xRead - Recurrent Respiratory Papillomatosis (October 2025)

Article

https://doi.org/10.1038/s41467-025-56729-6

Table 3 | INO-3107 signi fi cantly activates multiple innate and adaptive functional networks in responder airways

associated toxicities 32 – 35 . INO-3107 treatment was well-tolerated and allowed for partial or complete control of disease in the context of reducing surgical interventions during the monitoring period of the trial, with a four-dose regimen spanning only 9 weeks. Limitations of this study should be acknowledged. This study was single-arm and not placebo-controlled. The decision to proceed with the surgical intervention was the same during the 52-week pre- and post-treatment periods and was left to the individual investigators and patients, introducing some variability into the study from site to site. Additionally, as the study was conducted in 32 patients, study size should be considered. The current study focused on RRP disease in adults only; future investigations could include studies of INO-3107 in the pediatric population. For correlative analyses, end of study tissue was not available from all complete responders and the contribution of the induction of CD4 + T-cell responses, while previously described after treatment with INO-3107 36 , are not expanded on here as the current report is focused on CD8 + T-cell-based contribution to the proposed mechanism of action. The induction of anti-HPV-6 and HPV-11 immune responses has been identi fi ed in patients who have shown varying degrees of response to adjuvant antibody therapy inclusive of PD-1/PDL-1, TGF β , andVEGFA 10,37 – 39 , identifying a likely connection between the immune system and control of the disease. This concept is further supported by work from other groups using T-cell inducing immunotherapy for the treatment of RRP, which have shown promising clinical results, although the application of viral vectors for the induction of anti-HPV immune responses may have been limited by anti-vector immune responses and a dif fi cult to treat papilloma microenvironment 19 .Here, we report not only the induction of HPV-6 and HPV-11 targeted immune responses, but also track newly identi fi ed T-cell clonal populations from the blood into patient airways after INO-3107 treat ment. Post-treatment biopsies showed an increased in fl ammatory state, consisting of interferon signaling pathways and innate immune cells as well as CD8 + T-cell signatures, inclusive of cytotoxic immune responses. As the lack of such response is believed to be involved in maintaining papilloma disease state 9 – 13 , it was expected and con fi rmed that the induction of these responses by INO-3107 were identi fi ed as being associated with clinical bene fi t in our study. The maintenance of this immune response should, therefore, be expected to be associated with a prolonged impact on the disease state, although this remains to be con fi rmed. Of interest, patients who did not show a decrease in surgical intervention frequency while on this trial still showed immu nological changes in their papilloma tissue with respect to these parameters, albeit less than patients who responded clinically. As the DNA platform allows multiple administrations without the risk of generating anti-vector immunity, and as it has been previously shown that adding “ booster ” doses outside the primary series of DNA thera pies can augment immune responses 40 , the possibility remains open for re-administration of INO-3107 to augment the immune response to further improve clinical response. Such an undertaking would also allow for continued research into immune responsiveness in RRP patients over time, which may have broader implications for under standing the mechanisms underpinning changes in disease state. The results presented here make a strong rational case for use of INO-3107 as a non-surgical approach to RRP treatment from both a clinical and mechanistic standpoint. To that end, future clinical trials are being planned to further address prolonged ef fi cacy, immune response, and redosing to expand the potential impact of this approach for the bene fi t of RRP patients. Methods Study design and patients The study was conducted in accordance with the Declaration of Hel sinki, Good Clinical Practice guidelines, and local and national reg ulatory requirements. The protocol was reviewed and approved by the

Functional network T cell homeostasis

p value 5.58e -23 6.78e -22 1.38e -12 6.27e -21 3.89e -13 5.73e -11 4.79e -07 6.50e -15

Activation z -score

3.377

3.480 2.462 2.685 3.076 2.806 2.496

T cell development

T cell migration

Quantity of T lymphocytes

Differentiation of T lymphocytes 7.55e -17

Interaction of T lymphocytes

Binding of T lymphocytes

2.614

Adhesion of T lymphocytes

2.436

Migration of antigen present ingcells Cell movement of antigen pre senting cells Interaction of antigen present ingcells Binding of antigen present ingcells Recruitment of antigen present ingcells Quantity of antigen present ingcells Response of antigen present ingcells Immune response of antigen presenting cells Traf fi cking of antigen present ingcells Chemotaxis of antigen present ingcells Engulfment of antigen present ingcells Migration of dendritic cells Response of dendritic cells Recruitment of macrophages Phagocytosis by macrophages

3.52e -13

2.445

3.37e -12

2.673

1.33e -11

2.479

1.00e -10

2.309

1.37e -10

2.284

5.85e -07

3.021

9.26e -07

2.510

1.33e -06

2.176

3.03e -06

2.715

1.00e -05

3.173

Cell movement of dendritic cells 7.63e -09

2.207

9.14e -09 7.74e -05 1.12e -09 6.53e -06 1.23e -05 2.39e -20 7.78e -23 1.20e -19 2.04e -19 6.95e -28 4.94e -25 2.89e -21

2.087

2.000

2.013

2.772

2.207 3.342 3.494 3.450 2.896

Chemotaxis of macrophages

Adhesion of immune cells

Lymphocyte homeostasis

Lymphopoiesis

Lymphocyte migration

2.716

Activation of lymphocytes

2.113

Quantity of lymphocytes

2.267

Cytotoxicity of lymphocytes

Cell movement of lymphocytes 4.82e -18

2.643 2.998

2.79e -13 2.78e -11

Interaction of lymphocytes

Binding of lymphocytes 2.710 Various outputs of Ingenuity Pathway Analysis depicting innate and adaptive immune functional networks, with associated p valuesand z -scores determined using one-sided Fisher ’ s exact test, seen in airways of Responders ( n = 12) after INO-3107 treatment.

from a long-term study of RRP patients undergoing recurrent surgeries reported an increased incidence of permanent damage to vocal cords and airway as a direct function of surgical intervention and speci fi cally highlighted the need for non-surgical interventions in RRP patients 8 . Additionally, while non-surgical interventions such as monoclonal antibodies to various targets have been attempted in the treatment of RRP, they have shown variable impact with respect to control of dis ease once therapy is removed, and continued therapy has known

Nature Communications | (2025)16:1518

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