L-ICON3 Suppresses Endometriosis in a Murine Model

As described above, endometriosis was induced in C57BL/6 mice ( n  = 36). Mice were allowed to develop endometriosis for 6 weeks after transplant and then divided randomly into three groups ( n  = 12 per group). One control group was treated with vehicle (PBS), another control group with IgG (0.5 mg/kg body weight), and a third group with L-ICON3 (0.5 mg/kg body weight) for 6 weeks. After 6 more weeks of treatment, mice were sacrificed and underwent laparotomy to evaluate the endometriosis lesions as shown in Fig.  1 for PBS (1B), IgG (1C), and L-ICON3 (1D). Figure  2 shows the histomorphology of H & E-stained endometriosis lesions from mice treated with PBS (A), IgG (B), and L-ICON3 (C). Cystic fluid represents the largest area of the lesion and was not resorbed after treatment. Endometriosis thickness was significantly reduced more than 2.5-fold in PBS vs. L-ICON3 treated animals ( p  < 0.01) and 4.5-fold (after IgG vs. L-ICON3 treatment (p  < 0.0001) as shown in Fig.  2 D. The endometriosis area was also decreased 2.5-fold in the L-ICON3 treated group compared to PBS or IgG ( p  < 0.05). Fig. 2 L-ICON3 treatment reduced endometriosis epithelial thickness lesion. H&E-stained tissue sections from endometriotic lesions with endometrial tissue plus fluid-filled cysts from mice treated with PBS ( A ), IgG ( B ), or L-ICON3 ( C ). A significant reduction in endometriotic lesion thickness was seen in L-ICON3 treated mice compared to PBS and IgG treatments ( D ). The thickness of endometriosis tissue between the outer and inner layers was used to calculate the endometriosis thickness. A 500 μm bar is placed to the right of each lesion as a measure of relative scale. Each bar represents the mean ± SEM of endometrial thickness. * p  < 0.01, between L-ICON3 and PBS and **** p  < 0.0001, between L-ICON3 and IgG treatments using student’s t-test with Welch’s correction L-ICON3 treatment reduced endometriosis epithelial thickness lesion. H&E-stained tissue sections from endometriotic lesions with endometrial tissue plus fluid-filled cysts from mice treated with PBS ( A ), IgG ( B ), or L-ICON3 ( C ). A significant reduction in endometriotic lesion thickness was seen in L-ICON3 treated mice compared to PBS and IgG treatments ( D ). The thickness of endometriosis tissue between the outer and inner layers was used to calculate the endometriosis thickness. A 500 μm bar is placed to the right of each lesion as a measure of relative scale. Each bar represents the mean ± SEM of endometrial thickness. * p  < 0.01, between L-ICON3 and PBS and **** p  < 0.0001, between L-ICON3 and IgG treatments using student’s t-test with Welch’s correction The effect of L-ICON3 on TF protein levels was measured by immunohistochemical staining. Figure  3 A shows representative images of tissue sections stained using an anti-TF antibody. The intensity of staining is reduced significantly in lesions treated with L-ICON3 compared to IgG. H-score was used to determine a semiquantitative measure of protein expression as shown in Fig.  3 B. In L-ICON3 treated mice, TF protein levels were significantly decreased 2-fold ( p  < 0.02) compared to the IgG treated group. Fig. 3 Tissue factor (TF) expression is reduced by L-ICON3. ( A ) Shows representative IHC images of TF expression in lesions from mice with endometriosis treated with either IgG or L-ICON3. TF staining is significantly reduced in L-ICON3 treated mouse group compared to the IgG treated group. Original magnification: 20×. (B) Bar graph shows TF H-score is significantly reduced in lesions from mice treated by L-ICON3 compared to IgG. TF is stained by anti-TF antibody. Two people blind to the treatment groups determined the H-score. Data represents mean and SEM. * p  = 0.02 between IgG vs. L-ICON3 Tissue factor (TF) expression is reduced by L-ICON3. ( A ) Shows representative IHC images of TF expression in lesions from mice with endometriosis treated with either IgG or L-ICON3. TF staining is significantly reduced in L-ICON3 treated mouse group compared to the IgG treated group. Original magnification: 20×. (B) Bar graph shows TF H-score is significantly reduced in lesions from mice treated by L-ICON3 compared to IgG. TF is stained by anti-TF antibody. Two people blind to the treatment groups determined the H-score. Data represents mean and SEM. * p  = 0.02 between IgG vs. L-ICON3 The effect of L-ICON3 on CD3, CD4, CD8, and CD56 expression was measured by immunohistochemical staining. Figure  4 A shows the representative images of tissue sections stained by anti-CD3, anti-CD4, anti-CD8, and anti-CD56 antibodies respectively. The number of cells stained for the above antibodies was significantly increased in the treatment (L-ICON3) group versus control (IgG) for each antibody. The number of cells stained per area was calculated showed in Fig.  4 B as follows: anti-CD3 (2.5-fold; p  = 0.04), anti-CD4 (12.9-fold; p  = 0.002), anti-CD8 (13.8-fold; p  = 0.04), and anti-CD56 (6.5-fold; p  = 0.02). We next determined the expression levels of IL-6 and TNF-α, inflammation markers, and caspase7, an apoptosis marker, to identify potential effects of L-ICON3 treatment on these processes. There was no significant effect of L-ICON3 on Interleukin 6 (IL-6), TNF-α, or Caspase 7 expression in lesions from mice with endometriosis treated with L-ICON3 compared to control group IgG (Fig.  5 ). Though there was a trend towards increased TNF-α, and caspase7 staining in L-ICON3 treatment, no significant change observed either in the intensity of protein staining (Fig.  5 A) or H-scores (Fig.  5 B). Fig. 4 Immune cells were increased in L-ICON3 treatment Group. ( A ) Shows the representative IHC images of CD3, CD4, CD8, and CD56 expression in lesions from mice with endometriosis treated with IgG or L-ICON3 stained by the respective antibodies. The staining of all CD3, CD4, CD8, and CD56 are significantly increased in L-ICON3 treated group compared to IgG treated group. Original magnification: 20×. (B) Bar graph shows the number of positive cells per area are significantly increased in lesions from mice treated by L-ICON3 compared to IgG. Two people blind to the treatment groups determined the number of positive cells per area. Data represents SEM. * p  < 0.05 between IgG vs. L-ICON3 Immune cells were increased in L-ICON3 treatment Group. ( A ) Shows the representative IHC images of CD3, CD4, CD8, and CD56 expression in lesions from mice with endometriosis treated with IgG or L-ICON3 stained by the respective antibodies. The staining of all CD3, CD4, CD8, and CD56 are significantly increased in L-ICON3 treated group compared to IgG treated group. Original magnification: 20×. (B) Bar graph shows the number of positive cells per area are significantly increased in lesions from mice treated by L-ICON3 compared to IgG. Two people blind to the treatment groups determined the number of positive cells per area. Data represents SEM. * p  < 0.05 between IgG vs. L-ICON3 Fig. 5 No change in IL-6, TNF-α, and Caspase7. ( A ) Shows the representative IHC images of IL-6, TNF-α, and caspase7 expression in lesions from mice with endometriosis treated with IgG or L-ICON3 stained by the respective antibodies. There is no significant changes observed in staining between the two groups. Original magnification: 20×. (B) Bar graph shows no significant difference between the two groups. Two people blind to the treatment groups determined the H-score. Data represents SEM. p  > 0.05 between IgG vs. L-ICON3 No change in IL-6, TNF-α, and Caspase7. ( A ) Shows the representative IHC images of IL-6, TNF-α, and caspase7 expression in lesions from mice with endometriosis treated with IgG or L-ICON3 stained by the respective antibodies. There is no significant changes observed in staining between the two groups. Original magnification: 20×. (B) Bar graph shows no significant difference between the two groups. Two people blind to the treatment groups determined the H-score. Data represents SEM. p  > 0.05 between IgG vs. L-ICON3

Endometriosis was induced in mice utilizing a syngeneic endometriosis protocol previously established by our laboratory [ 24 ]. In brief description, donor mice were sacrificed using carbon dioxide and uterine horns from each female donor mouse were used for transplantation. The lumen of each horn was opened longitudinally and then divided into four equal fragments of approximately 3 mm in length. Recipient mice were then anesthetized using inhaled isoflurane (Covetrus, Dublin, OH, USA) and a midline laparotomy was performed. Buprenorphine (0.1 mg/Kg body weight), an analgesic drug, was given subcutaneously before starting the surgery and lidocaine (5 mg/Kg body weight) was given subcutaneously as local anesthesia. Four uterine fragments were then transplanted into each recipient mouse, with two fragments on either side of the peritoneum 1 cm apart, using 5 − 0 polyglactin suture (Vicryl; Ethicon, Somerville, NJ, USA). The peritoneum was closed using 5 − 0 polyglactin suture and the skin was closed with 4 − 0 polyglactin suture. Meloxicam (5 mg/Kg body weight) was given subcutaneously once in a day post-surgery for 72 h. This model was first validated using four mice with successful induction of endometriotic lesions by four weeks postoperatively. Next additional mice ( n = 36) underwent surgery for the induction of endometriosis and were allowed to develop endometriosis for six weeks.

Female C57BL/6 mice six-to-eight-week-old were obtained from Charles River Laboratories (Wilmington, MA, USA) and maintained at the Yale School of Medicine animal facility. Five mice were housed per cage and kept in alternating 12-hour day light and dark cycles with access to food and water ad libitum . All experiments were carried out according to the protocol (#2023–07113) approved by the Institutional Animal Care and Use Committee (IACUC) at Yale University.

The 36 mice with surgically induced endometriosis were allowed to develop endometriosis for 6 weeks and then randomly divided into three groups of 12 mice in each. One group was treated with recombinant L-ICON3 protein obtained from Dr. Zhiwei Hu from Ohio State University, Columbus, OH. L-ICON3 was given twice per week to the mice at a dose of 0.5 mg/Kg body weight (50 µl) by retroorbital injection for 6 weeks. The second and third control groups were treated with either PBS or an IgG isotype control. All treated mice were then sacrificed by cervical dislocation after six weeks of treatment to evaluate the endometriosis lesions. The timeline for the induction and treatment of endometriosis is shown in Fig.  1 A. Fig. 1 Induction and treatment of endometriosis by L-ICON3, IgG isotype control and PBS. ( A ) Depiction of the timeline of induction of endometriosis ( n  = 36), lesion formation followed by treatment with L-ICON3 (0.5 mg/kg body weight), IgG (0.5 mg/kg body weight), or PBS in 50 µl by retro-orbital injections, twice a week for 6 weeks ( n  = 12 mice per group). Mice peritoneal cavity shows endometriotic lesions treated with PBS ( B ), isotype control IgG ( C ), or L-ICON3 ( D ) Induction and treatment of endometriosis by L-ICON3, IgG isotype control and PBS. ( A ) Depiction of the timeline of induction of endometriosis ( n  = 36), lesion formation followed by treatment with L-ICON3 (0.5 mg/kg body weight), IgG (0.5 mg/kg body weight), or PBS in 50 µl by retro-orbital injections, twice a week for 6 weeks ( n  = 12 mice per group). Mice peritoneal cavity shows endometriotic lesions treated with PBS ( B ), isotype control IgG ( C ), or L-ICON3 ( D )

Three measurements of each lesion were taken: length, width, and height. Volumes were calculated using the following formula: volume = (smallest diameter) 2 x largest diameter/2 [ 25 ]. Lesions were then fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned by the Pathology Department at Yale School of Medicine. Lesion tissue sections were stained with hematoxylin and eosin (H&E) and evaluated under light microscopy. To assess the effect of L-ICON3 on endometriosis, histological assessment of lesions was used to measure the area and thickness of the endometriosis tissue within the lesion. Lesion histological area was calculated by subtracting the total cystic area(s) within each lesion from the total lesion area. Epithelial thickness was measured at four predefined locations (12, 3, 6, and 9 o’clock positions) around each lesion, and the mean value was used for subsequent analyses. Both lesion histological area and epithelial thickness were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Measurements were independently performed by two investigators blinded to treatment group allocation, and the averaged values were used for statistical analysis. Tissue sections of five micrometers (µM) were cut from paraffin embedded lesions and placed on glass slides. Tissue sections on slides were steamed for 15 min in sodium citrate (pH 6.0) and blocked with 5% goat serum. Slides were then incubated with anti-tissue factor antibody (catalog # sc-37444, Santa Cruz Biotechnology, Dallas, TX, USA, 1:250 dilution), anti-TNF-α antibody (catalog # sc-57468, Santa Cruz Biotechnology, 1:250 dilution), anti-IL6 antibody (catalog # NBP2-16957, Novus Biologicals, Littleton, CO, USA, 1:250 dilution), anti-caspase 7 antibody (catalog # ab255818, Abcam, Waltham, MA, USA, 1:2500 dilution), anti-CD3 antibody (Abcam, catalog #ab16669, 1:1000 dilution), anti-CD4 antibody (Abcam, catalog # ab183685, 1:1000 dilution), anti-CD8 antibody (Abcam, catalog #ab217344, 1:2000 dilution), and anti-CD56 antibody (catalog #MA5-11563, Invitrogen, Waltham, MA, USA, 1:250 dilution) which were diluted in 2% goat serum at 4 °C. Slides with tissue sections were then incubated with goat anti-mouse IgG (1:200 dilution, #115-035-003; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 1-hour at room temperature. Vectastain ABC kit with peroxidase (PK-400; Vector Laboratories, Newark, CA, USA) was used for detection and slides were counterstained with hematoxylin. Negative controls were obtained by omitting primary antibody. H-scores were assigned according to percentage of cells at each staining intensity (0 to 3+) according to the equation: H-score = (0 x % cells staining 0) + (1 x % cells staining 1+) + (2 x % cells staining 2+) + (3 x % cells staining 3+). H-scores were independently assessed by two laboratory members blinded to treatment group and the results were averaged. GraphPad Prism 9.4.0 software (GraphPad Software, San Diego, CA, USA) was used for statistical analyses. Quantitative data was tested for normality using the Shapiro-Wilk test. Student’s t -test was used for normally distributed data and the Mann-Whitney U test was used for non-parametric data. A p value of < 0.05 was considered statistically significant.

Our study is the first to demonstrate that systemic L-ICON3 treatment reduced both the macroscopic and microscopic size of endometriotic lesions, suppressed TF expression and increased immune cell infiltration in endometriosis. Collectively, our results support the potential of L-ICON3 as a promising therapeutic strategy for endometriosis, warranting further study.

In this study, we demonstrate that treatment with L-ICON3 significantly reduces the area and thickness of endometriosis lesions when compared to controls. Prior studies with ICON and ICON1 have demonstrated a similar effect, whereby a reduction in the size of macroscopic endometriotic implants was noted in mice and baboons [ 8 , 19 , 21 ]. This is the first study to demonstrate that treatment with L-ICON3 not only reduces macroscopic endometriosis lesion size but also reduces the thickness of endometriosis lesions on a microscopic level. This is also the first study to demonstrate that treatment with any ICON reduces the expression of TF in endometriotic lesions compared to controls. Although L-ICON3 is known to bind TF, the effect of this treatment on TF expression in endometriotic lesions has not been evaluated in prior studies. Our findings suggest that once treatment with L-ICON3 is administered, the ongoing expression of TF is decreased. Furthermore, our study demonstrates a significant increase in CD3, CD4, CD8, and CD56 positive immune cells in mice treated with L-ICON3 versus those treated with IgG isotype control. This finding is consistent with the study by Scheerer et al. [ 26 ], which characterized the infiltration of several immune cell types into endometriotic lesions. Therefore, reduced lesion size and endometriosis wall thickness is associated with altered immune cell infiltration into the lesions. The increased numbers of T cells in ectopic and eutopic endometrial tissue in patients with endometriosis versus those without suggests a level of chronic inflammation [ 26 ]. Specifically, increased CD3, CD4, and CD8 cells have been seen in peritoneal and ectopic endometrial implants [ 26 ]. Interestingly, NK cells are absent or present in very low quantities in endometriosis lesions [ 26 ]. The increase in NK cells in animals treated with L-ICON3 versus IgG is most likely due to L-ICON3 binding to TF and activating a NK chemotactic and cytolytic cellular response resulting in the destruction of endometriosis cells [ 16 , 18 , 22 ]. In our study, the comparable levels of TNF-α and IL-6 suggest no overall change in inflammation; rather there is likely a specific destruction of endometriosis cells. In prior studies, ICON has been shown to specifically target pathological TF expression as opposed to physiological TF expression in normal vessels. This is partly because endothelial cells do not express TF unless there is disruption of the normal vasculature [ 16 ]. This was further highlighted in the studies by Krikun et al. where aberrant TF expression was seen in ectopic endometrium endothelium, but not within the normal intrauterine endometrial tissue [ 8 , 21 ]. Hu et al. (1999) demonstrate how earlier versions of the ICON molecules are designed to bind TF localized specifically to tumor vasculature (specifically melanoma cells). To decrease the risk of disseminated intravascular coagulation or coagulopathy arising from binding TF, a mutation was introduced into the targeting domain of the fVII which inhibits the proteolytic activity that triggers the coagulation cascade [ 16 ]. Notably, no coagulation dysfunction was observed in either our current study or prior studies utilizing the ICON molecule. In keeping with other studies utilizing ICON, there were also no adverse effects noted in our study [ 8 , 16 , 18 , 19 , 21 , 22 ]. Limitations of our study include the use of an animal model of endometriosis rather than human endometriosis samples as well as the limited sample size. Furthermore, although immunohistochemistry staining demonstrated reduced TF expression and increased T and NK cell recruitment in mice treated with L-ICON3, direct causality of each of these molecular and cellular changes in reducing endometriosis have not been definitively established here.

To make recombinant L-ICON3 protein, an expression plasmid vector pcDNA3.1(+) encoding human L-ICON3 cDNA was transfected transiently into suspension Expi293F mammalian expression system using Expi293F Transfection Kit following the manufacturer’s instructions (Thermo Fisher, Waltham, MA, USA). The supernatant of serum free Expi293F cell culture supplemented with 5 µg/ml vitamin K1 (Sigma Aldrich, St. Louis, MO, USA) was harvested on day 4 or 5 days post transient transfection when the cell viability was above 50%. Recombinant L-ICON3 protein was purified from the supernatants of SFM by a 5 ml HiTrap rProtein G FF affinity column (Cytiva, Marlborough, MA) on BioLogic LP chromatography system (Bio-Rad Laboratories, Hercules, CA), as similarly described by Hu et al. [ 22 ]. The concentration of L-ICON3 protein was determined by Protein Assay Reagent (Bio-Rad Laboratories) using bovine serum albumin standards (Pierce, Rockford, IL, USA).

Endometriosis is a chronic, systemic inflammatory disease affecting 5–10% of reproductive-aged women [ 1 – 3 ]. The clinical manifestations include dysmenorrhea, non-menstrual pelvic pain, and infertility, with a significant impact on the physical and mental well-being of patients [ 1 ]. It is defined as the presence of endometrial-like tissue outside the uterus [ 3 ]. Different phenotypes of endometriosis have been proposed, including superficial peritoneal endometriosis, deep infiltrating endometriosis, and ovarian endometriomas [ 4 ]. On a molecular level, endometriosis is characterized by local inflammation, tissue injury fibrosis, angiogenesis, and neurogenesis [ 4 ]. Current medical treatments include analgesia, hormonal contraceptives for disease suppression, and gonadotropin-releasing hormone analogues (GnRHa). Surgical excision of endometriosis can also be performed to remove lesions and restore normal anatomy, which can both improve symptoms of pelvic pain and chances of successful pregnancy, however, formation of adhesions and disease recurrence is common. Hormonal treatments, including contraceptives and GnRHa, disrupt gonadotropin production, have significant side effects and are not effective treatment options for patients desiring pregnancy [ 4 ]. Currently, there are no non-hormonal treatment options or therapies targeting the immune alterations seen in this disease. TF also known as coagulation factor III, tissue thromboplastin, or CD142 is a cell-membrane-bound glycoprotein and a member of the class II cytokine receptor family [ 5 , 6 ]. TF is expressed throughout the body on both mesenchymal and epithelial cells. Following vascular disruption of endothelial cells, perivascular bound tissue factor binds to circulating Factor VIIa and results in the generation of thrombin and propagation of the coagulation cascade [ 7 , 8 ]. Tissue factor can also act as a signaling molecule whereby upon binding to fVIIa, protease-activated receptor (PAR-2) is cleaved, resulting in downstream inflammation, tumor progression, and angiogenesis [ 8 – 10 ]. Activation of TF through the PAR-2 signaling can lead to cytokine production, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukins, and angiogenic growth factors such as vascular endothelial growth factor (VEGF) [ 11 ]. Thus, TF remains crucial in both physiological and aberrant coagulation, pathological angiogenesis, and inflammation. Interestingly, tissue factor-null embryos display disruption of the yolk sac vasculature and die at embryonic day 10.5, highlighting the importance of tissue factor in early angiogenesis [ 8 , 12 ]. The role of TF in the endometrium has not yet been fully elucidated. In normal endometrial tissue, tissue factor expression varies throughout the menstrual cycle; during the luteal phase, progesterone significantly increases TF expression in decidualized stromal cells. However, there is minimal TF expression in glandular epithelial cells throughout the menstrual cycle [ 13 , 14 ]. This suggests that there is an intrinsic cyclicity to TF expression and perhaps an underlying poorly understood signaling/angiogenic role that is physiological to menstruation. Previous studies comparing both eutopic and ectopic endometrial tissue from women with endometriosis show marked elevation of TF expression in the glandular epithelial cells compared to those without the disease [ 8 ]. Krikun et al. (2008) demonstrated increased TF signaling receptor PAR-2 activity in endometriotic lesions compared to control lesions, thus highlighting the possible signaling role of TF in aberrant inflammation and angiogenesis that is seen with ectopic endometriosis lesions [ 8 ]. Lin et al. (2012) also demonstrated abnormal upregulation of TF and PAR-2 in eutopic and ectopic endometrium in women with endometriosis [ 15 ]. They compared TF and PAR-2 mRNA expression in ectopic and eutopic endometrial tissue in women with and without endometriosis and found increased expression of TF in ectopic and eutopic endometrium in patients with endometriosis throughout the menstrual cycle compared to controls. PAR-2 expression was increased in the ectopic endometrium throughout the menstrual cycle in women with endometriosis compared to those without. However, in eutopic endometrium, PAR-2 expression was increased only during the secretory phase compared to controls [ 15 ]. This alludes to a degree of aberrant TF and PAR-2 expression/signaling in ectopic endometrium compared to eutopic endometrium in patients with endometriosis. ICON, the first ligand-based TF-targeting antibody-like immunoconjugate molecule, is composed of coagulation active site mutated full length factor VII (K341A), the natural ligand for TF, and fused to human IgG1Fc [ 16 – 18 ]. It has been investigated as a novel antibody immunotherapy for several unmet human diseases, notably cancer [ 18 ], endometriosis [ 19 ], and age-related macular degeneration (AMD) [ 20 ], in preclinical animal models and in early phase clinical trials, in patients with AMD or uveal melanoma, where tissue factor is implicated. Compared with anti-TF antibody, the ICON molecule binds to tissue factor with higher affinity. In endometriosis, ICON binds TF expressed on endometriotic neovasculature and has been shown to reduce endometriosis in mice without any adverse effects, including thrombosis, teratogenicity or reduced fertility [ 21 ]. Krikun et al. (2010) implanted human endometrial tissue into a murine model of endometriosis and treated the mice with ICON followed by assessment of endometriosis and fertility [ 21 ]. They found that ICON treatment significantly decreased endometriosis lesions and vascularization compared with controls. Mice treated with ICON demonstrated normal fertility compared with controls. The live birth rate was 8.25 per litter for controls and 8.5 per litter for ICON treatment, with normal progression of all pregnancies and normal heart, lung, brain, liver, kidney, spleen, bone, muscle, and gastrointestinal tract development on histopathological analysis [ 21 ]. In non-human primates, treatment with ICON showed a significant reduction of red endometriosis lesions. Hufnagel et al. (2018) surgically induced endometriosis in baboons followed by intraperitoneal injection of ICON [ 19 ]. The volume of red, blue and white endometriosis lesions was compared pre- and post-treatment in both the ICON and control groups in a blinded fashion. In the animals treated with ICON, there was a significant reduction in red endometriotic lesions, however, there were no changes seen in the number of blue or white lesions, or dense/filmy adhesions [ 19 ]. The ICON molecule (also called ICON-1 for the first generation ICON) has been improved by replacing the full length fVII with the first 152 amino acid residue (light chain) of mature fVII peptide and/or replacing IgG1Fc with human IgG3Fc, resulting in the development of second and third generation versions, called L-ICON1 [ 22 ] and L-ICON3 (for light chain ICONs), respectively. While L-ICON1 (GenBank accession no. KX760097 ) is a fusion of human fVII light chain to an IgG1 Fc, L-ICON3 is a fusion of hfVIIL to an engineered human IgG3Fc (GenBank accession no. KY223609 ). L-ICON1 has been shown to treat triple-negative breast cancer cells in mouse models [ 22 ]. Most importantly, ICON1 has been tested in phase 1 trials in humans and shown to be safe and effective. Wells et al. (2018) injected ICON-1 into the intra-vitreous space of 18 patients with neovascular age-related macular degeneration. ICON1 was safe and well-tolerated at all doses, and no adverse events, including coagulation defects, were noted. Higher doses of treatment (300 µg) appeared to produce the most beneficial biological outcomes in terms of decreased central retinal thickness and choroidal neo-vascularization [ 23 ]. Given the safety and efficacy of ICON and its potential to treatment endometriosis, this study evaluates the effect of L-ICON3 on macroscopic and microscopic lesions and TF expression in a murine model of endometriosis. We also aimed to assess the possible mechanisms of action of how L-ICON3 may affect endometriosis by performing immunohistochemistry staining of various molecules implicated in the inflammatory processes associated with endometriosis.