for DBCO BP Fluor 647, 12 ± 3 min for DBCO-PEG4-DM1, 42 ± 10 min for DBCO-PEG4-ValCit-MMAE, 77 ± 13 min for polyA15 CCC oligo, 27 ± 4 min DBCO-PEG15, 16 ± 2 min for DBCO-PEG4-biotin, and 25 ± 8 min DBCO-PEG4-DSPE (Figure 2C-I). Mass spectrometry confirms the sequential conjugation and desired product formation (Supplementary Figure 7).To enable assembly of antibody-enzyme conjugate using this technology, we expressed a recombinant enzyme with the docking peptide sequence fused into the C-terminal of the peroxidase C1A. The activity of the engineered HRP-P4 enzyme is > 60 U/mg, as measured via an enzymatic reaction with the colorimetric 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Figure 2J and Supplementary Figure 8). The introduction of peptide to the HRP enzyme doesn’t interfere with its activity and provides a handle for assembly with receiving peptide.

Coiled-coil mediated assembly of antibody–payload conjugates and their functions. We then assembled the antibody conjugates by simply mixing the antibody-receiving peptide (αHER2-P3) with the payload modified docking peptide in PBS for one hour followed by spin column centrifugation to isolate the desired product (Figure 3A). To confirm the conjugation and payload’s activity, we performed functional assays. The antibody conjugate with the BP Fluor 647 payload absorbs at 647 nm while the native antibody does not (Supplementary Figure 9). Based on absorbance measurements at 280 nm and 647 nm, the fluorophore-to-antibody ratio is 2.02, consistent with the designed coiled-coil assembly. Additionally, mixing αHER2-P3 with a solution containing P1, P2, P3 and P4 peptides labeled with different fluorophores (P1-BP Fluor 647; P2-TAMRA 555; P3-BP Fluor 594 and P4-BP Fluor 488) results in a conjugate with an absorbance in the BP Fluor 488 region only, indicating selective assembly of P4-BP Fluor 488 and αHER2-P3 (Supplementary Figure 10). These data are consistent with the results with the coiled-coils themselves 53. The antibody-fluorophore conjugate specifically targets ErbB2/HER2-positive cells in an ErbB2/Her2+ dependent manner as determined by flow cytometry using this fluorescent tag (Supplementary Figure 11).

To evaluate the αHER2-HRP conjugate activity, we conducted a direct ELISA using ErbB2/Her2 protein immobilized on a plate and TMB as the HRP substrate (Figure 3B). The αHER2-HRP conjugate catalyzes the oxidation of TMB in a dose-dependent manner, demonstrating that, despite the presence of a large payload, αHER2-HRP retains its binding affinity to ErbB2/Her2 post-conjugation, while also maintaining HRP enzymatic activity. For the antibody-PEG5k-DSPE lipid, mPEG10k polymer, and biotin conjugates we performed indirect ELISA assays using anti-PEG secondary antibodies or streptavidin-HRP for payload detection. All the assays confirm the successful conjugation of antibodies with different payloads (Supplementary Figure 12). For oligonucleotide-loaded antibodies, we mixed the single-stranded DNA-loaded (polyA15 GGG) antibody with a complementary TAMRA fluorophore-labeled strand (polyT15 CCC-TAMRA) and incubated the mixture to allow hybridization followed by purification via spin filtration. The results confirm successful hybridization of the complementary oligos, as indicated by the TAMRA fluorophore absorbance of the final product, demonstrating the capability of formation of a double-stranded oligonucleotide structure post conjugation to the αHER2 antibody (Figure 3C and Supplementary Figure 13). Finally, we assessed the cytotoxicity of the synthesized αHER2-MMAE conjugates using the MTS assay against three cell lines with different ErbB2/Her2 expression levels: 4T1 (Negative ErbB2/Her2-), A549 (Low ErbB2/Her2+), SKOV-3 (Medium-high ErbB2/Her2++) and SKBR3 (High ErbB2/Her2+++). We treated cells with varying concentrations of ADCs, unloaded αHER2 antibodies, or free MMAE drug for 72 hours (Figure 3D and Supplementary Figure 14). Treatment with the ADC shows a dose-dependent and ErbB2/Her2 expression-dependent decrease in cell viability for SKBR-3 (ADC EC50 ~ 0.002 uM), SKOV-3 (ADC EC50 ~ 0.09 uM) and A549 cell lines, while showing minimal toxicity in ErbB2/Her2 negative 4T1.

Dual-payload loaded antibody-conjugates. An additional beneficial feature of this coiled-coil design is that two or more, similar or different, entities can be attached to the antibody by extending the coil-coiled structure with the same peptide or an orthogonal peptide sequence set. To demonstrate the assembly of dual-payload loaded antibodies, we fused receiving peptide P1 followed with another receiving peptide P3 sequence into the heavy chain of trastuzumab resulting in antibodies with two sequential orthogonal conjugation sites (Figure 3E and Supplementary Figure 15). As a model system, we chose DBCO-modified BP Fluor 488 (Fluor488) and DBCO-modified BP Fluor 647 (Fluor647), which we conjugated to azide-modified P2N3 and P4N3 peptides respectively resulting into P2-Fluor488 and P4-Fluor647 conjugates. Coil peptide P1 forms an orthogonal coiled-coil structure with peptide P2, while peptide P3 forms a dimer with peptide P4. Next, we mixed P2-Fluor488 or P4-Fluor647 with the antibodies separately. After one hour incubation and spin-filtering purification we obtained the conjugates bearing either Fluor488 or Fluor647 fluorophores. The absorbance spectra of the conjugation products exhibit maxima at 280 nm, corresponding to protein absorbance, and either at 488 nm or 647 nm, depending on which payload loaded indicating that the extended peptide tag structure does not interfere with payload loading (Figure 3F,G). We then mixed both P2-Fluor488 and P4-Fluor647 with the antibodies bearing P1 and P3 peptides. The corresponding peptide pairs self-assemble leading to formation of dual-loaded antibody conjugates both with Fluor488 and Fluor647 (Figure 3H). This approach offers the opportunity to increase the drug-antibody ratio or load two different payloads in a defined stoichiometric ratio and at a specific location on the antibody, enabling the development of new co-delivery strategies.

Stability of antibody-payload conjugates. To evaluate the stability of the proposed conjugation method, we compared the stability of conjugates formed via conventional thiol-maleimide covalent linkage with those generated through coiled-coil-mediated conjugation. In addition to the P3 and P4 pair (4 repeating amino acid heptad pattern, total 28-amino-acid-long coil peptides) described above, we investigated two additional peptide pair designs based on prior work on coiled-coil interactions. Woolfson et al., report that increasing the number of heptads in a coiled-coil structure increases the stability affording nM or lower Kd values 39. Also Xia et al., and Lee et al. show that the presence of complementary reactive groups capable of forming a bond (e.g., a disulfide) between the peptides stabilizes the coiled-coils 50,59. Based on this knowledge, for the first design, we extended the P3 and P4 peptide sequence by one coiled-coil heptad pattern, creating 35 amino acids long peptides (P3 5mer and P4 5mer N3, see Supplementary Table 1). We used the same repeating units for the second design except that we replaced the isoleucine with a cysteine residue in the last heptad unit of each peptide to allow a disulfide bond formation between coils (P3 5mer-cys and P4 5mer-cysN3, see Supplementary Table 1). A MALDI laser ionization spectrum of the coiled-coil lacking cysteines (P3 5mer:P4 5mer) shows a primary peak at a m/z of ~ 5,000 – the expected molecular weight of single peptide - consistent with disruption of the coiled-coil noncovalent interactions. In contrast, the spectrum for the cysteine-containing dimer (P3 5mer-cys:P4 5mer-cys) reveals a high-intensity peak at m/z of ~ 10,000. This peak reduces upon addition of TCEP consistent with cleavage of the disulfide bond between the peptides with more resulting single peptides present – analogous to the results obtained with coiled-coil lacking cysteines (Supplementary Figure 16). We produced αHER2-P3 5mer and αHER2-P3 5mer-cys antibodies (Supplementary Figure 17) and assembled them with corresponding docking peptides conjugated to DBCO BP Fluor 647. We then incubated the antibody-fluorophore conjugates (AFCs) in human serum at 37°C in 5% CO2 atmosphere. We collected serum samples over a 28-day period and analyzed for intact AFC levels using an ELISA assay that detects the payload portion of AFC. The AFC prepared by the standard thiol-maleimide hinge conjugation technique degrades by 25%, while the coiled-coil based conjugates showed varying stability levels depending on the coil design (Figure 4A,B). The P3-P4 N3 coils (short 4-heptad sequence) disassemble over the first few days, with only 13% of the AFC intact by the end of the study. In contrast, the AFCs prepared using P3 5mer, P4 5mer N3 and P3 5mer-cys, P4 5mer-cysN3 coil pairs are stable with only a 4% loss in structure over 4 weeks, outperforming the conventional conjugation approach (Figure 4C,D). From a design perspective, we selected the 5-heptad construct with the disulfide bond for the subsequent in vivo experiments because it possesses two stabilizing mechanisms.

Efficacy of antibody-drug conjugates in vivo. Given our interest in ovarian cancer and the dearth of available treatments 60–63, we prepared MMAE loaded ADC conjugates to assess the overall therapeutic potential of the coiled-coil conjugation. We conjugated P4 5mer-cysN3 to DBCO-PEG4-Val-Cit-PAB-MMAE (Figure 4E and Supplementary Figure 18A), where the Val-Cit-PAB portion ensures scar-less drug release 64. MMAE-peptide loading to the αHER2-P3 5mer-cys antibody results in a SEC trace shift (Supplementary Figure 16B) with drug-antibody ratio of two as determined by UV-vis absorbance at 247 nm and 280 nm 65,66 (Supplementary Figure 16C). An indirect ELISA using anti-MMAE secondary antibodies additionally confirms the modification of αHER2-P3 5mer-cys with MMAE drug compared to unloaded antibodies (Supplementary Figure 19).

First, we evaluated the pharmacokinetic (PK) profile of the ADC in vivo. We intravenously (i.v.) administrated native mAb (trastuzumab), unloaded αHER2-P3cys mAb, and MMAE loaded ADC at dose of 10 mg/kg to healthy Balb/c mice. We collected blood samples at predefined time points and measured the concentration of mAb in the blood by an ELISA assay. The half-life for trastuzumab (native αHER2) is 6.5 hours (95% CI 5.5-7.3 h), similar to the unloaded ADC (αHER2-P3 5mer-cys) and MMAE loaded ADC conjugates at 6.8 (95% CI 6.2-7.4 h)and 6.5 (95% CI 5.0-7.7 h) hours respectively (Figure 4F). The coiled-coil peptide tag does not negatively affect antibody PK and clearance in the mouse.

To assess tissue biodistribution of the antibody conjugate, we used an orthotopic xenograft model of ErbB2/Her2-positive human ovarian tumors by implanting SKOV-3 cells intraperitoneally into immunodeficient BALB/c nude mice. When tumors established, we i.v. administered a BP Fluor 647-loaded AFC at a dose of 10 mg/kg. At 1 hour, 1 day, 3 days, 5 days, and 7 days post-injection, we euthanized the animals and quantified the fluorescent signal in the tumor and major organs by IVIS. (Figure 4G,H and Supplementary Figure 20). The AFC accumulates in the tumor throughout the 7-day study period, along with liver and kidneys at the earlier time points. These results are consistent with the literature 67,68 and demonstrate the ability of the coiled-coil based antibody conjugate to deliver a payload to ErbB2/Her2-positive tumors.

To identify an effective dose, we performed a dose–response study. We intraperitoneally administered 2 × 10⁶ SKOV-3-Fluc cells to immunodeficient BALB/c nude mice and treated two weeks post-implantation with ADC doses of 1, 3, 5, or 10 mg/kg, or with PBS as a negative control (Supplementary Figure 21). Treatment with the ADC at 1 mg/kg does not result in IVIS signal reduction. Doses of 3 and 5 mg/kg show a modest reduction in IVIS signal, whereas treatment at 10 mg/kg leads to reduction in IVIS signal consistent with tumor regression. Based on these results, we selected a dose of 10 mg/kg for the subsequent in vivo efficacy studies. For comparison, the effective dose for disitimab vedotin is between 5 and 10 mg/kg 69,70, and for trastuzumab-vedotin is between 3 and 10 mg/kg 71,72 in gastric and ovarian cancer models in mice.

Using the established orthotopic xenograft model (Figure 4I), we performed an efficacy study and i.v. administered a single dose of 10 mg/kg of trastuzumab (native αHER2 antibodies) or 10 mg/kg of coiled-coil ADC (n = 5 per group). Additionally, we administered PBS as the negative control and two positive controls: 1) trastuzumab vedotin and 2) disitamab vedotin — the latest HER2-targeting ADC to advance to Phase III clinical trials 73. Trastuzumab vedotin and disitamab vedotin consist of the trastuzumab and hertuzumab antibody, respectively, and, similar to our ADC, uses the cytotoxic payload MMAE linked via a Val-Cit cleavable linker. However, both commercial antibodies employ cysteine-based conjugation, resulting in approximately four MMAE molecules per antibody. To match the drug dose of the positive control groups, we also administered a double dose of coiled-coil ADC (20 mg/kg, n = 5 per group). The antibody vehicle controls for this study are trastuzumab mAb and disitamab mAb. All groups tolerated the treatment with no significant changes in body mass (weight loss of >15%) (Figure 4J and Supplementary Figure 22) or observable adverse effects on animal behavior at earlier time points. However, at week 7, the PBS treatment group and one trastuzumab-treated animals exhibited hunched posture and signs of gait rigidity. We euthanized the mice on week 7 post single dose treatment, and tumor size and mass were measured. Compared to the PBS treated animals, all treatment groups significantly reduce tumor size and mass (Figure 4K-M and Supplementary Figure 23). Mice treated with 10 mg/kg coiled-coil ADC exhibit a significant reduction in tumor size, with a 42-fold decrease in tumor mass and 37-fold decrease in tumor volume (m = 13±9 mg, V = 24±18 mm3) compared to the untreated control (m = 542±322 mg, V = 880±479 mm3). In contrast, those receiving the antibody vehicle controls (trastuzumab mAb or disitamab mAb) show only an 10-fold or 5-fold and 5-fold or 6-fold reduction in tumor mass and volume respectively (m = 52±27 mg, V = 166±81 mm3 for trastuzumab and m = 78±32 mg, V = 145±97 mm3 for disitamab mAb). Treatment with 20 mg/kg ADC, 10 mg/kg trastuzumab vedotin, or 10 mg/kg disitamab vedotin completely eradicates tumor in one mouse from each group, with average tumor masses of 5±3 mg, 6±7 mg, and 2±3 mg, respectively, with a similar trend in the tumor volume: V = 6±5 mm3, V = 9±11 mm3 and V = 4±4 mm3, respectively (Figure 4K-M and Supplementary Figure 23). Disitamab vedotin and trastuzumab vedotin are highly efficacious, consistent with its elevated drug-to-antibody ratio for both ADCs and enhanced affinity for the HER2 target of disitamab vedotin 74. Our comparable results from coiled-coil ADC treatment group bodes well for further use of the coiled-coil conjugation method for antibody conjugation.

Finally, we confirmed the dependency of the therapeutic efficacy of the ADC on ErbB2/Her2 expression levels in vivo. We compared ErbB2/HER2 expression in human lung A549 and SKOV-3 cell lines, as well as in their corresponding tumor tissues. SKOV-3 cells exhibit an 83-fold higher ErbB2/Her2 targeting at the cellular level compared to isotype control and an 11-fold increase for tumor tissues, whereas for A549 both for the cell line and tumor tissue the ErbB2/Her2 targeting is modest, with 1.6-fold and 2.5-fold increases, respectively (Supplementary Figure 24). We then established an A549 xenograft model via subcutaneous implantation. At the 10 mg/kg i.v. dose, neither the trastuzumab antibody (mAb), MMAE coiled-coil ADC, nor disitamab vedotin demonstrate any significant tumor growth inhibition, consistent with the low ErbB2/Her2 expression levels observed in A549 cells and tumors (Supplementary Figure 26).

Methods
Data representation. Data represented as mean ± s.d. with biological replicates.

Cells. SKOV-3 (cat.# HTB-77), SKBR-3 (cat.# HTB-30), A549 (cat.# CRM-CCL-185) and 4T1 (cat.# CRL-2539) cell lines were obtained from American Type Culture Collection (ATCC, USA). Cell media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin was used to maintain the cell lines: McCoy's 5A Medium (ATCC 30-2007) for SKBR-3 and SKOV-3, F-12K Medium (ATCC 30-2004) for A549 and RPMI 1640 (ATCC 30-2001) for 4T1. The cell lines were cultured at 37 °C in a humidified CO2 (5%) incubator. Expi-293F cells (cat.# A14527, Gibco™) were cultured in Expi293™ Expression Medium (Gibco™) at 37 °C under constant shaking (120 r.p.m.) in a humidified CO2 (8%) incubator.

Plasmids. The pcDNA3.1(-) backbone (Invitrogen, cat.# V79520) was pre-cut with EcoRV and BamHI restriction enzymes (NEB) and purified using the QIAquick PCR Purification Kit (Qiagen). The 5′ ends were dephosphorylated with Quick CIP (NEB) to prevent re-ligation. The gBlocks of interest were synthesized by Integrated DNA Technologies (IDT) and inserted into the prepared pcDNA3.1(-) backbone via Gibson Assembly (NEB). The assembled plasmids were transformed into NEB Stable Escherichia coli and purified using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research). Sequence confirmation was performed by Sanger sequencing using CMV-F and BGH-R primers (Genewiz).

Protein expression and purification. Native αHER2 and receiving-peptide modified αHER2 antibodies, as well as HRP-P4 conjugates were produced in Expi293F expression system (Gibco™) according to manufactures’ protocol. Expi293F cells were co-transfected with a mixture of light and heavy chains containing plasmids for mAb production or with a single plasmid containing HRP-5xHis docking peptide fusion sequence. Proteins were harvested on day 6-7 by centrifugation at 3,220 g for 20 min. The supernatant was filtered through a 0.22-µm membrane for subsequent purification and mixed 1:1 with equilibration buffer. mAbs were purified using a gravity-flow column (Biorad) packed with rProtein A Sepharose Fast Flow resign (Cytiva), according to the resin manufacturer protocol. Phosphate-buffered saline (pH=7.4) was used as equilibration and wash buffer, 0.2M glycine-HCl (pH=2.5) and 1M TRIS-HCl (pH=9.0) were used as elution and neutralization buffers respectively. HRP-P4 was purified using HisPur™ Ni-NTA Resin (Thermo Fisher Scientific) according to the resin manufacturer protocol. High-salt phosphate buffer (20 mM sodium phosphate, 300 mM sodium chloride) with 10 mM imidazole (pH=7.4) was used as equilibration buffer, high-salt phosphate buffer with 25 mM imidazole (pH=7.4) and high-salt phosphate buffer with 250 mM imidazole (pH=7.4) were used as wash and elution buffers respectively. Collected proteins were concentrated and buffer exchanged to PBS with centrifugal filters (10kDa or 50 kDa molecular weight cut-off, MilliporeSigma). The concentration of all proteins was determined by absorbance at 280 nm using sequence predicted extinction coefficients (mAb ε280 =216,340 M-1cm-1; and HRP-P4 ε280 =15,065 M-1cm-1).

Proteins characterization.
Size exclusion chromatography (SEC-RI). Antibodies were analyzed using an AdvanceBio SEC column (130Å, 2.7 µm, 4.6 x 150 mm Agilent Technologies). The column was equilibrated and operated with phosphate-buffered saline (PBS, pH 7.4) as the mobile phase at room temperature under isocratic conditions at 0.7 mL/min flow rate, and elution was monitored by RI detector (Agilent 1260 Infinity II).

SDS-PAGE gel. Produced proteins (αHER2, αHER2-coil peptide, SHRP-P4) were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). 1 μg of each protein was mixed with gel loading buffer (reducing or non-reducing) and loaded into wells of a 4-15% precast gel (BioRad). The gel was subjected to 120 constant volts for 40-60 minutes. Following this, the gel was stained with Coomassie Blue (Thermo Fisher Scientific).

Circular Dichroism (CD). Antibodies and peptides were prepared in PBS buffer (pH 7.4) at the concentration of 0.2 g/L. The measurements were performed on CS/2 Chirascan (Applied Photophysics) CD spectrometer using a 1 mm QS 100 (Hellma) cuvette. PBS solution was used as a blank. To study the stability of the coiled-coil assembly at different pH levels, the peptides were buffer exchanged using 3 kDa MWCO spin filters (MilliporeSigma) into a PBS solution adjusted with HCl to pH 5.4, 6.4, or 7.4. For the overtime stability study of the PC3-P4 coiled-coil assembly, the peptides were incubated at 37 °C between measurements. The proportion of intact coiled-coil assemblies relative to single α-helices was estimated by calculating the ratio of ellipticity at 222 nm to that at 208 nm 79.

Recombinant antibodies antigen binding assay. The ability of recombinant antibodies to recognize ErbB2/Her2 protein was evaluated using indirect ELISA. Nunc MaxiSorp 96-well ELISA plates were coated overnight at 4 °C with 1 μg/mL of recombinant human ErbB2/Her2 protein (Cat. #10126-ER, R&D Systems Inc.) in 0.05 M carbonate–bicarbonate buffer (pH 9.6). Plates were then blocked with 5% bovine serum albumin (BSA) in PBS and washed with PBS containing 0.05% Tween-20 (PBS-T) between each step, following a standard ELISA protocol. Serial dilutions of native αHER2 (Human ErbB2/Her2 Antibody, Cat. # MAB9589-100, R&D Systems Inc) or antibodies fused to a receiving peptide (αHER2-P3) were prepared in blocking buffer (starting at 3 ug/mL). Wells without antigen plated were used as negative controls. Rabbit anti-Human IgG (H+L) Secondary Antibody (1:8000, Invitrogen, Cat. #A18903) was used as the secondary antibody. After washing, 100 μL of TMB substrate (1-Step™ TMB ELISA Substrate Solution, Thermo Scientific™) was added to each well. Following incubation, the reaction was stopped with 100 μL of 2N sulfuric acid. Absorbance was measured at 450 nm using a microplate reader (SpectraMax iD3, Molecular Devices).

In vitro FcRn recognition assay. The interaction between recombinant antibodies with fused receiving peptides was studies according to the SinoBiological activity protocol for recombinant human FCGRT & B2M heterodimer protein (Cat. # CT071-H27H-B, SinoBiological). Nunc MaxiSorp 96-well ELISA plates were coated overnight at 4 °C with 2 μg/mL of native αHER2 or antibodies fused to receiving peptide αHER2-P3 in 0.05 M carbonate–bicarbonate buffer (pH 9.6). Plates were then blocked with 5% bovine serum albumin (BSA) in PBS and washed with PBS containing 0.05% Tween-20 (PBS-T) between each step. Biotinylated recombinant human FcRn (FCGRT & B2M) heterodimer (SinoBiological, Cat. #CT071-H27H-B) was serially diluted two-fold (starting at 5 µg/mL) in dilution buffer (PBS, pH 6.0, containing 0.2% Tween-20 and 0.1% BSA) and incubated in an ELISA plate for 1 hour at 37 °C.. A solution of streptavidin-HRP (1:40, R&D Systems Inc. part # 893975) in dilution buffer (0.5% BSA in PBST, pH=6.0) was used for detection. After washing steps, 60 μL of TMB solution (1-Step™ TMB ELISA Substrate Solutions, Thermo Scientific™) was added to the wells. The reaction was stopped with 60 μL of 2N sulfuric acid. Absorbance was measured at 450 nm using a microplate reader (SpectraMax iD3, Molecular devices).

Biolayer Interferometry (BLI). P2N3, P3N3 and P4N3 peptides were first biotinylated with DBCO-PEG4-biotin. Streptavidin SA tips were baslined in BLI buffer (28 mM Tris-HCl pH 8.0, 5 mM MgCl, 4.25% Glycerol, 25 mM NaCl, 1.67 mg/mL BSA) and dipped into 100 nM docking peptide solution for 60 s. Then the peptide coated tips were baselined in BLI buffer again and dipped into wells containing αHER2-P3 (12.5 nM, 25 nM, 50 nM and 150 nM) or native αHER2 (100 nM) or αHER2-P2 (100 nM). After reaching the equilibrium tips were dipped in BLI buffer for complex dissociation. The assay was performed using ForteBio OctetRed96 and data analysis was performed in Octet Analysis Studio 13.0. Tips with no docking peptide loading were used as association control and tips with no dipping to antibody solutions were used as dissociation control. Trace Y-Axes were aligned to the last 5 s of the second baseline step and data was fit to a Mass Transport model.

Docking peptide synthesis. Docking peptides were synthesized by ABclonal (Woburn, MA) using solid phase synthesis with >95 % purity and verified by HPLC and mass spectrometry by the manufacturer.

Payload loading to docking peptide. The DIBCO bearing payloads were dissolved according to manufacturer recommendations BP Fluor 647 DBCO in DMF, BroadPharm; DM1-PEG4-DBCO in DMF, BroadPharm; DBCO-PEG4-ValCit-MMAE in DMF, BroadPharm; DBCO-NSH-polyA15 GGG in water, Integrated DNA Technologies; DBCOC-mPEG10k and DBCO-PEG2k-OH in water, Biopharma PEG; DBCO-biotin in DMF, BroadPharm; DBCO-PEG4-DSPE and DBCO-PEG4-DSPE in DMF:H2O:EtOH 1:1:2, BroadPharm. The small excess of DBCO bearing payload (1.2 eq.) was added dropwise to a solution of P4 docking peptide in PBS (20 ug). The absorbance at 310 nm was measured immediately using NanoDrop spectrophotometer.

MALDI-TOF analysis of peptides and antibodies. Antibodies, peptides, and their conjugates were analyzed using a Bruker Autoflex Speed MALDI-TOF mass spectrometer with sinapinic acid (SA) or CHCA (α-Cyano-4-hydroxycinnamic acid) as the matrix (10 mg/mL in 50% acetonitrile and 0.1% trifluoroacetic acid in water). Prior to analysis, samples were buffer-exchanged into ultrapure water using centrifugal filter units (50 kDa MWCO for antibodies and 3.5 kDa MWCO for peptides; Millipore). The resulting solutions were mixed 1:1 (v/v) with the SA or CHCA matrix solution immediately before spotting onto the MALDI target plate.

HRP-P4 docking peptide activity. Serial 10-fold dilutions of commercially available HRP (Sigma, CAS #9003-99-0) were prepared in phosphate-buffered saline (in PBS; 1 mg/L - 0.001 ug/L) as standards. A volume of 60 µL from each dilution or recombinant HRP-P4 was transferred into a well of a flat-bottom 96-well microplate. Subsequently, 60 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) solution (1-Step™ TMB ELISA Substrate Solutions, Thermo Scientific™) was added to each well and absorbance at 650 nm was measured immediately using a microplate reader (SpectraMax iD3, Molecular devices) at one-minute intervals. In 15 minutes the rection was stopped by addition 60 uL of 2N sulfuric acid and absorbance at 450 nm was recorded. Recombinant HRP-P4 activity was quantified by comparing the end point values of recombinant HRP samples to a standard curve generated using serial dilutions of a commercially available HRP standard.

Antibody-payload assembly. Solution of αHER2-docking peptide (1 eq., in PBS) was mixed with corresponding payload-modified peptide (2.2 eq., in PBS) and incubated for 1 hour at RT or overnight at 4°C to let the coiled-coil peptides to self-assembly. The solution was centrifuged using a spin filter to isolate the conjugated antibody-payload.

Conjugates functional assays.
HRP conjugates functional assay. Activity of the αHER2-HRP conjugates was assessed by direct ELISA. Nunc MaxiSorp 96-well ELISA plates were coated overnight at 4 °C with 2 μg/mL of recombinant human ErbB2/Her2 protein (Cat. #10126-ER, R&D Systems Inc.) in 0.05 M carbonate–bicarbonate buffer (pH 9.6). Plates were then blocked with 5% bovine serum albumin (BSA) in PBS and washed with PBS containing 0.05% Tween-20 (PBS-T) between each step, following a standard ELISA protocol. The αHER2-HRP conjugates were serially diluted (two-fold) in blocking buffer (2 μg/mL - 1 ng/mL, conjugates concentration determined via nanodrop using ε280 =237,385 M-1cm-1) and incubated in ELISA plate for 1 hour at 37 °C. After washing steps, 60 μL of TMB solution (1-Step™ TMB ELISA Substrate Solutions, Thermo Scientific™) was added to the wells. After 30 minutes of incubation, the reaction was stopped with 60 μL of 2N sulfuric acid. Absorbance was measured at 450 nm using a microplate reader (SpectraMax iD3, Molecular devices).

DNA oligo conjugates functional assay. A solution of antibodies conjugated to 5’-DBCO-NSH-polyA15 GGG-3’ DNA oligo (αHER2-P3-P4-polyA15 CCC, 1 eq., 0.17 mM, 27 uL in PBS) was mixed with the complementary 5’-TAMRA-CCCpolyT15-3’ (2.1 eq., 0.37 mM, 13 uL in water). The mixture was incubated at 34.4 °C for 30 minutes in a thermal cycler (T10I0 Thermal Cycler, Bio-Rad) to allow oligo hybridization. The product was purified by sequential washing using an Amicon Ultra 30 kDa MWCO spin column until no TAMRA absorbance is detected in the flowthrough (3 times). αHER2-P3 with no P4-polyA15 CCC were used as a negative control. The absorbance spectra of the resulting conjugate were measured using NanoDrop spectrophotometer. DNA oligos were synthesized by Integrated DNA Technologies (IDT).

MMAE conjugates functional assay. Activity of the αHER2-MMAE conjugates was assessed by MTS cell viability assay. SKBR-3 cells were plated at a density of 5,000 cells per well, A549 cells were plated at a density of 2,500 cells per well, SKOV-3 cells were plated at a density of 2,000 cells per well, and the 4T1 were plated at a density of 1,000 cells per well the day before treatment. Next day, cell media was removed, and the cells were treated with varying concentrations of mAb (αHER2-P3), ADC (αHER2-MMAE) or MMAE diluted in the cell media. The MMAE was serially diluted starting from 2.5 uM in media (100 uL per well) and the ADC concentration was selected to match the MMAE dose (assuming two MMAE molecules per antibody). mAb concentration matched ADC concentration. After 72 hours cell media was removed, and solution of MTS was added. The signal development (30-60 min) was monitored with microplate reader (SpectraMax iD3, Molecular devices) as absorbance at 490 nm. Media only and untreated cells were used as controls.

Conjugates plasma stability. AFCs were prepared using conventional hinge cysteine conjugation or coiled-coil–based strategy for subsequent stability studies. Hinge cysteine conjugation was performed via TCEP reduction (2.75 eq.). The reduction was carried out in 1 M sodium borate buffer containing 25 mM NaCl and 1 mM EDTA for 90 minutes at 37 °C. Without purification the antibodies were cooled to 0 °C and reacted with 5 equivalents of maleimide - BP Fluor 647 overnight at 4 °C covered with foil wrap. The resulting conjugates were purified using a centrifugal spin filter with a 10 kDa MWCO. Then, the conjugates were reoxidized using 2 eq. of DTNB for 20 minutes and subsequently re-purified using a 50 kDa MWCO spin filter to obtain the final product. Coiled-coil AFC were prepared as described previously using short coiled-coil peptides pairs P3-P4N3 or long coiled-coil peptides P3 5mer-P4 5merN3 or cysteine modified P3 5mer-cys-P4 5mer-cys-N3. The AFCs (5 µg) were dissolved in 0.5 mL of human plasma and incubated at 37 °C under constant shaking conditions. Aliquots (10 µL each) were collected at predetermined time points. flash frozen in liquid nitrogen and stored -80 °C for downstream analysis. The samples were analyzed using indirect ELISA assay using 2 μg/mL of recombinant human ErbB2/Her2 protein (Cat. #10126-ER, R&D Systems Inc.) as the antigen and anti-AF647 antibodies secondary antibodies (1:10000, mouse anti-Alexa Fluor 647 Antibody Immunology Consultatnt Laboratory, Cat. #M647-65A-400; HRP labeled with Abcam – HRP Conjugation Kit - Lightning-Link® Cat. #ab102890). Flash-frozen AFCs collected prior to plasma incubation (day 0) were used as an intact AFC standard.

Animal studies. All animal experiments described in this study were performed in accordance with protocol (PROTO201900105) that was reviewed and approved by the Institutional Animal Care and Use Committee of Boston University. All mice were maintained with a 12-h light/dark cycle at 20–26 °C and 30–70% humidity in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.

Pharmacokinetic studies and serum preparation. Non-tumor bearing Balb/c mice (6-7 weeks old) were treated with 10 mg/kg of native mAb (αHER2) or unloaded ADC (αHER3-P3 5mer-cys) or MMAE conjugated ADC (αHER3- P3 5mer-cys -P4 5mer-cys-MMAE) intravenously (100 µL, PBS solution). Blood was collected post-treatment via tail vein bleeding at 30 minutes, 1 day, and 3 days, and via cheek bleeding at the 7-day time point. Serum was isolated by centrifuging blood in a benchtop centrifuge at 1,000 g for 10 min at room temperature. Serum was then transferred to a new Eppendorf tube and stored at −20 °C for downstream analysis. The data was analyzed using indirect ELISA assay detecting total amount of human IgG and standard curve.

Efficacy study. Female NSG mice (6–8 weeks old) were obtained from the Jackson Laboratory (#005557). The orthotopic xenograft model of SKOV-3 was established by injecting SKOV-3 cells (2 × 106 cells in 0.2 ml PBS) intraperitoneally. Mice were administered a single dose of 100 µl of PBS or trastuzumab or ADCs at a dose of 10 mg/kg or 20 mg/kg in 100 µl PBS solution by i.v. injection in the tail vein. Animals were monitored regularly for changes in body weight and body conditions. The study was terminated week 7 post-treatment when animals began to exhibit severe hunched posture and signs of gait rigidity. Tumor tissues were collected, their mass and weight were recorded ex vivo. The formula, volume = 1/2 (length × width2), was used to calculate the tumor volumes. Disitamab vedotin (cat.# HY-P9985), disitamab (cat.# HY-P99854), trastuzumab (cat.# HY-P9907) and trastuzumab vedotin (cat.# HY-164992) were purchased from MedChemExpress.

Biodistribution. SKOV-3 tumor-bearing BALB/c nude mice were administered a single intravenous dose of 10 mg/kg of the AFC (100 µL, PBS solution) on day 60 following tumor cell inoculation. Animals were sacrificed at predetermined time points—1 hour, 1 day, 3 days, 5 days, and 7 days post-injection (n=3 per time point). Major organs were harvested, and fluorescence signals were quantified ex vivo using IVIS Spectrum (PerkinElmer) to assess the biodistribution of the AFC. Untreated animal was used as a control. Total flux analysis was performed with Living Image® 4.8.2.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors report coiled-coil-based antibody drug conjugates (ADCs). In heavy chains of IgG, they introduced various receiving peptides based on glutamate (E)-containing coil peptide. Then, docking peptides based on lysine (K)-containing coiled coil were employed to conjugate drugs onto IgG. The coiled coil-based conjugation was popularly used for the conjugation of biomoleculecules including small molecules, peptides, proteins, and even cells. Actually, similar strategy had been already used for the production of ADCs, as published in Bioconjugate Chemistry, 2025, 36, 8, 1670-1682 (Use of Coiled-Coil Affinity Peptides to Manufacture Antibody Conjugates). The previous Bioconjugate Chemistry paper used different sequences (EVSALEK-KVSALKE pairs), but also based on coiled coil structure based on K and E-rich peptides for conjugation of anti-HER2 IgG and fluorescence tags as model drugs. Although the previous paper did not show the therapeutic efficacy, high accumulation of ADCs on HER2-positive tumors was shown in the paper. Since there are no conceptual breakthrough in the conjugation strategy nor outstanding therapeutic efficacy in this paper, I think that this study is not suitable for the publication in Nature Communications. More specific journals related to pharmaceutics would be better choices.", "reviewer_2": "\"A Supramolecular Self-assembly Approach to Site-Specific Antibody Conjugates via a Coiled-coil Peptides Platform\" by Ringaci et al.\nThe study introduces a coiled-coil affinity peptide platform for site-specific antibody conjugation, allowing uniform and modular loading of diverse payloads (e.g., drugs, enzymes, oligonucleotides, fluorophores) under mild aqueous conditions. The HER2 antibody (trastuzumab) is genetically modified to include receiving peptides at the C-terminus of the Fc region and preserves antibody functionality, including antigen and FcRn receptor binding. Payloads are attached to docking peptides via azide-alkyne cycloaddition, then assembled with the antibody through coiled-coil interaction.\nThe authors show that coiled-coil conjugates exhibit greater serum stability than conventional hinge cysteine conjugates and that their lead coiled-coil ADC compares favorably to the best-in-class HER2-targetting clinical ADC disitamab vedotin.\nIn summary, the paper describes an elegant method allowing for facile antibody conjugation that could ultimately find applicability in the clinic. I think the paper could however be improved by addressing a few points as described below.\nMajor comments\n1. Manufacturability: the authors should mention the αHER2 titers obtained in harvested culture medium for all constructs described in the paper (including the αHER2-P1-P3 and αHER2-P3-5mer-cys constructs) and yields obtained following pA purification. This is important to demonstrate manufacturability and applicability of the approach for larger-scale needs (e.g. commercial).\n2. Immunogenicity: the authors should discuss potential immunogenicity in human of the coiled-coils they used in their study.\n3. Disulfide bridge: to increase stability of the antibody conjugate in the presence of serum and in vivo, the authors replaced an isoleucine for a cysteine in the last heptad of both the receiving and docking peptides (both having five heptads). While formation of the ADC is clearly demonstrated (Fig SI15), proper formation of the disulfide bridge is not demonstrated. The authors should provide evidence for the formation of a disulfide bridge between αHER2-P3-5mer-cys and P4-5mer-cys-MMAE.\n4. Peptide design rationale: Also, the authors should discuss why they chose peptides with 5 heptads for disulfide bond formation. The stabilizing impact of the disulfide bond would have been more evident by comparison to the stability data of the four heptads construct shown in Fig 4B).\nMinor comments\n1. Provide more details on mabs and HRP-P4 purification, especially composition of wash and elution buffers?\n2. αHER-P2 shows significant tailing on SEC (Fig 1C) – could the authors provide an explanation for this?\n3. αHER-P4 shows significant level of aggregates by SEC (Fig 1C) – please discuss if this is linked to mab stability (impact of the P4?).\n4. Fig SI19: The authors mention the “dashed line indicates the treatment day”, but no dashed line is apparent on the figure.\n5. Lane 290 “Then tumors established…” should be “When tumors established…”\n6. Lane 340: “employ this new coiled-coil approach to prepare a fluorophore labeled antibody possessing fluorophores for tumor targeting” the term “fluorophore” is redundant – please rephrase.\n7. Lane 343: “the fluorophore labeled antibody is unstable after in vivo administration with dissociation of the fluorescent payload”. This sentence is misleading. Baniahmad observed early but only partial dissociation with a majority of the coiled-coil mab-CF750 remaining in the tumor over 10 days post-injection. Please correct the sentence to reflect this.", "reviewer_3": "This manuscript by Ringaci and colleagues reports on an approach to conjugate payloads to antibodies via a coiled-coil fusion domain. The overarching principle is that one half of the coiled-coil heterodimer unit can be fused onto the Fc domain of an antibody, while the other half of the coiled-coil heterodimer unit can be conjugated to a ‘payload’. The approach leverages existing knowledge for designing heterodimer coiled-coils through charge-complementary interfaces.\nAs shown here, a benefit of this system is that the payload can be of many forms – a small molecule (e.g., drug or fluorophore), another protein, a polymer, or a nucleic acid oligomer. The simplicity of the charge-complementary coiled-coil interface is intended to address limitations of existing antibody-drug conjugation strategies, which often employ chemistries or conditions that can adversely affect antibody function. The authors provide compelling evidence that their conjugation strategy does not disrupt antibody-antigen interactions, nor Fc domain interactions with Fc receptors, nor the activity of the payload. This versatility exemplifies the significance of this approach, as it underscores the broad potential utility. Further, the data strongly suggest a nearly quantitative payload conjugation efficiency at a predictable 2:1 ratio, which is critical for ensuring well-defined and reproducible antibody and payload dosing.\nMy initial impression based on the abstract was that the overall strategy was implausible even with the most robust coiled-coil heterodimer without some means to chemically ‘staple’ the domain together via a stable covalent bond. Indeed, as detailed by the authors in the manuscript, others have tried and failed with an approach that only relies on the non-covalent coiled-coil motif. The authors address this through well-established strategies, including increasing the number of heptads and introducing a disulfide bridge at the interface between the alpha-helices, with ultimately the latter showing to be effective in vivo. However, much of the work is centered around characterizing the coiled-coil unit lacking these stabilizing elements.\nConcerns were noted with the SPR traces in Figure 1F, wherein the unbinding phase appears exceptionally slow. This is a well-known issue with SPR for bi-/multi-valent interactions, where the high density of immobilized capture agent can amplify interactions due to statistical binding and chelation events that do not truly reflect the “solution-state interaction” of relevance to this system. Consistent with this, the authors report a Km of 6 x10^-10 M, which is significantly stronger than what would be expected for a coiled-coil of their design, as it exceeds even the strongest antibody-antigen interactions. To strengthen the manuscript, the authors are encouraged to include studies that provide an apparent dissociation constant in a more relevant dilute solution-state interaction. One could envision that this could be achieved by way of a competition assay in which fluorescently-labeled coiled-coil payload is outcompeted by an unlabeled peptide. Such insights would help rationalize the poor stability of their first-generation coiled-coil design, as reported in Figure 4A. Further, the studies would be particularly strengthened by a control experiment that explicitly demonstrates the specificity/selectivity of the coiled-coil interaction. This is particularly prudent for the studies that suggest dual loading of different payloads in Figure 3.\nSome concerns are noted with the pharmacokinetics studies, most significant of which are the claims about clearance and FcRn binding. Trastuzumab is a humanized IgG1 monoclonal antibody, wherein the entire Fc region is of human origin. It is well established that human IgG1 has an approximately 2.5-fold higher affinity for mouse FcRn than for human FcRn, which is known to confound pharmacokinetics studies. The authors are encouraged to not overstate the results of their study, which only show that the modification does not affect the clearance rate in a mouse model when compared to the parent antibody. There is no data presently shown that unequivocally demonstrate recycling in vivo. Indeed, a half-life of approximately 6 h is quite short for a monoclonal antibody therapy that is being effectively recycled.\nThe data in Figure 4g seem to suggest that more antibody accumulates in the tumor for the engineered variant when compared to the parent antibody (see day 5 and 7 data). This is unexpected given that no effort was made to improve the ‘targeting’ effectiveness of the antibody itself. This largely seems to be mediated by one outlier in the cohort that has an unusually high signal at both day 5 and day 7. This begs the question of which animal was the source of the tissues that are used to represent accumulation in Figure 4h. The absence of any intensity scale in Figure 4h makes it difficult to interpret the images accurately.\nThe data in Figure 4i-m provide a compelling demonstration that the engineered coiled-coil ADC is effective in vivo. However, the comparable effectiveness of the coiled-coil ADC and the gold-standard disitimab vidotin leave open the question of whether the new approach has in fact addressed a limitation of existing methods, which is at the core of this study. The authors state in the conclusion, “The coiled-coil composition controls conjugate stability and structures with stability beyond that of conventional hinge cysteine conjugation which is known to prematurely release payload.” This is at best speculative and seems to be rooted in their data shown in Figure 4a, in which they synthesized a “conventionally prepared ADC” and evaluated its stability against the coiled-coil ADC. While the data in Figure 4a suggest that the conventionally prepared ADC may be less stable than the coiled-coil ADC in this model, the stability experiment should instead be set up to compare their coiled-coil ADC against disitimab vidotin head-to-head. There is some reasonable expectation that disitimab vidotin is manufactured using a robust and reliable process that may not be accurately reflected by their \"conventionally prepared ADC\" method. Additionally, to show an advance beyond the gold-standard, the experiments in Figure 4k-m need to have a cleaner set of controls to account for the effects of both the antibody and the payload, given that (1) trastuzumab provides some therapeutic effect on its own and (2) disitimab vidotin is based on a different antibody (hertuzumab) with a different profile than trastuzumab. Recommendations here would include a dose-response profile comparing the coiled-coil ADC versus disitimab vidotin, as well as inclusion of a group treated with hertuzumab alone to provide a baseline for antibody-only effectiveness."}, "author_response": "We appreciate the time and attention the reviewers gave to the manuscript and the helpful comments and suggestions. Please find below the detailed point-by-point answers to the reviewers’ questions. As required where the text of the manuscript has been rewritten or new text added, the parts are marked up in blue.\nEditorial Comments\nComment 1. Abstract should be 150 words or less\nResponse 1: Abstract is 146 words\nComment 2: Figure legends need a title\nResponse 2: Figure legends contain a title\nComment 3: Please include the NR reporting summary\nResponse 3: NR reporting summary included\nReviewer #1\nComment 1: “In this manuscript, the authors report coiled-coil-based antibody drug conjugates (ADCs). In heavy chains of IgG, they introduced various receiving peptides based on glutamate (E)-containing coil peptide. ……….Since there are no conceptual breakthrough in the conjugation strategy nor outstanding therapeutic efficacy in this paper, I think that this study is not suitable for the publication in Nature Communications. More specific journals related to pharmaceutics would be better choices.”\nResponse 1: We respectively disagree and novelty is present in the article. The text acknowledging the previous work remains in the manuscript and has not been edited in the revised manuscript.\nThe novelty aspects of our work include:\n Use of coiled-coil supramolecular structures for antibody drug conjugation;\n Site-specific conjugation of anti-cancer agents, polymers, enzymes, or fluorophores to an antibody;\n Conjugation of two or more similar or different payloads to the antibody;\n Separation of the antibody preparation and conjugation engineering/chemistry to enable facile coupling to the antibody; and,\n Demonstration of in vivo efficacy in a human ovarian cancer xenograft model comparable to a best-in-class ADC.\nReviewer #2\nComment: The study introduces a coiled-coil affinity peptide platform for site-specific antibody conjugation, allowing uniform and modular loading of diverse payloads (e.g., drugs, enzymes, oligonucleotides, fluorophores) under mild aqueous conditions. The HER2 antibody (trastuzumab) is genetically modified to include receiving peptides at the C-terminus of the Fc region and preserves antibody functionality, including antigen and FcRn receptor binding. Payloads are attached to docking peptides via azide-alkyne cycloaddition, then assembled with the antibody through coiled-coil interaction.\nThe authors show that coiled-coil conjugates exhibit greater serum stability than conventional hinge cysteine conjugates and that their lead coiled-coil ADC compares favorably to the best-in-class HER2-targetting clinical ADC disitamab vedotin. In summary, the paper describes an elegant method allowing for facile antibody conjugation that could ultimately find applicability in the clinic. I think the paper could however be improved by addressing a few points as described below.\nResponse: Thank you for the positive comments and noting the elegant method present in the manuscript.\nComment 1: Manufacturability: the authors should mention the αHER2 titers obtained in harvested culture medium for all constructs described in the paper (including the αHER2-P1-P3 and αHER2-P3-5mer-cys constructs) and yields obtained following pA purification.\nResponse 1: We agree with the reviewer on the importance of manufacturability of antibodies.\nWe have included antibody titers in cell media on day 7 post two different independent transfection rounds of Expi293F cells for all constructions used in the paper to supplementary information. To access the antibody titers, we used antibody-sensitized microspheres agglutination-based assay and described it in detail in methods section. We did not observe an obvious correlation between antibody design and titer levels. The obtained titers were in the typical academic lab range 1 and comparable to those of the unmodified mAb (aHER2-O), indicating that peptide incorporation does not impair antibody production. Further optimization for industry scale production will likely include adjusting vector design, transfection and culture conditions, as is standard. We have added text describing these results on pages 3 and 4, and included the following data in supplementary information and to the methods section as follows:\n“Incorporation of the peptide at the C terminal end of the antibody does not impair antibody production, as titers are similar between peptide modified and native antibodies and ranged from 40 to 80 mg/L (Table SI2, SI3).”\nTable SI2. Exemplary titers of antibody production in cell media on day 7 post Expi293F cells\nAntibody Production scale, mL of cell culture Production scale, mL of cell culture Titer in cell media, mg/L Titer in cell media, mg/L\naHER2-P1 30 70.9 50 76.0\naHER2-P2 30 50.1 50 40.9\naHER2-P3 30 40.1 50 81.8\naHER2-P4 30 53.4 50 84.0\naHER2-O 30 68.4 50 82.7\naHER2-P1-P3 30 37.1 50 98.9\naHER2-P3-5mer 30 54.3 50 70.6\naHER2-P3-5mer-cys 200 120.3 200 55.8\n“Antibody titer determination. Expi determination. Expi293F cell culture media samples were harvested on day 7 post ExpiFectamine transfection. Samples were centrifuged to remove cells (3,200 g, 25 min) and the supernatants were stored at −20°C until analysis. Antibody titers were determined using an agglutination-based antibody-sensitized microspheres assay (Easy-Titer™ Human IgG (H+L) Assay Kit; Thermo Fisher Scientific) according to the manufacturer’s protocol. Cell culture media samples were diluted 200-fold in assay buffer, and 20 µL of each diluted sample were incubated with 20 µL of microsphere beads for 5 min under shaking conditions. Following incubation, 100 µL of the kit blocking reagent was added, and samples were incubated for additional 5 min under shaking conditions. Human ErbB2/Her2 (Trastuzumab) antibody, clone # Hu5 (R&D Systems) was used as a standard. Microspheres agglutination was quantified by measuring absorbance at 405 nm using a microplate reader (SpectraMax iD3, Molecular Devices).”\nAdditionally, we also provide purification yields to supplementary information. The antibodies were purified using protein A gravity columns and buffer exchanged to PBS via spin filtering (3 times, 50 kDa cut off). The yields were determined with NanoDrop using absorbance at 280 nm and ε =210,000 M-1 cm-1. While titers values are typical, purification yields are modest for both coil-modified trastuzumab and native trastuzumab indicating the need of further optimization for large scale manufacture.\nTable SI3. Exemplary antibody purification yields. The yields were determined via protein absorbance at 280 nm, post purification and buffer exchange using nanodrop (ε =210,000 M-1 cm-1).\nAntibody Purification yield, mg per L of cell culture Purification yield, mg per L of cell culture\naHER2-P1 11.0 13.8\naHER2-P2 8.5 9.7\naHER2-P3 4.5 5.5\naHER2-P4 7.6 9.4\naHER2-O 6.5 8.7\naHER2-P1-P3 4.4 14.6\naHER2-P3-5mer 6.9 10.1\naHER2-P3-5mer-cys 22.2 14.15\nComment 2: Immunogenicity: the authors should discuss potential immunogenicity in human of the coiled-coils they used in their study.\nResponse 2: We thank the reviewer for the comment about the potential immunogenicity in humans. Future experiments in humans will directly address this question, however data in the literature suggests that it will be minimal. The work published by Ljubetič et al. in Nature Biotechnology 2017 2 shows that coiled-coil protein origami formed in part by modified P3 and P4 peptides are minimally immunogenic and inflammatory in vitro and in vivo. Finally, short peptides are typically not immunogenic, as noted in the literature 3,4. We have added the following discussion to the manuscript on page 15 as well as the references to the revised manuscript.\n“Another potential limitation of a coiled-coil design is that it may induce a host immune response even though short peptides are usually non-immunogenic 74,75. Ljubetič et al. report that a complex coiled-coil protein origami structure, formed in part from modified-like P3 and P4 peptides, induces minimal immune activation in vitro and caused no signs of inflammation or other side effects in vivo 76. Therefore, we do not expect our coiled-coils to be strongly immunogenic, but this will need to be tested.”\nComment 3: Disulfide bridge: to increase stability of the antibody conjugate in the presence of serum and in vivo, the authors replaced an isoleucine for a cysteine in the last heptad of both the receiving and docking peptides (both having five heptads). While formation of the ADC is clearly demonstrated (Fig SI15), proper formation of the disulfide bridge is not demonstrated. The authors should provide evidence for the formation of a disulfide bridge between αHER2-P3-5mer-cys and P4-5mer-cys-MMAE.\nResponse 3: We thank the reviewer for the comment. Discerning one disulfide linkage over other disulfide linkages present in the antibody is particularly challenging. A direct method to observe this disulfide bond is likely via X-ray crystallography which is beyond the scope of this paper and the short-turnaround time request. This is an experiment we will attempt in the future.\nThus, we focused on the peptides themselves. We performed MALDI-TOF to detect peaks at m/z ~10,000 and 5,000, corresponding to the assembled coiled-coil structure and monomeric peptide, respectively. MALDI laser ionization can disrupt noncovalent interactions; and the most abundant peak observed for the coiled-coil lacking cysteines (P3 :P4 ) corresponded to m/z ~ 5,000 – the expected molecular weight of single peptide. In contrast, for the cysteine-containing dimer (P3 -cys:P4 -cys), a high-intensity peak at m/z ~ 10,000 was detected. This peak disappeared upon addition of the reducing agent TCEP, indicating the cleavage of a disulfide bond between the peptides and more single peptides present – analogous to the results obtained with coiled-coil lacking cysteines. We have added new text to the revised paper on page 11; 1) describing the new data collected on the coiled-coil peptides, and, 2) presenting the supplementary figure shown below.\n“A MALDI laser ionization spectrum of the coiled-coil lacking cysteines (P3 :P4 ) shows a primary peak at a m/z of ~ 5,000 – the expected molecular weight of single peptide - consistent with disruption of the coiled-coil noncovalent interactions. In contrast, the spectrum for the cysteine-containing dimer (P3 -cys:P4 -cys) reveals a high-intensity peak at m/z of ~ 10,000. This peak reduces upon addition of TCEP consistent with cleavage of the disulfide bond between the peptides with more resulting single peptides present – analogous to the results obtained with coiled-coil lacking cysteines.”\n“Figure SI16. MALDI-TOF spectra demonstrating A. dissociation of non-covalent P3 :P4 dimer upon laser ionization; B. maintaining dimeric structure of covalently linked P3 -cys:P4 -cys coiled-coil pair; C. dimeric structure disrupted when covalent bond is reduced with TCEP. Single peptide predicted molecular weight is ~4.8 kDa and coiled-coil pair predicted molecular weight is ~10 kDa. Laser power was set to 100%. ”\n“MALDI-TOF disulfide bond experiment. 4 ug of P3 or P3 -cys peptide were mixed with 4 ug of P4 or P4 -cys correspondingly (in 3-5 uL of PBS solution). The peptide pairs were incubated for 30 minutes at room temperature to allow coiled-coil dimerization. Upon incubation 0.5 uL of 0.05M TCEP water solution was added to reduce the disulfide bond in P3 -cys:P4 -cys coiled-coil pair. The samples without purification were mixed 1:1 (v/v) with SA matrix solution immediately before spotting onto the MALDI target plate.”\nComment 4: Peptide design rationale: Also, the authors should discuss why they chose peptides with 5 heptads for disulfide bond formation. The stabilizing impact of the disulfide bond would have been more evident by comparison to the stability data of the four heptads construct shown in Fig 4B).\nResponse 4: The published report by Woolfson et al., shows that increasing the number of heptads increases the stability with 4 or more heptads affording nM or lower Kds. Similarly, Xia et al., and Lee et al. show that the presence of complementary reactive groups (e.g., a disulfide bond) increases the stability of the coiled-coil via bond formation 5–7. Building on these results, we investigated the 4 and 5 heptads. Our results from the in vitro stability assay in human plasma show that both 5 heptads with and without the disulfide bond were stable, performed similarly over the 28-day period, and superior to the 4 heptad without a disulfide bond. We agree with the reviewer that the stabilizing impact of the disulfide on the 4 heptad is missing, however the stabilizing effect of a disulfide on a coiled-coil structure is known 8–10. Importantly both 5-heptad systems are viable candidates for the in vivo studies. We selected the 5-heptad construct with the disulfide bond for the subsequent in vivo experiments because it has two stabilizing mechanisms.\nWe have clarified this discussion and rationale on pages 11 and 12 of the revised manuscript.\n“Woolfson et al., report that increasing the number of heptads in a coiled-coil increases the stability with 4 heptads affording nM or lower Kd values. 39 Also Xia et al., and Lee et al. show that the presence of complementary reactive groups capable of forming a stabilizing bond (e.g., a disulfide) between the peptides stabilizes the coiled-coils 50,59 stabilized coiled-coil regions through increasing the number of heptads 39 or\n“The P3 -P4 N coils disassemble over the first few days, with only 13% of the AFC intact by the end of the study. In contrast, the AFCs prepared using P3 , P4 N and P3 -cys, P4 -cysN coil pairs are stable with only a 4% loss in structure over 4 weeks, outperforming the conventional conjugation approach (Figure 4C,D). From a design perspective, we selected the 5-heptad construct with the disulfide bond for the subsequent in vivo experiments because it possesses two stabilizing mechanisms.”\nComment 5: Provide more details on mabs and HRP-P4 purification, especially composition of wash and elution buffers?\nResponse 5: We have included more details on the purification to the methods section, please see page 18.\n“Protein expression and purification. Native αHER2 and receiving-peptide modified αHER2 antibodies, as well as HRP-P4 conjugates were produced in Expi293F expression system (Gibco™) according to manufactures’ protocol. Expi293F cells were co-transfected with a mixture of light and heavy chain containing plasmids for mAb production or with a single plasmid containing HRP-docking peptide-5xHis fusion sequence. Proteins were harvested on day 6 by centrifugation at 3,220 g for 20 min. The supernatant was filtered through a 0.22-µm membrane for subsequent purification and mixed 1:1 with equilibration buffer. mAbs were purified using a gravity-flow column (Biorad) packed with rProtein A Sepharose Fast Flow resign (Cytiva) according to the resin manufacturer protocol. Phosphate-buffered saline (pH=7.4) was used as equilibration and wash buffer, 0.2M glycine-HCl (pH=2.5) and 1M TRIS-HCl (pH=9.0) were used as elution and neutralization buffers respectively. HRP-P4 was purified using HisPur™ Ni-NTA Resin (Thermo Fisher Scientific) according to the resin manufacturer protocol. High-salt phosphate buffer (20 mM sodium phosphate, 300 mM sodium chloride) with 10 mM imidazole (pH=7.4) was used as equilibration buffer, high-salt phosphate buffer with 25 mM imidazole (pH=7.4) and high-salt phosphate buffer with 250 mM imidazole (pH=7.4) were used as wash and elution buffers respectively. Collected proteins were concentrated and buffer exchanged to PBS with centrifugal filters (10kDa or 50 kDa molecular weight cut-off, MilliporeSigma). The concentration of all proteins was determined by absorbance at 280 nm using sequence predicted extinction coefficients (mAb ε =216,340 M-1cm-1; and HRP-P4 ε =15,065 M-1cm-1).”\nComment 6: αHER-P2 shows significant tailing on SEC (Fig 1C) – could the authors provide an explanation for this? αHER-P4 shows significant level of aggregates by SEC (Fig 1C) – please discuss if this is linked to mab stability (impact of the P4?).\nResponse 6: We repeated the size-exclusion chromatography experiment. We attribute the aggregates observed for αHER-P4 to storage, as freshly prepared antibodies did not show this tailing. We used freshly prepared materials for all in vitro and in vivo experiments. We have not optimized the storage conditions, and this will be a future study. However, peak tailing for αHER-P2 remained slightly pronounced more than for the other samples. We hypothesize that this behavior arises from secondary ionic interactions caused by the highly positively charged P2 peptide, whose heptad repeat sequence is lysine-rich. The experimental details and corresponding text have been updated accordingly. Please see page 4.\n“The peptide-modified antibodies, except αHER2-P2, elute slightly earlier (αHER2-P1 t=9.2 6.95 min; αHER2-P2 t=10.0 7.15 min; αHER2-P3 t=9.2 7.03 min; αHER2-P4 t=9.2 7.00 min) than the native αHER2 antibodies (t=10.1 7.15 min) during SEC chromatography, consistent with an increase in mass due to peptide insertion (Figure 1C). The slower elution of αHER2-P2 is likely attributed to the lysine-rich P2 sequence, which also contributes to peak tailing. Antibodies modified with the P3 peptide display a more uniform peak and were used for further experiments.”\n“ … C. SEC-RI-UV traces at 280 nm of native trastuzumab (αHER2) and trastuzumab fused to different receiving peptides (αHER2-P1, αHER2-P2, αHER2-P3, αHER2-P4)…”\nComment 7: Fig SI19: The authors mention the “dashed line indicates the treatment day”, but no dashed line is apparent on the figure.\nResponse 7: Thank you for the catching this and we have updated the figure:\n“Figure SI25. Average tumors size after different treatments in female NSG mice engrafted subcutaneously with 2 million A549 tumor cells and treated on day 23 (average tumor volume ~175 mm3) with either PBS or coiled-coil ADC or mAb (αHER2-P3 -cys) or commercial ADC (disitamab vedotin) at the dose 10 mg/kg. The dashed line indicates the treatment day.”\nComment 8: Lane 290 “Then tumors established…” should be “When tumors established…”\nResponse 8: We corrected the typo on page 13.\n“Then When tumors established, we i.v. administered a BP Fluor 647-loaded AFC at a dose of 10 mg/kg. “\nComment 9: Lane 340: “employ this new coiled-coil approach to prepare a fluorophore labeled antibody possessing fluorophores for tumor targeting” the term “fluorophore” is redundant – please rephrase.\nResponse 9: We rephrased the sentence on page 15.\n“Baniahmad et. al., 73 employ this new coiled-coil approach to prepare a fluorophore labeled antibody possessing fluorophores for tumor targeting.”\nComment 10: Lane 343: “the fluorophore labeled antibody is unstable after in vivo administration with dissociation of the fluorescent payload”. This sentence is misleading. Baniahmad observed early but only partial dissociation with a majority of the coiled-coil mab-CF750 remaining in the tumor over 10 days post-injection. Please correct the sentence to reflect this.\nResponse 10: Thank you and we agree. We corrected the sentence on page 15.\n“the fluorophore labeled antibody undergoes partial payload dissociation is unstable after in vivo administration with dissociation of the fluorescent payload.”\nReviewer #3\nComment: This manuscript by Ringaci and colleagues reports on an approach to conjugate payloads to antibodies via a coiled-coil fusion domain. The overarching principle is that one half of the coiled-coil heterodimer unit can be fused onto the Fc domain of an antibody, while the other half of the coiled-coil heterodimer unit can be conjugated to a ‘payload’. The approach leverages existing knowledge for designing heterodimer coiled-coils through charge-complementary interfaces.\nAs shown here, a benefit of this system is that the payload can be of many forms – a small molecule (e.g., drug or fluorophore), another protein, a polymer, or a nucleic acid oligomer. The simplicity of the charge-complementary coiled-coil interface is intended to address limitations of existing antibody-drug conjugation strategies, which often employ chemistries or conditions that can adversely affect antibody function. The authors provide compelling evidence that their conjugation strategy does not disrupt antibody-antigen interactions, nor Fc domain interactions with Fc receptors, nor the activity of the payload. This versatility exemplifies the significance of this approach, as it underscores the broad potential utility. Further, the data strongly suggest a nearly quantitative payload conjugation efficiency at a predictable 2:1 ratio, which is critical for ensuring well-defined and reproducible antibody and payload dosing.\nResponse: We thank the reviewer for the positive comments and highlighting the benefits and versatility of this methodology which “address limitations of existing antibody-drug conjugation strategies, which often employ chemistries or conditions that can adversely affect antibody function” as well as noting the broad potential utility.\nComment 1:\nMy initial impression based on the abstract was that the overall strategy was implausible even with the most robust coiled-coil heterodimer without some means to chemically ‘staple’ the domain together via a stable covalent bond. Indeed, as detailed by the authors in the manuscript, others have tried and failed with an approach that only relies on the non-covalent coiled-coil motif. The authors address this through well-established strategies, including increasing the number of heptads and introducing a disulfide bridge at the interface between the alpha-helices, with ultimately the latter showing to be effective in vivo.\nResponse 1: We started this project to ask the question if supramolecular chemistry can be utilized to expand or improved ADC synthesis, as it offers a number of advantages. We were equally excited to see the results come to fruition with an ADC construct which performed in vivo.\nComment 2: Concerns were noted with the SPR traces in Figure 1F, wherein the unbinding phase appears exceptionally slow. This is a well-known issue with SPR for bi-/multi-valent interactions, where the high density of immobilized capture agent can amplify interactions due to statistical binding and chelation events that do not truly reflect the “solution-state interaction” of relevance to this system. To strengthen the manuscript, the authors are encouraged to include studies that provide an apparent dissociation constant in a more relevant dilute solution-state interaction. One could envision that this could be achieved by way of a competition assay in which fluorescently-labeled coiled-coil payload is outcompeted by an unlabeled peptide.\nResponse 2: The trace in Figure 1F is from a BLI experiment and like SPR is a technique where one substrate is immobilized. We agree with the reviewer that immobilization of one of the agents, could amplify the Kd value. Therefore, to complement our BLI data we performed a MicroScale Thermophoresis experiment capable of quantifying molecular interactions when both molecules are in the solution. These data confirm that the Kd values are in nonamolar range which is also consistent with the reported values for similar E/K coiled-coil systems in Plaper et al. 2021 11. The results of the experiment are included in the manuscript main text, supplementary information, and the details are described in the method section along with the reference by Plaper on pages 4, 5, and SI methods.\n“The calculated dissociation constant (Kd) is 6 x 10-10 M, indicating a highly stable structure and similar in magnitude as that reported by Plaper et al.”\n“Repeating the affinity experiment using microscale thermophoresis (MST), an immobilization-free assay format, affords similar Kd values in nanomolar range (Figure SI5).”\n“Microscale Thermophoresis (MST) Assay. The P4N peptide was first labeled with DBCO-BP 647 fluorophore and diluted to 30 nM in MST assay buffer (PBS with 0.05% tween 20). A serial 2-fold dilution of the aHER3-P3 was prepared starting from 200 nM in MST assay buffer (0.05% Tween20 in PBS). 10 uL of labeled peptide and 10 uL of each antibody dilution were mixed and incubated at room temperature for 2 minutes. The mixtures were subsequently loaded into Monolith Premium Capillaries (Nanotemper Technologies Inc; cat# MOK025) and measurements were performed using a red fluorescence channel.”\n“Figure SI5. A. MST dose-response curves for independent measurements and B. Exemplary MST traces for the binding interaction between aHER3-P3 and P4 peptide labeled with DBCO-BP647 fluorophore. The concentration of P4-BF647 peptide is kept constant at 50 nM, while the aHER3-P3 concentration varies from 400 nM to 0.0122 nM. The fitting and K values calculation d was done automatically by MO.AffinityAnalysisv2.3 software. The calculated K values are 6.0 nM; 8.7 nM; 30 nM and 5.8 nM for each experiment respectively. Blue band on MST traces represents cold region start at -1 s and cold region end at 0 s; red band represents hot region start at 4 s and hot region end at 5 s.”\nComment 3: Further, the studies would be particularly strengthened by a control experiment that explicitly demonstrates the specificity/selectivity of the coiled-coil interaction.\nResponse 3: We thank the reviewer for raising this important point. The specificity of coiled-coil structure formation was previously demonstrated by Helena Gradisar and Roman Jerala in the article “De-novo design of orthogonal peptide pairs forming parallel coiled-coil heterodimers”. The authors designed and performed an extensive peptides specificity experiments using circular dichroism, SEC and melting temperatures prediction for all possible peptide combinations (36 total, including homodimers and mismatched heterodimers). Gradisar and Jerala show coiled-coils form exclusively between designed partner peptides, while all mismatched combinations remain largely unfolded.\nTo complement their findings, we performed the BLI assay to demonstrate the specificity of the coiled-coil formation if one of the coil peptides is fused to an antibody. Please see Figure SI5 which includes specificity controls. The data demonstrate the binding of αHER2-P3 to different specific (P4N ) and non-specific peptides (P2N ). The BLI response was observed for the specific pair combination only (αHER2-P3 and P4N ), while there was no interaction detected for P2N + αHER2-P3, P3N + αHER2-P3 or P4N + αHER2 pairs. Please see the text on Pag 5\n“The interaction between the docking and receiving peptides is specific as antibodies without a receiving peptide and those with a non-complementary receiving peptide tail, do not elicit a BLI response with the docking peptide (see Figure SI6).”\n“Figure SI6. Binding affinity of αHER2-P3 to P4N peptide biotinylated with DBCO-PEG -biotin 4 measured by biolayer interferometry (experimental curve) and specificity controls: 1) target docking peptide and mAb fused to non-specific receiving peptide (P4N + αHER2-P2); 2) no docking peptide and mAb fused to specific receiving peptide (0 + αHER2-P3); 3) target docking peptide and native mAb (P4N + αHER2); 4) self-interaction control - receiving peptide and mAb fused to the same receiving peptide (P3N + αHER2-P3); 5) non-specific docking peptide and mAb fused to target receiving peptide (P2N + αHER2-P3). ”\nWe also demonstrated the selectivity of the coiled-coil interaction by running a competition assay. aHER2-P3 was mixed with a solution of P1, P2, P3 and P4 peptides labeled with different fluorophores (P1-BP Fluor 647; P2-TAMRA 555; P3-BP Fluor 594 and P4-BP Fluor 488). To avoid self-assembly of free peptides (P1 with P2 and P3 with P4) in the solution we preincubated aHER2-P3 with P1-BP Fluor 647 and P4-BP Fluor 488 for 3 minutes and then added the solution of P2-TAMRA 555 and P3-BP Fluor 594 peptides. The mixture of aHER2-P3 and 4 different peptides was incubated for 2 h at room temperature. Then the mixture was purified by spin filtering (50 kDa MWCO) until flow through has no fluorophores present (3-4 times). The resulting conjugate exhibits the absorbance only at BP Fluor 488 region suggesting selective assembly of P4-BP Fluor 488 and aHER2-P3. These data have been added to the SI, please see Figure SI10 and referenced on Page 8.\n“Additionally, mixing αHER2-P3 with a solution containing P1, P2, P3 and P4 peptides labeled with different fluorophores (P1-BP Fluor 647; P2-TAMRA 555; P3-BP Fluor 594 and P4-BP Fluor 488) results in a conjugate with an absorbance in the BP Fluor 488 region only, indicating selective assembly of P4-BP Fluor 488 and aHER2-P3 (Figure SI10). These data are consistent with the results with the coiled-coils themselves.”\n“Figure SI10. A. Schematic diagram illustrating selectivity of coiled-coil mediated aHER2-P3 loading with a fluorophore by competition assay. aHER2-P3 is mixed with a set of fluorophores labeled peptides (P1-BP Fluor 647; P2-TAMRA 555; P3-BP Fluor 594 and P4-BP Fluor 488). To avoid self-assembly of free peptides (P1 with P2 and P3 with P4) in the solution, aHER2-P3 was preincubated with P1-BP Fluor 647 and P4-BP Fluor 488 for 3 minutes and then solution of P2-TAMRA 555 and P3-BP Fluor 594 peptides was added. Post 2 h incubation and spin filtering purification aHER2-P3 antibody conjugate exhibits the absorbance only at BP Fluor 488 region suggesting selective assembly of P4-BP Fluor 488 and aHER2-P3. B. UV-Vis absorbance spectra of unloaded antibodies C. UV-Vis absorbance spectra of aHER2-P3 antibodies mixed with specific P4-BP Fluor 488 and non-specific peptides (P1-BP Fluor 647; P2-TAMRA 555; P3-BP Fluor 594) post spin filter purification. The dashed lines represent the absorbance peak maxima corresponding to BP Fluor 488, TAMRA 555, BP Fluor 594 and BP Fluor 647. The resulting conjugates post purification absorb only in BP Fluor 488 region indication the selectivity of coiled-coil loading.”\n“Selectivity competition assay. aHER2-P3 (30 ug) was mixed with a solution of P1, P2, P3 and P4 peptides labeled with different fluorophores (P1-BP Fluor 647; P2-TAMRA 555; P3-BP Fluor 594 and P4-BP Fluor 488, 3 ug of each peptide). To avoid self-assembly of free peptides (P1 with P2 and P3 with P4) in the solution we preincubated aHER2-P3 with P1-BP Fluor 647 and P4-BP Fluor 488 for 3 minutes and then added the solution of P2-TAMRA 555 and P3-BP Fluor 594 peptides The mixture of aHER2-P3 and 4 different peptides was incubated for 2 h at room temperature. Then the mixture was purified by spin filtering (50 kDa MWCO) until flow through has no fluorophores present (3-4 times). The absorbance of resulting conjugates was recorded with nanodrop at 280 nm, 488 nm, 555 nm, 594 nm and 647 nm.”\nComment 4: Some concerns are noted with the pharmacokinetics studies, most significant of which are the claims about clearance and FcRn binding. Trastuzumab is a humanized IgG1 monoclonal antibody, wherein the entire Fc region is of human origin. It is well established that human IgG1 has an approximately 2.5-fold higher affinity for mouse FcRn than for human FcRn, which is known to confound pharmacokinetics studies. The authors are encouraged to not overstate the results of their study, which only show that the modification does not affect the clearance rate in a mouse model when compared to the parent antibody. There is no data presently shown that unequivocally demonstrate recycling in vivo. Indeed, a half-life of approximately 6 h is quite short for a monoclonal antibody therapy that is being effectively recycled.\nResponse 4: Thank you for the comment and we agree. We have edited the pharmacokinetics results description as follows as shown on page 12.\n“…The half-life for trastuzumab is 6.5 hours (95% CI 5.5-7.3 h), similar to the unloaded ADC and MMAE loaded ADC conjugates at 6.8 (95% CI 5.0-7.7 h) and 6.5 (95% CI 6.2-7.4 h) hours respectively (Figure 4F). The coiled-coil peptide tag does not negatively affect antibody PK and clearance in the mouse. These data along with the in vitro results showing that the antibody binds the FcRn receptor (Figure 1G) suggests that coiled-coil peptide modification of the C-terminus does not significantly affect FcRn binding.”\nComment 5: The data in Figure 4g seem to suggest that more antibody accumulates in the tumor for the engineered variant when compared to the parent antibody (see day 5 and 7 data). This is unexpected given that no effort was made to improve the ‘targeting’ effectiveness of the antibody itself. This largely seems to be mediated by one outlier in the cohort that has an unusually high signal at both day 5 and day 7.\nResponse 5: We have clarified the figure description. Figure 4G does not compare accumulation of mAb and ADC. The figure shows the accumulation of BP Fluor 647–loaded AFC in SKOV-3 tumor-bearing mice over a 7-day period in the tumor tissue and major organs. We have edited the text to be clearer on page 13.\n“…The AFC accumulates in the tumor throughout the 7-day study period, along with liver, kidneys and lungs at the earlier time points. These results are consistent with the literature 12,13 and demonstrate the ability of coiled-coil based antibody conjugate to enhance accumulation of deliver a payload to ErbB2/Her2-positive tumors…”\nThe Reviewer also mentioned the outliner in the cohort that contributes to the higher average value on day 5 and 7. We agree with the reviewer and have repeated the in vivo experiment. We implanted SKOV-3 cells intraperitoneally into immunodeficient BALB/c nude mice and administered a BP Fluor 647-loaded AFC at a dose of 10 mg/kg. At 1 hour, 1 day, 3 days, 5 days, and 7 days post-injection, we euthanized the animals and quantified the fluorescent signal in the tumor and major organs by IVIS. The new dataset shows no outliers. The updated figures are included in the manuscript and supplementary information as follows:\n“…G. Ex vivo organ biodistribution of AFC injected i.v. at 10 mg/kg dose into SKOV-3 tumor bearing BALB/c nude mice at different time points (n=3 per time point). The total radiant efficiency was calculated using IVIS software keeping area of ROI the same across all the samples…”\n“Figure SI17. Ex vivo organ biodistribution of coiled-coil AFC injected i.v. at 10 mg/kg dose into SKOV-3 tumor bearing Balb/c nude mice at different time points (n=3 per time point). The total radiant efficiency was calculated using IVIS software keeping area of ROI the same across all the samples. Letters indicate: T – tumor, L – lungs, H – heart, Li – liver, S – spleen, K – kidneys.”\nComment 6: The absence of any intensity scale in Figure 4h makes it difficult to interpret the images accurately.\nResponse 6: Thank you for bringing this to our attention. We have updated the figure with the latest results, included an intensity scale in Figure 4h and adjusted the figure description:\n“ H. Representative fluorescent picture of ex vivo organs post AFC injection on day 5. Color scale: min = 1.05×109, max = 1.1×1010 (p⁄sec/cm2⁄sr / µW⁄cm2).”\nComment 7: The data in Figure 4i-m provide a compelling demonstration that the engineered coiled-coil ADC is effective in vivo. However, the comparable effectiveness of the coiled-coil ADC and the gold-standard disitimab vidotin leave open the question of whether the new approach has in fact addressed a limitation of existing methods, which is at the core of this study. …….While the data in Figure 4a suggest that the conventionally prepared ADC may be less stable than the coiled-coil ADC in this model, the stability experiment should instead be set up to compare their coiled-coil ADC against disitimab vidotin head-to-head. There is some reasonable expectation that disitimab vidotin is manufactured using a robust and reliable process that may not be accurately reflected by their \"conventionally prepared ADC\" method.\nResponse 7: We agree and acknowledge that disitimab vidotin is manufactured using a robust and reliable process which is more stringent than one in an academic lab. Further, as it has entered clinical trials, significant preclinical testing has been done by the company. We have only found one manuscript that describes the in vitro serum stability results, and the authors are from a university. 14 They report serum stability over one day with loss of less than 1% MMAE for disitimab vidotin via ELSIA. We performed a head-to-head comparison of the coiled-coil ADC and disitamab vedotin following a standard established ELISA method 15,16. At the 24-hour time point we observed a small decrease in stability with free MMAE (<1%) for both disitimab vidotin and our ADC. This result agrees with published data 14 . At longer time periods out to day 30, we see no change in signal for the coiled-coil ADC indicating that MMAE is not released, however the ELISA signal increases for the disitimab vidotin over time (both IgG and MMAE content). This is likely due to aggregation or some other phenomenon (and not MMAE release). Aggregation is a recognized problem with ADCs especially those with 4+ drugs and is known to depend on both the antibody scaffold and the conjugated drug 17,18. To address this issue, we replaced the cytotoxic payload with a fluorophore and changed the antibody scaffold to match the antibody in coiled-coil ADC, which was included in the original manuscript (Figure 4A). We applied TCEP/DTNB conjugation protocol similar to published and patented protocol for disitimab vidotin 19,20 . This approach allowed us to focus on evaluating the conjugation strategy itself rather than directly comparing our system to a commercial product. If you would like us to develop an alternative method for assessment of disitimab vidotin stability, please let us know.\nFigure 1. Serum stability of disitamab vedotin (blue) and coiled-coil ADC (red) in human serum incubated at 37 °C. Total IgG and MMAE content was assessed by indirect ELISA detecting using anti-IgG secondary antibodies or anti-MMAE secondary antibodies respectively. Signal was normalized to intact ADC (no incubation in serum, flash frozen at zero time point).\nComment 8: Additionally, to show an advance beyond the gold-standard, the experiments in Figure 4k-m need to have a cleaner set of controls to account for the effects of both the antibody and the payload, given that (1) trastuzumab provides some therapeutic effect on its own and (2) disitimab vidotin is based on a different antibody (hertuzumab) with a different profile than trastuzumab. Recommendations here would include a dose-response profile comparing the coiled-coil ADC versus disitimab vidotin, as well as inclusion of a group treated with hertuzumab alone to provide a baseline for antibody-only effectiveness.\nResponse 8: We thank the Reviewer for both suggestions and have performed both in vivo experiments.\nBecause the formation of palpable solid tumors in the SKOV-3 model requires a prolonged period (50-60 days), we performed the dose escalation study and assessed tumor growth using IVIS bioluminescence imaging with SKOV-3-Fluc cells. We used an animal model similar to that employed in the biodistribution study (nude mice, intraperitoneal injection of 2 × 10⁶ cells) and treated animals with the coiled-coil ADC at doses of 1, 3, 5, or 10 mg/kg. Treatment with the ADC at 1 mg/kg did not result in tumor growth suppression. Doses of 3 and 5 mg/kg maintained stable luminescence signals, whereas treatment at 10 mg/kg led to a decrease in luminescence signal, indicating a decrease in number of tumor cells. Based on these results, a dose of 10 mg/kg was selected for subsequent efficacy studies. The dose response profile for disitimab vidotin is published 20 and the effective dose is between 5 and 10 mg/kg in mice (gastric and ovarian cancer models) 19–21, and we referenced this report in the revised manuscript as well as the data for trastuzumab-vedotin. We included these new findings into the manuscript on page 13, and SI information as follows:\n“To identify an effective dose, we performed a dose–response study. We intraperitoneally administered 2 × 10⁶ SKOV-3-Fluc cells to immunodeficient BALB/c nude mice and treated two weeks post-implantation with ADC doses of 1, 3, 5, or 10 mg/kg, or with PBS as a negative control (Figure SI21). Treatment with the ADC at 1 mg/kg does not result in IVIS signal reduction. Doses of 3 and 5 mg/kg show a modest reduction in IVIS signal, whereas treatment at 10 mg/kg leads to reduction in IVIS signal consistent with tumor regression. Based on these results, we selected a dose of 10 mg/kg for the subsequent in vivo efficacy studies. For comparison, the effective dose for disitimab vedotin is between 5 and 10 mg/kg 69,70, and for trastuzumab-vedotin is between 3 and 10 mg/kg 71,72 in gastric and ovarian cancer models in mice.”\nDose response\ncoiled-coil ADC\n10\n1 mg/kg\n3 mg/kg\n1 p=0.03 5 mg/kg\np=0.02 10 mg/kg\n0.1\n0.01\n0 10 20 30\nDay post treatment\nFigure SI21. Dose selection study. Quantification of the BLI signal of tumors after different treatments in female nude mice engrafted intraperitoneally with 2 million SKOV-3 tumor cells and treated on day 14 with either PBS or coiled-coil ADC at the dose: 1 mg/kg, 3 mg/kg, 5 mg/kg or 10 mg/kg. Statistical significance was assessed using an unpaired, one-tailed t-test.\n“Dose response. Female BALB/c nude (6–8 weeks old) were obtained from the Jackson Laboratory (#:002019). The orthotopic xenograft model of SKOV-3 was established by injecting SKOV-3-fluc cells (2 × 106 cells in 0.2 ml PBS) intraperitoneal and allowed to develop for 14 days. Mice were administered a single dose of 100 µl of PBS or ADCs at doses of 1 mg/kg, 3 mg/kg, 5 mg/kg or 10 mg/kg in 100 µl PBS solution via intravenous injection into the tail vein. Tumor progression was assessed weekly by IVIS bioluminescence imaging. D-Luciferin potassium salt (Revvity Health Sciences Inc.) was diluted to 0.38 mg/mL in PBS and injected intraperitoneally (200 µL) under anesthesia. Animals were imaged 10 min after luciferin administration.”\nWe agree that an additional control group would be useful to tease out the performance of the new ADC. We performed an additional in vivo study and included the suggested hertuzumab control as requested as well as the commercial ADC based on trastuzumab antibody. Similarly to trastuzumab, disitamab monoclonal antibody (hertuzumab) significantly contributes to tumor growth inhibition. Please see page 13, 14 and revised Figures 4K-M and SI 22-23.\n“Using the established orthotopic xenograft model (Figure 4I), we performed an efficacy study and i.v. administered a single dose of 10 mg/kg of trastuzumab (native αHER2 antibodies) or 10 mg/kg of coiled-coil ADC (n = 5 per group). Additionally, we administered PBS as the negative control, and two positive controls: 1) trastuzumab vedotin and 2) disitamab vedotin — the latest HER2-targeting ADC to advance to Phase III clinical trials 22 — as the positive control. Trastuzumab vedotin and disitamab vedotin consists of the trastuzumab and hertuzumab antibody, respectively, and, similar to our ADC, uses the cytotoxic payload MMAE linked via a Val-Cit cleavable linker. However, both commercial antibodies employ cysteine-based conjugation, resulting in approximately four MMAE molecules per antibody. To match the drug dose of the positive control groups, we also administered a double dose of coiled-coil ADC (20 mg/kg, n = 5 per group). The antibody vehicle controls for this study are trastuzumab mAb and disitamab mAb.”\n“In contrast, those receiving the antibody vehicle controls (trastuzumab mAb or disitamab mAb) show only an 8-fold or 5-fold and 5-fold or 6-fold reduction in tumor mass and volume respectively (m = 52±27 mg, V = 166±81 mm3 for trastuzumab and m = 78±32 mg, V = 145±97 mm3 for disitamab mAb). Treatment with 20 mg/kg ADC, 10 mg/kg trastuzumab vedotin, or 10 mg/kg disitamab vedotin completely eradicates tumor in one mouse from each group, with average tumor masses of 5±3 mg, 6±7 mg, and 2±3 mg, respectively, with a similar trend in the tumor volume: V = 6±5 mm3, V = 9±11 mm3 and V = 4±4 mm3, respectively (Figure 4K-M and SI23). Disitamab vedotin and trastuzumab vedotin are highly efficacious, consistent with its elevated drug-to-antibody ratio for both ADCs and enhanced affinity for the HER2 target relative to trastuzumab of disitamab vedotin 23 ”\n“ Figure 4 … K. Images of harvested tumors at study termination at day 48 post treatment. L. Recorded tumor size and M. tumor mass. Statistical significance is indicated as follows: ns, non-significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005 ****P ≤ 0.0005 (unpaired t-test).”\n“Figure SI23. Images of harvested tumors at study termination at week 7 post treatment. Female NSG mice were engrafted with 2 million SKOV-3 tumor cells by intraperitoneal injection and treated on day 14 post tumor inoculation with single dose of PBS (100 uL) or disitamab (monoclonal antibody, 10 mg/kg), disitamab vedotin (commercial ADC, 10 mg/kg) or traztuzumab-vedotin (commercial ADC, 10 mg/kg), n= 5 mice per group. All mice were euthanized when PBS mice treated mice exhibited severe hunching posture and signs of morbidity.”\n“Figure SI22. Average mouse body weight measured twice a week (±SD) for PBS, disitamab (mAb, 10 mg/kg), disitamab vedotin (commercial ADC, 10 mg/kg) and trastuzumab-vedotin (commercial ADC, 10 mg/kg) treatment group demonstrating no significant difference between the groups over the observation period.”\nReferences\n1. Gebhardt, L. et al. A Robust, High-Titer, Semi-Automated, and In-Culture Antibody-Capturing Transient CHO Platform Technology. Antibodies 14, (2025).\n2. Lapenta, F. et al. Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo. Nat. Biotechnol. 35, (2017).\n3. Wu, Y. & Collier, J. H. α-Helical coiled-coil peptide materials for biomedical applications. Wiley Interdiscip. Rev. Nanomedicine Nanobiotechnology 9, 1–17 (2017).\n4. Rudra, J. S., Tripathi, P. K., Hildeman, D. A., Jung, J. P. & Collier, J. H. Immune responses to coiled coil supramolecular biomaterials. Biomaterials 31, 8475–8483 (2010).\n5. Thomas, F., Boyle, A. L., Burton, A. J. & Woolfson, D. N. A set of de novo designed parallel heterodimeric coiled coils with quantified dissociation constants in the micromolar to sub-nanomolar regime. J. Am. Chem. Soc. 135, 5161–5166 (2013).\n6. Wang, J., Yu, Y. & Xia, J. Short peptide tag for covalent protein labeling based on coiled coils. Bioconjug. Chem. 25, 178–187 (2014).\n7. Ahn, J. H. et al. Reversible Protein Conjugation on Live Cell Surfaces by Specific Recognition between Coiled-Coil Motifs of Natural Amino Acid Sequences. Biomacromolecules 21, 3539–3546 (2020).\n8. Kraatz, S. H. W., Bianchi, S. & Steinmetz, M. O. Combinatorial use of disulfide bridges and native sulfur-SAD phasing for rapid structure determination of coiled-coils. Biosci. Rep. 38, (2018).\n9. Chernyatina, A. A. & Strelkov, S. V. Stabilization of vimentin coil2 fragment via an engineered disulfide. J. Struct. Biol. 177, 46–53 (2012).\n10. Papers, J. B. C. & Doi, M. Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel. J. Biol. Chem. 288, 17968–17975 (2013).\n11. Plaper, T., Aupič, J., Dekleva, P., Lapenta, F. & Keber, M. M. Coiled‑coil heterodimers with increased stability for cellular regulation and sensing SARS ‑ CoV ‑ 2 spike protein ‑ mediated cell fusion. Sci. Rep. 11, 9136 (2021).\n12. Kuo, W. Y. et al. Noninvasive assessment of characteristics of novel anti-HER2 antibodies by molecular imaging in a human gastric cancer xenograft-bearing mouse model. Sci. Rep. 8, 1–9 (2018).\n13. Park, S., Nedrow, J. R., Josefsson, A. & Sgouros, G. Human HER2 overexpressing mouse breast cancer cell lines derived from MMTV.f.HuHER2 mice: characterization and use in a model of metastatic breast cancer. Oncotarget 8, 68071–68082 (2017).\n14. Li, L. et al. Conjugating MMAE to a novel anti-HER2 antibody for selective targeted delivery. Eur. Rev. Med. Pharmacol. Sci. 2, 12929–12937 (2020).\n15. Anami, Y. et al. Glutamic acid-valine-citrulline linkers ensure stability and efficacy of antibody-drug conjugates in mice. Nat. Commun. 9, (2018).\n16. Pei, M. et al. Enzyme-linked immunosorbent assays for quanti fi cation of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera. J. Pharm. Anal. 12, 645–652 (2022).\n17. Durbin, K. R., Nottoli, M. S., Catron, N. D., Richwine, N. & Jenkins, G. J. High-Throughput, Multispecies, Parallelized Plasma Stability Assay for the Determination and Characterization of Antibody − Drug Conjugate Aggregation and Drug Release. ACS Omega 2, 4207–4215 (2017).\n18. Gieseler, H., Johann, F. & Steffen, W. “Negative” Impact: The Role of Payload Charge in the Physicochemical Stability of Auristatin Antibody − Drug Conjugates. J. Pharm. Sci. 113, 2433–2442 (2024).\n19. Li, H. et al. An anti-HER2 antibody conjugated with monomethyl auristatin E is highly effective in HER2-positive human gastric cancer. Cancer Biol. Ther. 17, 346–354 (2016).\n20. Jianmin, F. et al. Anti-Her2 antibody and conjugate thereof Pub . No .: US 2016 / 0304621 A1. (2016).\n21. Wang, L. et al. Preclinical pharmacokinetics , distribution , metabolism and excretion of disitamab vedotin. ADMET DMPK 13, 1–13 (2025).\n22. Galsky, M. D. et al. Phase 3 open-label, randomized, controlled study of disitamab vedotin with pembrolizumab versus chemotherapy in patients with previously untreated locally advanced or metastatic urothelial carcinoma that expresses HER2 (DV-001). J. Clin. Oncol. 42, TPS717–TPS717 (2024).\n23. Shi, F. et al. Disitamab vedotin: an"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors made substantial revision to the manuscript, addressing most of the reviewer’s comments.\nMy only remaining comment is related to the disulfide-linked coiled-coil pair. The authors used a surrogate approach by analyzing free peptides or peptides-cys (not the antibody-coil-cys + complementary coil-cys) to demonstrate disulfide-bridge formation within the cysteine-containing coiled-coils. By MALDI-TOF analysis, the authors have show that some disulfide bridges are forming, but to a limited extent: according to Figure SI16 panel B, it seems that less than 60% of the peptides formed a disulfide bridge after a 30 minutes incubation. I am wondering if bridge formation would be even less efficient when mixing antibody-coil-cys with complementary coil-cys, raising again the question about the benefit/necessity of adding a disulfide bridge to further stabilize the complexes as it may also increase heterogeneity of the final ADC due to incomplete disulfide bridge formation. I think the paper would benefit having a more elaborate discussion about this aspect.", "reviewer_3": "The authors have satisfactorily addressed my critiques in the revised version of this manuscript. I believe that this manuscript will be a welcomed addition to the coiled-coil literature."}, "author_response": "We appreciate the time and attention the reviewers gave to the manuscript and encouraging feedback. Please find below the detailed point-by-point answers to the reviewers’ questions. As required where the text of the manuscript has been rewritten or new text added, the parts are marked up in blue.\nReviewer #2\nComment: The authors made substantial revision to the manuscript, addressing most of the reviewer’s comments.\nMy only remaining comment is related to the disulfide-linked coiled-coil pair. The authors used a surrogate approach by analyzing free peptides or peptides-cys (not the antibody-coil-cys + complementary coil-cys) to demonstrate disulfide-bridge formation within the cysteine-containing coiled-coils. By MALDI-TOF analysis, the authors have show that some disulfide bridges are forming, but to a limited extent: according to Figure SI16 panel B, it seems that less than 60% of the peptides formed a disulfide bridge after a 30 minutes incubation. I am wondering if bridge formation would be even less efficient when mixing antibody-coil-cys with complementary coil-cys, raising again the question about the benefit/necessity of adding a disulfide bridge to further stabilize the complexes as it may also increase heterogeneity of the final ADC due to incomplete disulfide bridge formation. I think the paper would benefit having a more elaborate discussion about this aspect.\nResponse: We thank the reviewer for acknowledging the revisions made to the manuscript. We agree that if disulfide bridge formation does not occur it may also increase heterogeneity of the final ADC. We also recognize that the structures without the cysteine bridge show stability in plasma over prolonged period of time. We have added the following section to the discussion addressing this limitation, please see page 12.\n“…and, thus, we lengthened the coiled-coil region and introduced a disulfide linkage between the peptides to improve stability. As shown in Figure 4, the extended coiled-coil sequence itself provides a robust structure as does the one with the disulfide linkage, as a supplemental stabilization strategy. Incorporation of these two changes in the coiled-coil design affords a functional ADC for in vivo use. However, if incomplete disulfide linkage forms (e.g., unfavorable pH or steric limitations) the cysteines may cause heterogenicity introducing a possible limitation to the ADC. “\nReviewer #3\nComment: The authors have satisfactorily addressed my critiques in the revised version of this manuscript. I believe that this manuscript will be a welcomed addition to the coiled-coil literature.\nResponse: We thank the reviewer for their positive feedback and for recognizing our work as a valuable addition to the coiled-coil literature."}]

Decision letter