DESC:Bispecific Agonists (BAs) toward IL-7R and EGFR promotes healing of the wounds
FIELD OF THE INVENTION
The present invention relates to the field treatment of wounds and associated infections, and more particularly, to the treatment of acute or chronic wounds as well as immune suppressive conditions in patients having with or without comorbidities (diabetes, transplant patients, major surgeries, repeat infections, etc.). The present invention pertains specifically to Bispecific Agonists (BAs) targeting towards IL-7R and EGFR. The Bispecific Agonists (BAs) have been demonstrated to restore immune homeostasis, promote the proliferation, secretion, or migration of various wound healing factors and tissues. As a result, wound healing can be significantly achieved at a faster rate. Further, the Bispecific agonists (BAs) offer promising avenues for combating bacterial, fungal, or viral infections, particularly in cases where untreated or treatment resistant infections could escalate to life-threatening conditions like sepsis or necessitate organ amputations. Bispecific Agonists ((BAs) poss unique biochemical and functional properties that enable them to engage with multiple targets simultaneously, potentially enhances the immune response against pathogens by acting on gamma delta T cells, T cells, and innate immune cells like neutrophils, macrophages, etc., at the site of wound occurrence. Moreover, these agonists stimulate the proliferation of fibroblasts, keratinocytes etc., which aids in covering the wounded area and re-establishing the compromised skin barrier. Hence, Bispecific Agonists (BAs) plays a crucial role in mitigating the severity and spread of such infections, offering new hope in the battle against infectious diseases.
BACKGROUND OF THE INVENTION
Patients who are immunocompromised pose a unique and formidable challenge in wound care and healing. These patients may include children, older adults, organ transplant recipients, cancer patients, individuals with diabetes mellitus, burns, or HIV/AIDS. Immunocompromised patients are at increased risk of hypothermia, infection, and the development of poorly healing or recurring wounds. Many treatments for infection and interventions aimed at promoting wound healing depend on a robust immune system. However, in immunocompromised patients, alternative treatment approaches are necessary to compensate for their compromised immune responses.
As on 2021, over 422 million individuals worldwide are affected by diabetes, with 20% to 34% (85-126 million) of them experiencing diabetic wounds at least once. Approximately 50% to 70% (40-88 million) of these wounds progress to limb amputations.
Wound pathophysiology encompasses vascular, neuropathic, nephrotic, immune, and biochemical factors. Impaired wound healing arises from decreased production of growth factors and associated molecules such as cytokines and chemokines. This leads to diminished production and repair of new blood vessels, weakening of the skin barrier, and reduced collagen production. Moreover, dysfunction of NK cells, monocytes, macrophages, impaired neutrophil migration, and compromised T cell functionality and recruitment to the wound site contribute to the persistence of infected, unhealed wounds.
In diabetic foot infection patients, 38% to 48% exhibit immune suppression, with 5% to 18% experiencing microbiological recurrences.
The European Patent No. EP3111946B1 discloses compositions comprising platelet enriched plasma (PRP) for wound healing. The platelet enriched plasma (PRP) comprises transforming growth factor-ß (TGF-ß), fibrinogen, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor-a (TGF-a), vascular endothelial growth factor (VEGF), platelet thrombo-plastin, thrombospondin, coagulation factors, calcium, serotonin, histamine, and hydrolytic enzymes.
The PCT application No. PCT/US2021/030681 discloses compositions that are useful for inducing an immune response, treating an inflammatory response, treating a microbial infection, differentiating cells, wound healing, embryonic development, placental development, central nervous system development, or morphogenesis.
The US Patent No. US11376231B2 discloses compositions with fulvate fractions for the catalyzing cellular regeneration, including the healing, treatment, or prevention of skin disorders.
The US Publication No. 20210106651 discloses bio-mimetic formulations can comprise a carrier and recombinant peptides including basic fibroblast growth factor (bFGF) for wound healing.
The US Patent No. US8247384B2 discloses composition comprising a therapeutically effective amount of an anti-connexin 43 agent and a second wound healing composition comprising a therapeutically effective amount of a protein or peptide effective in promoting or improving wound healing.
Many prior arts offer compositions for wound healing treatment. However, none of these disclose the treatment of acute or chronic wounds, along with immune suppressive conditions, using molecular composition where the bi- or multi-functional activities of various proteins are expressed as a single polypeptide molecule while retaining full functionality.
Therefore, the present invention provides Bispecific Agonists (BAs) possessing agonistic properties of cytokines and growth factors. Active engagement of the subject’s immune system is crucial for wound healing, particularly in preventing severe measures like limb amputation, especially in immunocompromised patients, and averting life-threatening sepsis conditions.
SUMMARY OF THE INVENTION
The present invention is related to the field of treatment of wounds. More specifically, it pertains to the treatment of acute or chronic wounds as well as immune suppressive conditions in patients with Bispecific Agonists (BAs) having agonistic properties of cytokine and a growth factor.
In one aspect of the present invention provides Bispecific Agonist (BA) proteins including a cytokine and a growth factor to restore the immune homeostasis, proliferates, secretes or migrates various wound healing factors such that healing can be achieved at a significant rate.
In one aspect of the present invention, the BA proteins with a cytokine and a growth factor, consisting of immune cell modulator protein such as a cytokine (e.g. IL-2, IL-7, IL-15) linked with a growth factor (e.g. EGF, PDGF, VEGF, FGF) for the treatment of chronic wounds in immune compromised patients.
In one aspect of the present invention provides is related to the restoration of immune functionality by IL-7 and growth factors lead to accelerated recoveries in acute and chronic wounds Viz., surgical, burn, diabetic, accidental or antibiotic resistant conditions avoiding development of sepsis or gangrene.
In one aspect of the present invention, the Bispecific Agonists (BAs) targets IL-7R and EGFR.
In one aspect of the present invention provides a pharmaceutical composition for treatment of wounds, comprising: at least one Bispecific Agonist (BA) protein consisting of an immune cell modulator including a cytokine and a growth factor, the Bispecific Agonist (BA) protein comprising a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.
In one aspect of the present invention relates to correction of immune dysfunctionality aided by inclusion of growth factors as a fusion protein, as a bi or multi-functional molecule leading to accelerated wound healing. Bispecific Agonist (BA) proteins may be administered either topically, orally or by injectable routes.
In one aspect of the present invention relates to generation of single polypeptide chain having functionality of a cytokine and growth factor.
Further details and specification of the invention are described in the detailed description and accompanying figures, providing comprehensive insights into the operation and functionalities of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The above-mentioned and other features and advantages of this present disclosure, and the manner of attaining them, will become more apparent and the present disclosure will be better understood by reference to the following description of embodiments of the present disclosure taken in conjunction with the accompanying drawings, wherein:
FIG.1 shows a SDS PAGE gel image of Bispecific Agonist protein 1 (BA 1) cell culture supernatant loaded under reducing and non-reducing conditions;
FIG.2 shows a SDS PAGE gel image of Bispecific Agonist protein 2 (BA 2) cell culture supernatant loaded under reducing and non-reducing conditions;
FIG.3 shows a SDS PAGE gel image of Bispecific Agonist protein 3 (BA 3) cell culture supernatant loaded under reducing and non-reducing conditions;
FIG.4 shows a SDS PAGE gel image of Bispecific Agonist protein 4 (BA 4) cell culture supernatant loaded under reducing and non-reducing conditions;
FIG.5 shows a SDS PAGE gel image of purified Bispecific Agonist protein 3 (BA 3) obtained through the use of cation exchange columns. The samples are loaded under both reducing and non-reducing conditions, allowing for the assessment of protein purity. The SDS PAGE gel analysis indicates the purity of BA 3 is >95 %;
FIG.6 depicts RP HPLC analysis of BA 3 protein having a purity of > 87%;
FIG.7 depicts a western blot performed using Anti-EGF antibody;
FIG.8 depicts a western blot performed using Anti-IL-7 antibody;
FIG.9 depicts variants of BA have phosphorylated STAT5 in h PBMC CD45CD3+ and in CD45CD3- cells in a dose dependent manner where potency is determined as EC50;
FIG.10 depicts variants of BA have phosphorylated ERK1/2 in A431cells, epidermoid cancer cells in a dose dependent manner where potency is determined as EC50;
FIG.11 depicts the proliferation of Balb 3T3 fibroblast cells of mouse proliferating in a dose dependent manner as determined by MTT assay;
FIG.12 depicts Human fibroblasts post confluence was inflicted scratch wound and treated with various concentrations of BA and observed for scratch wound area closure from 0 h, 24 h and 48 h. The arrow head indicates the scratch wound area;
FIG.13 depicts significant increase in the migration of human fibroblast cells post treatment with BA 3 at different concentrations. Fibroblasts migrated to the scratch wounded area in a dose dependent manner where higher the dose greater the migration;
FIG.14 depicts the release kinetics of BA 3 encapsulated in Gelma Hydrogel and incubated with human plasma at 37 0 C. The quantification of released BA 3 protein is determined by Sandwich ELISA;
FIG.15 depicts calibration curve to quantify photo oxidation reaction where NADH converts to NAD upon exposure to UV light;
FIG.16 depicts Photo-oxidation of NADH upon interacting with out or with Gelma hydrogel loaded with bispecific Protein. The decrease in fluorescence intensity upon interaction with Gelma is insignificant with respective without Gelma interaction, indicating the concentration residual LAP in the Gelma does not cause photocatalytic reaction;
FIG.17 depicts comparison of LAP used in Glema (exposed to UV-VIS light) vs LAP not interacted with Gelma (not exposed to UV-VIS light) where there is significant decrease in the fluorescence in Gelma Interacted LAP;
FIG.18 depicts Spleen wet weight of the Rats post 72h of dose administration was measured and compared with control treatment. Statistical analysis shows a P value of 0.05 between control treated vs 0.25 mg/kg in terms of increase in spleen weight indicating the BA have stimulated immune cell proliferation;
FIG.19 depicts calibration curve generated by spiking known concentrations of BA in Rat plasma. Analysis done by 4PL sigmoidal curve where r2 value is 0.996;
FIG.20 depicts Plasma concentrations are estimated by Sandwich ELISA in the Rat plasma in ng/mL post administration of BA in Gelma hydrogel;
FIG.21 shows percent body weight change in Diabetic Rats treated with BA;
FIG.22 shows percent wound healing Diabetic Rats treated with BA;
FIG.23 shows increase in peripheral leukocyte and neutrophil populations in response to the BA treatment over the untreated controls; and
FIG.24 shows effect of BA administered intra-dermally in Gelma Hydrogel formulation, in which the leukocytes were depleted and excision wounds were inflicted , followed by infection (using faecal matter of the same mice) or non-infected in C57BL/6 mice. The administration of BA resulted in significant decrease in the wounded area in comparison to their respective controls.

DETAILED DESCRIPTION OF THE INVENTION
Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the invention that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the invention is intended to be illustrative, and not restrictive. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.
The present invention provides a pharmaceutical composition for treatment of both acute and chronic wounds as well as immune suppressive conditions in patients. The pharmaceutical composition includes Bispecific Agonist (BA) proteins aim to restore the immune homeostasis and, when necessary, promote the proliferation, secretion, or migration of various wound healing factors, leading to clinically significant healing outcomes.
Growth factors and immune modulators are currently utilized as complex mixtures derived from the physiology of cells or plasma factors enriched with growth factors and cytokines. However, these methods often necessitate the involvement of skilled personnel or hospitalizations. In contrast, the proposed invention provides pharmaceutical composition solution that can be administered through an over-the-cell (OTC) route, eliminating the need for specialized expertise or hospital visits.
In one embodiment of the present invention, the Bispecific Agonist (BA) proteins including a cytokine and a growth factor to restore the immune homeostasis, proliferates, secretes or migrates various wound healing factors.
The present invention provides Bispecific Agonist (BA) proteins for correcting immune dysfunctionality by incorporating cytokines or proliferating, migrating growth factors into a single polypeptide chain proteins, creating bi- or multi-functional molecules that accelerate wound healing. Bispecific Agonist (BA) proteins may be administered either topically, orally or by injectable routes. Till date there are no such molecules (bi functional or multi-functional) existing for the treatment of wounds (acute or chronic in nature) where compromised immune functionality is treated simultaneously with growth factors resulting in accelerated healing, avoiding sepsis and amputations.
Additionally, there is no existing molecule where the bi- or multi-functional activities of various proteins are expressed as a single polypeptide molecule while retaining full functionality. This is evident from the comparative potencies determined in various assay systems.
Transient Expression of Bispecific Agonist proteins using CHO-S Cells:
In one embodiment of the present invention, typically four variants of Bispecific Agonist proteins successfully expressed in CHO-S cells transiently as well as stably. The cell culture supernatants harvested on day 7 and day 9 are subjected to SDS-PAGE analysis, followed by Western Blot analysis. This approach allowed for the visualization and quantification of the expressed proteins, verifying their presence in the cell culture supernatants and providing insights into their molecular weight and purity. By evaluating expression levels and protein integrity at different time points, researchers have identified Bispecific Agonist proteins for various applications.
A total of 40.0 µL of culture supernatants from all four variants of Bispecific Agonist proteins collected on day 7 and day 9 are loaded onto a 4% to 20% gradient SDS-PAGE gel. The reduced sample buffer contains 250 mM DTT.
As shown in FIG.1 is a SDS PAGE gel image of Bispecific Agonist protein 1 (BA 1) cell culture supernatant loaded under reducing and non-reducing conditions. Where,
Lane M: Protein Marker,
Lane P: SDS PAGE Positive Control,
Lane 1: Cell culture supernatant from day 7 post-transfection under non-reducing conditions,
Lane 2: Cell culture supernatant from day 7 post-transfection under reducing conditions,
Lane 3: Cell culture supernatant from day 9 post-transfection under non-reducing conditions,
Lane 4: Cell culture supernatant from day 9 post-transfection under reducing conditions,
Lane 5: Cell culture supernatant from Negative Control post-transfection under non-reducing conditions, and
Lane 6: Cell culture supernatant from Negative Control post-transfection under reducing conditions.
The arrow depicted in FIG.1 indicates the presence of the protein of interest in the harvested samples. The negative control does not exhibit bands corresponding to the protein of interest. The theoretical molecular weight of Bispecific Agonist protein 1 (BA 1) based on primary sequence is 24852.33 Daltons.
The nucleotide sequence corresponding to the Bispecific Agonist protein 1 (BA 1) is a sequence having 95% or more of a sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2 or both, as presented in Table 12.
Further, amino acid sequence corresponding to the Bispecific Agonist protein 1 (BA 1) is a sequence having 95% or more of a sequence identity with SEQ ID NO: 9 as presented in Table 12.
As shown in FIG.2 is a SDS PAGE gel image of Bispecific Agonist protein 2 (BA 2) cell culture supernatant loaded under reducing and non-reducing conditions. Where,
Lane M: Protein Marker,
Lane P: SDS-PAGE Positive Control,
Lane 1: Cell culture supernatant from Negative Control post-transfection under non-reducing conditions,
Lane 2: Cell culture supernatant from Negative Control post-transfection under reducing conditions,
Lane 3: Cell culture supernatant from day 7 post-transfection under non-reducing conditions,
Lane 4: Cell culture supernatant from day 7 post-transfection under reducing conditions,
Lane 5: Cell culture supernatant from day 9 post-transfection under non-reducing conditions, and
Lane 6: Cell culture supernatant from day 9 post-transfection under reducing conditions.
The arrow depicted in FIG.2 indicates the presence of the protein of interest in the harvested samples. The negative control does not exhibit bands corresponding to the protein of interest. The theoretical molecular weight of Bispecific Agonist protein 2 (BA 2) based on primary sequence is 25615.45 Daltons.
The nucleotide sequence corresponding to the Bispecific Agonist protein 2 (BA 2 is a sequence having 98% or more of a sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4 or both, as presented in Table 12.
Further, amino acid sequence corresponding to the Bispecific Agonist protein 2 ((BA 2) is a sequence having 95% or more of a sequence identity with SEQ ID NO: 10 as presented in Table 12.
As shown in FIG.3 is a SDS PAGE gel image of Bispecific Agonist protein 3 (BA 3) cell culture supernatant loaded under reducing and non-reducing conditions. Where,
Lane M: Protein Marker,
Lane P: SDS-PAGE Positive Control,
Lane 1: Cell culture supernatant from day 7 post-transfection under non-reducing conditions,
Lane 2: Cell culture supernatant from day 7 post-transfection under reducing conditions,
Lane 3: Cell culture supernatant from day 9 post-transfection under non-reducing conditions,
Lane 4: Cell culture supernatant from day 9 post-transfection under reducing conditions,
Lane 5: Cell culture supernatant from Negative Control post-transfection under non-reducing conditions, and
Lane 6: Cell culture supernatant from Negative Control post-transfection under reducing conditions.
The arrow depicted in FIG.3 indicates the presence of the protein of interest in the harvested samples. The negative control does not exhibit bands corresponding to the protein of interest. The theoretical molecular weight of Bispecific Agonist protein 3 (BA 3) based on primary sequence is 26450.51 Daltons.
The nucleotide sequence corresponding to the Bispecific Agonist protein 3 (BA 3) is a sequence having 95% or more of a sequence identity with SEQ ID NO: 5 or SEQ ID NO: 6 or both, as presented in Table 12.
Further, amino acid sequence corresponding to the Bispecific Agonist protein 3 (BA 3) is a sequence having 95% or more of a sequence identity with SEQ ID NO: 11, as presented in Table 12.
As shown in FIG.4 is a SDS PAGE gel image of Bispecific Agonist protein 4 cell culture supernatant loaded under reducing and non-reducing conditions. Where,
Lane M: Protein Marker,
Lane P: SDS-PAGE Positive Control,
Lane 1: Cell culture supernatant from Negative Control post-transfection under non-reducing conditions,
Lane 2: Cell culture supernatant from Negative Control post-transfection under reducing conditions.
Lane 3: Cell culture supernatant from day 7 post-transfection under non-reducing conditions,
Lane 4: Cell culture supernatant from day 7 post-transfection under reducing conditions,
Lane 5: Cell culture supernatant from day 9 post-transfection under non-reducing conditions, and
Lane 6: Cell culture supernatant from day 9 post-transfection under reducing conditions.
The arrow depicted in FIG.4 indicates the presence of the protein of interest in the harvested samples. The negative control does not exhibit bands corresponding to the protein of interest. The theoretical molecular weight of Bispecific Agonist protein 4 (BA 4) based on primary sequence is 27881.98 Daltons.
The nucleotide sequence corresponding to the Bispecific Agonist protein (BA 4) is a sequence having 95% or more of a sequence identity with SEQ ID NO: 7 or SEQ ID NO: 8 or both, as presented in Table 12.
Further, amino acid sequence corresponding to the Bispecific Agonist protein 4 (BA 4) is a sequence having 95% or more of a sequence identity with SEQ ID NO: 12, as presented in Table 12.
Purification of Bispecific Agonist protein 3 using Cation exchange Spin Columns:
In another embodiment of the present invention, the cell culture supernatants are purified using Cation exchange spin columns. Acetate buffer at pH 5.6 was used to purify the proteins. The proteins were eluted at a Sodium chloride concentration of 300mM, 50 mM Sodium acetate pH 5.6 buffer. The storage temperature of purified protein is +2-8°celsius - 80°celsius. The purified proteins are loaded on SDS polyacrylamide gel to evaluate the integrity of proteins with respect to expected molecular weights (based on primary sequence).
In another embodiment of the present invention, the cell culture supernatants are purified using cation exchange spin columns. Then, acetate buffer at pH 5.6 is utilized for protein purification. The proteins are eluted at a sodium chloride concentration of 300 mM in a buffer containing 50 mM sodium acetate at pH 5.6 and obtained the purified protein. The storage temperature for purified proteins is maintained at +2-8°C - 80°C. Subsequently, the purified proteins are loaded onto SDS polyacrylamide gel to evaluate the integrity of proteins concerning the expected molecular weights based on the primary sequence.
FIG.5 depicts SDS PAGE gel image of purified Bispecific Agonist protein 3 (BA 3) using cation exchange column. Samples are loaded under reducing and non-reducing conditions. The SDS PAGE gel indicates purity of BA 3 is >95 %. Where,
M1: Protein marker,
R: Bispecific Agonist protein 3 in reducing conditions, and
NR: Bispecific Agonist protein 3 in non-reducing conditions.
In addition, the BA 3 is also analyzed by RP HPLC and found to have purity levels of 83 % as shown in FIG 6. FIG 6 depicts RP HPLC analysis of BA 3 protein having a purity of > 87%.
Determination of the identity of the expressed BA molecules in CHO cells by specific antigen-antibody binding (by Western blotting):
In another approach, the purification of cell culture supernatants is achieved through cation exchange columns, followed by loading the purified samples onto an SDS-PAGE gel. Subsequently, the protein bands are transferred onto a nitrocellulose membrane, which is then probed with primary antibodies, specifically Anti-EGF antibody and Anti-IL-7 antibody. Secondary antibodies conjugated with HRP, designed to bind to the respective primary antibodies, are utilized, and the resulting blot is developed using TMB substrate.
The target protein band in the purified protein sample are detected by Western blotting shown in the FIG.7 and FIG.8.
FIG.7 depicts a western blot performed using Anti-EGF antibody. Where,
Lane 1 and Lane 2: Product# 26616, Page ruler pre-stained protein ladder, Thermo Scientific,
Lane 3: Bispecific Agonist protein 1,
Lane 4: Bispecific Agonist protein 2,
Lane 5: Bispecific Agonist protein 3, and
Lane 6: Bispecific Agonist protein 4.
FIG.8 depicts western blot performed using Anti-IL-7 antibody. Where,
Lane 1 and Lane 2: Product# 26616, Page ruler pre-stained protein ladder, Thermo Scientific,
Lane 3: Bispecific Agonist protein 1,
Lane 4: Bispecific Agonist protein 2,
Lane 5: Bispecific Agonist protein 3, and
Lane 6: Bispecific Agonist protein 4.
Human PBMC Assays for activation of Lymphocytes:
In another embodiment of the present invention, partially purified variants of Bispecific Agonist proteins undergo testing for their ability to stimulate human PBMCs through pSTAT5 analysis.
Briefly, human PBMCs are subjected to multi-color flow-cytometry, wherein cells are labelled with specific antibodies to distinguish different cell populations, including CD45, CD3, and pSTAT5. The labelled cells are then analyzed using multi-color flow-cytometry to identify CD45+ cells, CD3+/- cells, and to determine the percentage of STAT5 phosphorylation among these populations. The phosphorylation of STAT5 in leukocytes serves as one of the primary mechanisms by which immune cells are activated for proliferation or TCR activations resulting in improved or restored immune functionality.
The results indicate that all the BA variants induce phosphorylation of STAT5 in both CD45+CD3+ and CD45+CD3- T lymphocytes. These findings demonstrate the functionality of all variants compared to their respective controls, as depicted in FIG. 9 and Table 1.

Test Compound Positive control BA 1 BA 2 BA 3 BA 4
EC50 in ng/mL
(pSTAT5 in CD45+ T lymphocytes) 0.064 0.073 0.043 0.021 0.080

Table 1: Potency of BA protein variants in phosphorylating STAT5 in hPBMC lymphocytes.
In another embodiment of the present invention, A431 cells (human epidermoid carcinoma cells) are utilized to assess the activation of ERK protein phosphorylation, which signifies the activation of signalling pathways crucial for cell proliferation, differentiation, and migration.
In brief, A431 cells obtained from ATCC are cultured, ensuring they do not exceed passage 10. These cells are then starved of serum and glucose while being stimulated for 15 minutes with various concentrations of BA variants. Following the incubation period, the reaction is halted by washing and staining the cells with anti-phosphorylated ERK antibodies to determine their potencies. Subsequently, the cells are analyzed using a flow-cytometer, incorporating appropriate controls to determine the percentage of pERK in response to varying doses, as depicted in FIG.10 and Table 2.
Test Article BA 1 BA 2 BA 3 BA 4
EC50 ng/mL
(pERK1/2 in A431 cells) 12.72 3.991 0.9861 1.081
Table 2: Potency of BA protein variants in phosphorylating ERK in epidermoid carcinoma cells, A431.

Efficacy of BA protein variants in 2-Dimentional (2D) Assays:
1. Proliferation of fibroblasts by MTT assay:
Further, in another embodiment of the present invention, the efficacy of BA 3 to proliferate the fibroblasts (mouse Balb/3T3 cells) has been evaluated by MTT assays. On the day of the experiment 3T3 cells not exceeding passage 10 are seeded to get 2000 cells/well/100 µL into a sterile 96 well flat bottom tissue culture plate. Cells are cultured at 37°C with 5% CO2 for 24 hours prior to addition of various concentrations of Bispecific Agonist single chain protein or epidermal growth factor (hEGF) alone, from 1000, 100, 10, 1, 0.1, 0.01, 0.001 & 0.0001 ng/mL in a DMEM culture medium in triplicates. Cells were cultured for 96 hours and analyzed by MTT viability assay to determine the metabolically active number of cells. MTT labeling reagent was added to the wells and incubated for 4 hours at 37°C in a humidified atmosphere at 37°C. Added 100 µL/well of Solubilization buffer and incubate overnight in a humidified atmosphere at 37°C. Measured the absorbance of the samples in the microplate using an ELISA reader at 575 nm wavelength. The proliferation potential of BA is compared with that of EGF alone in terms of EC50 as shown in the FIG.11 and Table 3.
S.No. Compounds EC50 (ng/mL) by MTT
1 BA 3 4.452
2 rhEGF 6.393

Table 3. Effect of BA 3 proliferation potential in comparison to that of EGF in mouse fibroblast cells.
The potency of the BA is very much comparable to that of the EGF alone treatment as shown in the Table 3. We are able to maintain the activity of EGF even in the single chain protein molecule having multiple functions.
2. Migration of human fibroblasts in to the wound area as determined by scratch wound assay:
Further, in another embodiment of the present invention, 2.5X 104 human dermal fibroblast cells (Passage No. 10) seeded in each well of 12-well TC- treated plate and incubated overnight in 5% CO2 incubator at 37°C. Post attaining confluence, cells were washed with sterile DPBS and added serum starve media (EMEM+0.1% BSA). Scratched the cell monolayer in a straight line using a 200 µl plastic pipette tip in each well. Post scratch a thorough wash of the cell monolayer with DPBS was done to remove detached cells and add 500µL of serum starve media with varying concentrations (0ng/ml, 1ng/mL, 10ng/mL, 100ng/mL and 500ng/mL) of bispecific protein. Microscopic images of the scratched area captured at 0h, 24h and 48h with 4X objective magnification shown in the FIG.12. Percent wound closure was calculated by imaging software ImageJ (NIH, USA).
Further, in another embodiment of the present invention, the treatment of scratch wound monolayer of human fibroblasts with various concentrations of BA has led to closure of the scratch wound in a dose dependent manner which is statistically significant as shown in the FIG.13.
Development of slow and sustained release Gelma hydrogel formulation for Bispecific proteins to treat wounds
Further, in another embodiment of the present invention, Gelma hydrogel is a novel biomaterial that has many applications in biomedical engineering and drug delivery. It is composed of gelatin, a natural polymer derived from animal or recombinantly expressed (in bacteria) collagen that is modified with methacrylic anhydride to introduce cross linkable groups. Gelma hydrogel can be formed by UV irradiation or chemical initiators, and its mechanical and physical properties can be tuned by varying the degree of methacrylation, the concentration of gelatin, and the crosslinking conditions. Gelma hydrogel is biocompatible and biodegradable, meaning that it does not cause adverse immune reactions and can be degraded by enzymes in the body. It also has excellent cell adhesion and proliferation properties, making it suitable for tissue engineering and regenerative medicine. One of the main advantages of Gelma hydrogel is its ability to encapsulate and release drugs or biomolecules in a controlled manner. This can be achieved by loading the hydrogel with various agents, such as antibiotics, anti-inflammatory drugs, growth factors, cytokines, or genes, and adjusting the release kinetics by manipulating the hydrogel parameters. Gelma hydrogel can thus be used as a carrier for localized and sustained delivery of therapeutic agents to treat various diseases and injuries.
To prepare the Gelma hydrogels, dissolved LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate) in PBS at three different concentrations: 0.25% (w/v), 0.05% (w/v), and 0.01% (w/v). Further heated the solutions at 55? until the LAP was fully dissolved. Weighed different amounts of Gelma powder to obtain final concentrations of 2.5 % or 5.0 % and dissolved in Phosphate buffered saline. Gelma and LAP solutions of different concentrations are cross linked by exposing to the UV-visible light (350 nm-400 nm) for one minute and incubated them at 37? for 24 hours in a 96- well plate as shown in Table 4.
1 2 3 4 5 6 7
2.5% Gelma 5% Gelma
A 0.25% LAP 0.25% LAP 0.25% LAP 0.25% LAP 0.25% LAP 0.25% LAP
B 0.05% LAP 0.05% LAP 0.05% LAP 0.05% LAP 0.05% LAP 0.05% LAP
C 0.01% LAP 0.01% LAP 0.01% LAP 0.01% LAP 0.01% LAP 0.01% LAP
Table 4: Percent of Gelma and LAP for Hydrogel preparation.
Further, in another embodiment of the present invention, the results of the UV exposure and incubation experiments showed that Gelma polymerization occurred rapidly and effectively at 0.25% and 0.05% LAP concentrations, but not at 0.01%. After one minute of UV exposure, Gelma polymerization was observed in the wells with higher LAP concentrations (0.25% and 0.05%), while no polymerization was detected in the wells with lower LAP concentration (0.01%). After 24 hours of incubation, the Gelma polymerization remained stable and formed hydrogels with different strengths (visual observation) depending on the LAP concentration. The wells with 0.01% LAP concentration did not show any signs of Gelma polymerization even after the incubation period. Based on these observations Gelma 0.25 % and LAP at 0.05 % are selected for encapsulating bispecific proteins. However, the present invention can use Gelma concentrations ranging from 0.25 to 10 % with any increment and LAP concentration from 0.05 to 1 % with any increment.
Further, the administration of BA can also be done using various other types of hydrogel formulations such as sodium alginates, nanoparticles etc.,
Determination of invitro release kinetics of BA 3 in plasma and PBS at day-0, day-2, day-3, day-6 and day 10.
The objective of this study is to determine the invitro release kinetics of BA 3 in plasma and phosphate-buffered saline (PBS) at different time points. The release kinetics will be measured using Sandwich ELISA at day-0, day-2, day-3, day-6 and day-10 after incubation of BA 3 with plasma or PBS at 37°C. The results will provide information on the stability, solubility and bioavailability of BA in biological fluids and tissues.
Study procedure:
To prepare Gelma hydrogels with different concentrations and BA doses were selected. LAP (Lithium phenyl-2, 4, 6 trimethylbenzoylphosphinate, a photoinitiator) was dissolved in PBS at 0.25% (w/v) and heated at 55? until fully dissolved. Then, Gelma was added to the LAP solution and mixed well to obtain three Gelma concentrations: 2.5%, 3.75% and 5% (w/v). Next, BA was added to each Gelma concentration at two doses: 200 ng and 100 ng per well (100 µL) in a 96-well plate. Three wells were used for each condition as replicates. For the blank samples, only plasma or PBS was added to the Gelma solutions. The plate was then exposed to UV light (350 nm-400 nm) for 1 minute to crosslink the Gelma hydrogels. After crosslinking, plasma and PBS were added to the designated wells. A protease inhibitor cocktail mix (1 µL per 100 µL of plasma sample) and sodium azide (0.02%) were added to the plasma samples to prevent protein degradation and as a preservative, respectively. At day 0, plasma samples were collected from three wells and stored at -80? in 1.5 ml tubes. The same procedure was repeated at day 2, day 3, day 6 and day 10. The plasma samples were analyzed for BA release.
The release kinetics of BA is determined employing Sandwich ELISA test where anti IL-7 antibody is used to capture BA while anti EGF antibody is used to detect the bispecific protein.
Yet, in another embodiment of the present invention, as shown in the enclosed figure, encapsulation of BA 3 demonstrated slow sustained release over a period of 10 days as observed. The increase in the percent of Gelma lead to greater detection of the BA 3 in dose dependent manner. The observed lower concentrations of BA 3 at lower Gelma concentrations may suggest that the protein may have released quickly in the hydrogel and degraded shown in FIG.14.
Administration of Gelma formulation is shown to be safe as determined by photo catalytic oxidation studies using NADH+:
Yet, in another embodiment of the present invention, Gelma (Gelatin Methacrylate) along with LAP, a common photoinitiator, facilitates photoactivated crosslinking of Gelma. The BA encapsulated in the Gelma hydrogel gets released in slow and sustained manner allowing enhancement of pharmacokinetic parameters in in vivo. However, LAP, one of the Lithium salts are shown to cause acute kidney injury, leading to adverse effects like renal tubulointerstitial nephropathy and nephrogenic diabetes insipidus. Free radicals generated from photo-excited LAP also exerts cytotoxicity. Successful therapeutic application of a drug loaded Gelma indeed relies upon minimizing residual LAP present in it. The detection of residual LAP can be achieved by utilizing LAP's ability to oxidize NADH to NAD+ under UV light exposure. The loss of NADH fluorescence in this process serves as a screening tool, allowing to track LAP’s photocatalytic activity and quantify its concentration via a dose-dependent NADH oxidation calibration curve. This fundamental technique is employed in the present study to elucidate the residual LAP remaining in the specially formulated Gelma loaded with the desired therapeutic protein.
Effect of residual LAP in Gelma hydrogels does not cause photo-oxidation:
Yet, in another embodiment of the present invention, LAP exhibits concentration dependent photo-oxidation effect on NADH when exposed to 60 seconds of U.V. light. As a result, the NADH fluorescence, measured at an excitation wavelength of 340 nm and an emission wavelength of 460 nm, gradually decreases with increasing LAP concentration. The recorded fluorescence intensity of NADH was highest at a LAP concentration of 0%, with a value of 1.08 x 107 (mean of n=3). The intensity decreased significantly to 3.2 x 105 (mean of n=3) at the highest LAP concentration of 0.05%. This reduction in fluorescence intensity indicates the potent photocatalytic activity of LAP as shown in the dose dependent curve shown in FIG.15.
As shown in FIG.15, a LAP-led NADH photo-oxidation calibration curve by plotting LAP concentration represented in log value) against NADH fluorescence recorded at 460nm emission wavelength, using a non-linear regression analysis (Sigmoidal 4PL interpolated curve) in GraphPad prism software version 10.2.2.
Photo-oxidation of NADH by residual LAP present in BA 3 loaded Gelma hydrogel formulation:
Further, in another embodiment of the present invention, added 100µL of 150 µM NADH solution prepared in PBS in each well with BA loaded Gelma hydrogel formulation. NADH added to empty well without hydrogel serves as the interaction control sample. Exposed 96 well plate to U.V. irradiation for 60 secs. Transfer the NADH solution to black microplate and immediately measure fluorescence reading at 340nm excitation and 460nm emission wavelengths using ELISA microplate reader.
NADH fluorescence, which had not interacted with Gelma, dropped from 1.09 x 107 (mean of n=3) to 9.2 x 106 (mean of n=3) on interacting with Gelma under 60 secs of U.V. light exposure. The fluorescence change indicates residual LAP in the Gelma, which causes photo-oxidation of NADH.
Yet, in another embodiment of the present invention, it is found to be non-significant (ns) compared to the control (Student’s unpaired two-tailed t-test), suggesting the presence of a shallow concentration of residual LAP in the Gelma shown in FIG.16.
Interpolating the NADH fluorescence readings obtained post 60 sec of NADH-Gelma interaction under 60 sec U.V. light exposure in the LAP-led NADH photo-oxidation calibration curve yields 0.00096 % or approximately 0.001% residual LAP. It is 50x lower than the initial LAP concentration, i.e., 0.05% used for crosslinking BA loaded Gelma formulation. The fall of LAP concentration is significant (**** = p<0.0001) compared to the initial LAP concentration used for Gelma crosslinking (Student’s unpaired two tailed-t test). Low residual LAP in BA encapsulated Gelma significantly improves its biocompatibility as shown in FIG.17.

Determination of injection site toxicity and Pharmacokinetic profile of BA:
Further, in another embodiment of the present invention, this study aimed to evaluate the administration of Gelma encapsulated BA intradermally to measure injection site reactions in dose dependent manner, to understand immune responses, measure of plasma levels of BA over a period of time to calculate the pharmacokinetic parameters in Wistar Male Rats. The doses tested in this study are 0.125mg/kg, i.d, 0.25mg/kg, i.d and 0.5mg/kg, i.d.
A total of 12 male rats were allotted to 4 groups having three rats in each group. Rats of G1, G2 and G3 groups were dosed with BA by intradermal route at 0.125, 0.25 and 0.5 mg/kg respectively. Animals of G4 group was not given anything and served as control. All animals were observed for cage side clinical signs once daily. Animals were observed for morbidity and mortality twice daily. Body weights were measured before randomization, on the day of dosing and before necropsy. Blood was withdrawn through retro orbital plexus of animals of all the treated groups at pre-determined time points (0, 0.5, 1, 2, 6, 24, 48 and 72 hours) in pre-labelled K2EDTA vials. A 300 µL of blood was collected at each time point. After blood sample collection, equivalent amount of saline was administered to the animals by intraperitoneal route. The blood samples were centrifuged at 4000 rpm for 10 minutes at 4 °C to obtain plasma. Plasma was separated with the help of micropipette and collected in pre-labelled vials. In addition, 2 mL blood was collected by heart puncture from all animals across the groups (G1 to G4) after 72-hour blood collection time point in pre-labelled K2EDTA vials and plasma was separated. Spleen from all animals across the groups were collected, weighed and measured (Length and width). Skin from the injection site of all animals was frozen in liquid Nitrogen and stored at -80 °C until further analysis.
Yet, in another embodiment of the present invention, there was no mortality across all the dose groups. Furthermore, no adverse clinical signs and body weight change observed in any of the animal across the groups during study. In addition, there was no local adverse reaction noted at the injection site in any animal across the treated groups. Histopathological observations in BA administration by intradermal route at 0.5mg/kg, i.d. induces, inflammatory cell infiltration in dermis and hypodermis, epidermal hyperplasia and Parakeratosis in skin (injection site) of animals. However, the observed changes are known contribute to the healing of wounds, thus demonstrating the efficacy of the BA to heal the wounds. Increase in spleen weight was observed in animals treated with BA at 0.25 mg/kg and 0.5 mg/kg i.d. when compared to control group animals.
Increased spleen weights is a sign of active immune cell proliferation which could be attributed to the functionality of IL-7 component of the BA shown in FIG.18.
Pharmacokinetic analysis of the Bispecific Agonist by Sandwich ELISA:
The plasma concentrations of BA were determined by Sandwich ELISA by generation of calibration curve in the relevant matrix. Sandwich ELISA was performed using Capturing antibody (anti IL-7) and Detection antibody (anti EGF antibody). Calibration curve was generated as shown in FIG.19 using Wistar rat plasma diluted 1:10 with PBS and spiking with BA standard protein by diluting to 10 different concentrations.
Calibrations standards are ULOQ (60 ng/mL) to LLOQ (0.312 ng/mL) plus blank sample.
Bio-analytical assays accuracy and precision:
Prepared Quality controls of 5 concentration levels ULOQ (60 ng/mL), HQC (46 ng/mL), Medium QC (6 ng/mL), LQC (0.936 ng/mL) and LLOQ (0.312 ng/mL) from BA spiked stock solution.
Plasma samples corresponding time points are subjected to bioanalysis and quantified available concentrations using Sandwich ELISA. Analysis of plasma samples reveled that administration of BA as gelma hydrogel intradermally has led to sustained release over a period of 72 h analyzed in this study. The peak concentration of the protein was detected at 5 minutes in S.C route of administration while intradermally at 30 min post administration while continued to detect BA within the quantification limits of this study even up to 72 h in the current study indicating sustained release is shown in the FIG.20.
Pharmacological Studies for determining the efficacy of Bispecific Agonist proteins:
Yet, in another embodiment of the present invention, all BA (1-4) demonstrating functionality in terms of phosphorylation of STAT5 and ERK in in vitro, are screened for efficacy by single dose administration in various chronic wound-diseased animal models as shown subsequently.
Streptozotocin (STZ) and Nicotinamide induced type 2 diabetic wound healing in Wistar rat model:
Rationale: Diabetes causes dysfunction of the immune responses as well as tissue repair. Further may also fails to control the spread of invading pathogens and impairs wound healing (Afiat Berbudi et. al; 2019). BA are expected to restore the immune responses by activation and migration of immune cells to the wound site. Further, BA promotes proliferation and migration of keratinocytes, fibroblasts, as well as stimulate wound healing factors.
Experimental Procedure includes, Healthy male Wistar rats, weight between 200-250 gm are labeled by permanent markers. Animals are administered STZ and nicotinamide to induce type 2 diabetes administration. The induction of diabetes is determined by measuring the blood glucose levels which needs to be >300 mg/dL. Rats that are having > 300mg/dL of blood glucose are subjected to wound infliction under Isoflurane anesthesia. Wound infliction is caused by 10 mm surgical punch on the dorsal surface above the tail region post clipping of hair, after attaining Hyperglycemic condition. Post 24 h after wound infliction test compound bispecific agonists are administered as q7dx2 dose regimen formulated in biocompatible Gelma hydrogels. Percentage of wound retraction is measured using vernier calipers thrice a week for monitoring the wound healing. At the end of treatment and post administration schedule, animals were further observed for wound healing and recovery of body weights for 15 days.
Further, in another embodiment of the present invention,
1. Successfully induced Diabetes Type 2 as measured by glucose levels of >350 mg/dL by Accu check.
? Diabetic Rats have lost their body weight by ~10 % over the naïve animals shown in FIG.21.
2. Infliction of 10 mm wounds in diabetic Rats has increased by>100 % over naïve Rats indicating a prolonged healing process owing to Type 2 diabetes.
3. Treatment of Diabetic Rats with BA variants (1, 2, 3 & 4) has resulted in enhanced healing /wound closure in comparison to vehicle shown in FIG.22.
? Among the BA protein variants, 1,2, 3 and 4 have shown greater wound healing over naïve wounded Rats
? Treatment of Diabetic Rats with BA variants 3 & 1 resulted in restoration of body weights similar to that of naïve animals.
4. Observed that all BA protein variants treated diabetic animals showed increased body weight and wound healing as compared to vehicle control as shown in FIG.22.
Burns wound healing in BALB/c mouse model:
Rationale: Immunosuppression caused in severe burn wounds is known to have phases of hyperimmune activity leading to inhibition of wound during early stages. Further, immune suppressive conditions are returned for extended durations leading to chronic wounds. BA proteins are expected to restore immune homeostasis and accelerate the wound healing.
Experimental procedure in brief includes 60 female BALB/c mice of 10-12 weeks old are subjected to burn wound following the protocol of Maria et.al. 2022, https://www.nature.com/articles/s41598-022-05768-w.
Yet, in another embodiment of the present invention, the infliction of burn wound / injury led to a slight increase in hyperactive immune populations in the thermal burn wound on Day 5. The presence of BA treatment reduced the circulating immune population through homing cells to peripheral tissues is shown FIG.23. The transient decrease in the circulating immune populations aids in controlling inflammatory process as well as promoting the healing. As it has been documented in our experiments, the restoration of the functionally active immune populations in later days helps to heal the burn wound by protecting from invading infections of the open wound. Thus, BA play dual role where transient suppression (due to homing of immune cells to lymphoid organs) followed by restoration of immune cell populations and enhanced functionality help in reducing the early hyperactivity of the immune system response followed by protection from the invading infectious agents such as bacteria, fungi or viruses.
Effect of Bispecific Agonist in Immune compromised infected wound model of C57BL/6 Mice model:
Rationale: Treatment of various ailments such as autoimmune diseases, surgical wounds, cancer etc., often involve suppression of the active immune system. Furthermore, certain pathological conditions such as diabetes, chronic immune suppression syndrome etc., also are responsible for chronic wounds. Often the immune suppression lead to infection of wounds with pathogens such as bacteria, fungi as well as virus. Owing to compromised immunity, the infection does spread and turn to life threatening conditions such as sepsis or amputations.
Experiment procedure includes C57BL/6 female mice administered cyclophosphamide, one of the clinically widely used immune suppression agent. Post 24 h of administration of Cyclophosphamide (200 mg/kg, i.p) to mice, blood samples are analysed for CBC analysis to understand the depletion status. Also, mice are inflicted with 6 mm excision wound by biopsy punch. Mice having depleted leukocyte population are randomized and grouped based on the body weight prior to infliction of 6 mm wound by biopsy punch. Post 24 h of wound infliction, mice are topically applied 100 µL of faecal bacteria cultured overnight and treated with 0.5 mg/kg BA in Gelma formulation through i.d route.
Yet, in another embodiment of the present invention, post administration of cyclophosphamide has led to decrease in the leukocyte population as shown in the Table 5 below:
RBC WBC Platelets
Control Mice (No treatment); N=3 9.47 2.68 8.47
Cyclophosphamide treatment; N=3 7.41 1.04 6.3
Table 5: Administration of cyclophosphamide to mice lead to depletion of immune cell populations
In comparison with Control mice (A1, A2 & A3), Cyclophosphamide administered mice found to have >50 % reduction in the WBC count post 24 h. As shown in Table 6 below, mice having depleted leukocyte population are randomized and grouped based on the body weight prior to infliction of 6 mm wound by biopsy punch. Mice (naïve) are not administered cyclophosphamide and so retained normal levels of leukocytes.
Group (N=8) Treatment
G1 Naïve wound
G2 Immunocompromised wound + Vehicle
G3 Immunocompromised wound + BA
G4 Immunocompromised wound + bacterial infection
G5 Immunocompromised wound + bacterial infection+BA
G6 Naïve wound + bacterial infection+ BA
Table 6: Treatment groups of mice depleted for leukocytes.
Post 24 h of wound infliction, mice are topically applied 100 µL of faecal bacteria cultured overnight followed by treatment with 0.5 mg/kg BA in Gelma formulation through i.d route or Gelma alone without BA. Mice were measured wound area using vernier callipers and compared among the groups as shown in the FIG.24 and Table 7.
Groups Statistics
Group Details % wound area changes on Day 6 post treatment
Mean SEM Stat Animals survived
G1 Naïve wound vs 53.47 2.35 **** 8
G2 Immunocompromised wound + Vehicle 20.49 1.19 - 8
G3 Immunocompromised wound + BA 45.00 1.98 **** 8
G4 Immunocompromised wound + bacterial infection 16.28 1.10 ns 6
G5 Immunocompromised wound + bacterial infection +BA 27.08 2.48 * 5
G6 Naïve wound + bacterial infection+ BA 33.20 2.95 **** 8
Table 7: Percent decrease in the wound area upon administration of BA in Glema Hydrogel.
Based on these results, a single dose of BA administration has showed significant decrease in the wound area in both non-infected and infected immune depleted mice over their respective controls. The decrease in wound area in immune depleted BA administered mice is very much comparable to that of in naïve mice. These results clearly establish that BA has potential to heal the chronic wounds under immune compromised conditions with or without infection to different extents.
Orientation of the combination molecules while linking with Linker sequence:
Bispecific agonist molecules comprising of Epidermal growth factor (EGF) and Interleukin-7 (IL-7) are combined with linker molecules known in prior art. However, the combination of two different molecules targeted towards two different pathways is unique in this invention. Furthermore, the orientation of combination molecules is shown in the sequences of the Bispecific agonist molecules 1, 2, 3 and 4. Orientations that have been tested include -
a) N terminal of IL-7 molecule is not connected to Linker sequence, only C-terminal is connected to N terminal of linker sequence, while EGF N terminal is connected to C terminal of linker sequence.
b) C terminal of EGF molecule is not connected to Linker sequence, only C-terminal is connected to N terminal of linker sequence, while IL-7 N terminal is connected to C terminal of linker sequence.
c) C-terminal of IL-7 is not connected while its N-terminal is connected to C terminal of Linker molecule, while N terminal of EGF molecule is not connected but its C terminal is connected to N terminal of Linker sequence.
d) C-terminal of EGF is not connected while its N-terminal is connected to C terminal of Linker molecule, while N terminal of IL-7 molecule is not connected but its C terminal is connected to N terminal of Linker sequence.
Mutational Analysis of the BA variants:
Based on the binding domains or interacting domains with their corresponding receptors (IL-7R & EGFR-R), the single chain polypeptide Bispecific Agonist molecule has been subjected to mutational analysis as shown for each in the Table 8 & 9.

S.No. Mutational position
(With respect to Bispecific Agonist EGF) Mutational position
(With respect to EGF protein)
1 R258K R41K
2 L264I L47I
3 R258K, L264I R41K, L47I
4 C250S, C259S C33S, C42S
5 C248S, C250S C31S, C33S
Table 8: EGF protein selective binding domain mutated amino acids.

S.No. Mutational Position
(With respect to Bispecific Agonist IL-7) Mutational Position
(With respect to IL-7)
1 W142F W142F
2 Y12F Y12F
3 L23I L23I
4 E137D E137D
5 N143Q N143Q
6 Y12F, L23I, E137D, W142F, N143Q Y12F, L23I, E137D, W142F, N143Q
Table 9: IL-8 protein selective binding domain mutated amino acids.
The impact of mutation of each of the amino acids in the BA protein has been conducted by protein-protein docking studies employing in-house generated bioinformatic tools where free energies are derived. Based on these studies BA mutants in IL-7 or EGF have not significantly altered their binding affinity towards their respective receptors. The free energies or docking scores ranged from -240 to -280 0 indicating that amino acids can be mutated without impacting binding affinities to their respective receptors. Furthermore, cell based phosphorylation assays where potencies of the each of the molecules has been derived are comparable to that of the individual proteins even in the Bispecific Agonist molecule, a single polypeptide chain expressing functionalities of two different pathways.
Table 10 and 11 below represent type of the sequence all the variants of Bispecific Agonist Proteins.
SEQ ID NO. 1 Bispecific Agonist 1
SEQ ID NO. 2 Bispecific Agonist 1 (Without signal sequence)
SEQ ID NO. 3 Bispecific Agonist 2
SEQ ID NO. 4 Bispecific Agonist 2 (Without signal sequence)
SEQ ID NO. 5 Bispecific Agonist 3
SEQ ID NO. 6 Bispecific Agonist 3 (Without signal sequence)
SEQ ID NO. 7 Bispecific Agonist 4
SEQ ID NO. 8 Bispecific Agonist 3 (Without signal sequence)
Table 10: Nucleotide Sequences
SEQ ID NO. 9 Bispecific Agonist 1 (Without signal sequence)
SEQ ID NO. 10 Bispecific Agonist 2 (Without signal sequence)
SEQ ID NO. 11 Bispecific Agonist 3 (Without signal sequence)
SEQ ID NO. 12 Bispecific Agonist 4 (Without signal sequence)
Table 11: Amino Acid Sequences
Table 12 below represents the sequence listings.
SEQ ID NO. 1 ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCTACCGGCGTGCACTCCAACTCTGATTCTGAGTGCCCTCTGAGCCACGACGGCTACTGTCTGCATGACGGCGTGTGTATGTACATCGAGGCCCTGGATAAGTACGCCTGCAACTGCGTCGTGGGCTACATCGGCGAAAGATGCCAGTACAGGGACCTGAAGTGGTGGGAATTGAGAGGTGGCGGAGGATCTGGCGGTGGTGGTTCTGGCGGAGGCGGTAGTGGCGGTGGCGGATCTGATTGTGACATCGAAGGCAAGGACGGCAAGCAGTACGAGTCCGTGCTGATGGTGTCTATCGACCAGCTGCTGGACTCCATGAAGGAAATCGGCTCCAACTGCCTGAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAAGAAGGCATGTTCCTGTTCAGAGCCGCCAGAAAGCTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCATCTGCTGAAAGTGTCTGAGGGCACCACCATCCTGCTGAACTGTACCGGCCAAGTGAAGGGCAGAAAGCCTGCTGCTTTGGGCGAAGCCCAGCCTACCAAGAGCCTGGAAGAGAACAAGTCCCTGAAAGAGCAGAAGAAGCTGAACGACCTCTGCTTCCTGAAGCGGCTGCTGCAAGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAAGAGCACTGA
SEQ ID NO. 2 AACTCTGATTCTGAGTGCCCTCTGAGCCACGACGGCTACTGTCTGCATGACGGCGTGTGTATGTACATCGAGGCCCTGGATAAGTACGCCTGCAACTGCGTCGTGGGCTACATCGGCGAAAGATGCCAGTACAGGGACCTGAAGTGGTGGGAATTGAGAGGTGGCGGAGGATCTGGCGGTGGTGGTTCTGGCGGAGGCGGTAGTGGCGGTGGCGGATCTGATTGTGACATCGAAGGCAAGGACGGCAAGCAGTACGAGTCCGTGCTGATGGTGTCTATCGACCAGCTGCTGGACTCCATGAAGGAAATCGGCTCCAACTGCCTGAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAAGAAGGCATGTTCCTGTTCAGAGCCGCCAGAAAGCTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCATCTGCTGAAAGTGTCTGAGGGCACCACCATCCTGCTGAACTGTACCGGCCAAGTGAAGGGCAGAAAGCCTGCTGCTTTGGGCGAAGCCCAGCCTACCAAGAGCCTGGAAGAGAACAAGTCCCTGAAAGAGCAGAAGAAGCTGAACGACCTCTGCTTCCTGAAGCGGCTGCTGCAAGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAAGAGCACTGA
SEQ ID NO. 3 ATGGGTTGGAGTTGCATCATCCTATTTCTAGTCGCTACCGCCACCGGCGTGCACTCCAACTCCGACTCTGAGTGTCCTCTGTCTCACGATGGCTACTGCCTGCATGATGGAGTGTGCATGTACATCGAGGCCCTGGACAAGTACGCCTGCAACTGCGTGGTGGGCTACATTGGCGAGAGATGTCAGTATAGAGATCTGAAGTGGTGGGAGCTGAGAGCCGAGGCTGCTGCCAAAGAAGCTGCCGCCAAAGAAGCCGCCGCTAAAGAAGCCGCAGCCAAGGCCGACTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGTCCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACTCCATGAAAGAGATCGGATCTAATTGTCTGAACAACGAGTTCAACTTCTTCAAGAGACACATCTGCGATGCCAACAAGGAAGGCATGTTTCTGTTCCGGGCTGCTCGCAAGCTGCGGCAGTTCCTGAAGATGAACAGCACAGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTCAATTGCACCGGCCAGGTGAAGGGTAGAAAGCCTGCTGCTCTGGGCGAAGCCCAGCCCACAAAGTCTCTGGAAGAGAACAAGAGCCTGAAAGAACAGAAGAAGCTGAACGACCTGTGCTTTCTCAAGCGGCTGCTGCAAGAGATCAAGACCTGTTGGAACAAGATCCTGATGGGCACCAAGGAGCACTGA
SEQ ID NO. 4 AACTCCGACTCTGAGTGTCCTCTGTCTCACGATGGCTACTGCCTGCATGATGGAGTGTGCATGTACATCGAGGCCCTGGACAAGTACGCCTGCAACTGCGTGGTGGGCTACATTGGCGAGAGATGTCAGTATAGAGATCTGAAGTGGTGGGAGCTGAGAGCCGAGGCTGCTGCCAAAGAAGCTGCCGCCAAAGAAGCCGCCGCTAAAGAAGCCGCAGCCAAGGCCGACTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGTCCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACTCCATGAAAGAGATCGGATCTAATTGTCTGAACAACGAGTTCAACTTCTTCAAGAGACACATCTGCGATGCCAACAAGGAAGGCATGTTTCTGTTCCGGGCTGCTCGCAAGCTGCGGCAGTTCCTGAAGATGAACAGCACAGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTCAATTGCACCGGCCAGGTGAAGGGTAGAAAGCCTGCTGCTCTGGGCGAAGCCCAGCCCACAAAGTCTCTGGAAGAGAACAAGAGCCTGAAAGAACAGAAGAAGCTGAACGACCTGTGCTTTCTCAAGCGGCTGCTGCAAGAGATCAAGACCTGTTGGAACAAGATCCTGATGGGCACCAAGGAGCACTGA
SEQ ID NO. 5 ATGGGTTGGAGTTGCATCATCCTATTTCTAGTCGCCACCGCTACAGGAGTGCACAGCAACAGCGACTCCGAGTGTCCTCTGTCTCATGATGGCTACTGCCTGCACGACGGCGTGTGCATGTACATCGAGGCCCTGGACAAGTACGCCTGCAATTGCGTGGTGGGCTATATCGGCGAGAGATGCCAGTACCGGGATCTGAAGTGGTGGGAGCTGAGAGCCCCTGCTCCAGCTCCTGCTCCCGCCCCTGCCCCCGCTCCCGCCCCTGCTCCTGCTCCTGCTCCTGCCCCTGCCCCTGCCCCCGCCCCTGCTCCAGCCCCTGACTGCGACATCGAGGGAAAGGACGGCAAGCAGTACGAGTCCGTGCTGATGGTGTCCATCGACCAGCTGCTGGATTCCATGAAAGAAATCGGCTCCAACTGTCTGAACAACGAGTTCAACTTCTTCAAGAGGCACATCTGCGATGCCAATAAAGAAGGCATGTTCCTGTTTAGAGCCGCCCGGAAGCTGAGACAGTTTCTGAAGATGAACTCTACCGGCGACTTCGACCTGCACTTGCTGAAAGTGTCTGAGGGAACCACCATCCTGCTGAACTGCACCGGCCAGGTGAAGGGCAGAAAGCCCGCTGCTCTGGGCGAAGCCCAGCCTACAAAGTCCCTGGAAGAGAACAAGTCTCTCAAAGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGCGGCTGCTCCAAGAGATCAAGACCTGTTGGAACAAGATCCTGATGGGCACCAAGGAACACTGA
SEQ ID NO. 6 AACAGCGACTCCGAGTGTCCTCTGTCTCATGATGGCTACTGCCTGCACGACGGCGTGTGCATGTACATCGAGGCCCTGGACAAGTACGCCTGCAATTGCGTGGTGGGCTATATCGGCGAGAGATGCCAGTACCGGGATCTGAAGTGGTGGGAGCTGAGAGCCCCTGCTCCAGCTCCTGCTCCCGCCCCTGCCCCCGCTCCCGCCCCTGCTCCTGCTCCTGCTCCTGCCCCTGCCCCTGCCCCCGCCCCTGCTCCAGCCCCTGACTGCGACATCGAGGGAAAGGACGGCAAGCAGTACGAGTCCGTGCTGATGGTGTCCATCGACCAGCTGCTGGATTCCATGAAAGAAATCGGCTCCAACTGTCTGAACAACGAGTTCAACTTCTTCAAGAGGCACATCTGCGATGCCAATAAAGAAGGCATGTTCCTGTTTAGAGCCGCCCGGAAGCTGAGACAGTTTCTGAAGATGAACTCTACCGGCGACTTCGACCTGCACTTGCTGAAAGTGTCTGAGGGAACCACCATCCTGCTGAACTGCACCGGCCAGGTGAAGGGCAGAAAGCCCGCTGCTCTGGGCGAAGCCCAGCCTACAAAGTCCCTGGAAGAGAACAAGTCTCTCAAAGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGCGGCTGCTCCAAGAGATCAAGACCTGTTGGAACAAGATCCTGATGGGCACCAAGGAACACTGA
SEQ ID NO. 7 ATGGGTTGGAGTTGCATCATCCTATTTCTAGTCGCCACCGCTACCGGCGTGCACTCCGACTGCGACATCGAGGGAAAGGACGGAAAGCAGTACGAGTCCGTGCTGATGGTGTCCATCGACCAGCTGCTGGATTCCATGAAAGAAATCGGCTCTAATTGTCTGAACAACGAGTTTAACTTCTTCAAGCGCCACATCTGCGATGCCAATAAGGAAGGCATGTTCCTGTTCAGAGCCGCCCGGAAGCTGAGACAGTTTCTGAAGATGAACAGCACAGGCGACTTCGACCTGCATCTGCTCAAGGTGTCTGAGGGCACAACCATCCTGCTCAACTGTACCGGACAGGTGAAGGGCAGAAAGCCCGCTGCCCTGGGCGAAGCCCAGCCTACCAAGTCTCTGGAAGAGAACAAGTCCCTGAAAGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGACTGCTGCAAGAGATCAAGACCTGTTGGAACAAGATCCTGATGGGCACCAAGGAACACGCCGAGGCCGCCGCCAAGGAAGCTGCCGCCAAAGAGGCTGCCGCTAAGGAGGCTGCTGCCAAGGCTCTGGAAGCTGAGGCCGCTGCTAAAGAAGCCGCCGCTAAAGAAGCTGCTGCTAAGGAGGCAGCCGCCAAGGCCAACAGCGACTCCGAGTGTCCTCTGTCTCACGACGGCTACTGCCTGCACGATGGCGTGTGCATGTACATCGAGGCCCTGGACAAGTACGCCTGCAACTGCGTGGTGGGCTATATCGGCGAGCGGTGCCAGTACAGAGATCTGAAGTGGTGGGAGCTGCGGTGA
SEQ ID NO. 8 GACTGCGACATCGAGGGAAAGGACGGAAAGCAGTACGAGTCCGTGCTGATGGTGTCCATCGACCAGCTGCTGGATTCCATGAAAGAAATCGGCTCTAATTGTCTGAACAACGAGTTTAACTTCTTCAAGCGCCACATCTGCGATGCCAATAAGGAAGGCATGTTCCTGTTCAGAGCCGCCCGGAAGCTGAGACAGTTTCTGAAGATGAACAGCACAGGCGACTTCGACCTGCATCTGCTCAAGGTGTCTGAGGGCACAACCATCCTGCTCAACTGTACCGGACAGGTGAAGGGCAGAAAGCCCGCTGCCCTGGGCGAAGCCCAGCCTACCAAGTCTCTGGAAGAGAACAAGTCCCTGAAAGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGACTGCTGCAAGAGATCAAGACCTGTTGGAACAAGATCCTGATGGGCACCAAGGAACACGCCGAGGCCGCCGCCAAGGAAGCTGCCGCCAAAGAGGCTGCCGCTAAGGAGGCTGCTGCCAAGGCTCTGGAAGCTGAGGCCGCTGCTAAAGAAGCCGCCGCTAAAGAAGCTGCTGCTAAGGAGGCAGCCGCCAAGGCCAACAGCGACTCCGAGTGTCCTCTGTCTCACGACGGCTACTGCCTGCACGATGGCGTGTGCATGTACATCGAGGCCCTGGACAAGTACGCCTGCAACTGCGTGGTGGGCTATATCGGCGAGCGGTGCCAGTACAGAGATCTGAAGTGGTGGGAGCTGCGGTGA
SEQ ID NO. 9 NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRGGGGSGGGGSGGGGSGGGGSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH
SEQ ID NO. 10 NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRAEAAAKEAAAKEAAAKEAAAKADCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH
SEQ ID NO. 11 NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH
SEQ ID NO. 12 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

Table 12
BA mutants in IL-7 or EGF have not significantly altered their binding affinity towards their respective receptors. Table 13 below represents the mutant variant sequences (SEQ ID NOs: 13-23).
SEQ ID NO. 13 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGEKCQYRDLKWWELR*

SEQ ID NO. 14 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDIKWWELR*

SEQ ID NO. 15 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGEKCQYRDIKWWELR*

SEQ ID NO. 16 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNSVVGYIGERSQYRDLKWWELR*

SEQ ID NO. 17 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYASNSVVGYIGERCQYRDLKWWELR*

SEQ ID NO. 18 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCFNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR*

SEQ ID NO. 19 DCDIEGKDGKQFESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR*

SEQ ID NO. 20 DCDIEGKDGKQYESVLMVSIDQILDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR*

SEQ ID NO. 21 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQDIKTCWNKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR*

SEQ ID NO. 22 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWQKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR*

SEQ ID NO. 23 DCDIEGKDGKQFESVLMVSIDQILDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQDIKTCFQKILMGTKEHAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKANSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR*

Table 13

SEQ ID NO. 13 Bispecific Agonist M1
SEQ ID NO. 14
Bispecific Agonist M2
SEQ ID NO. 15 Bispecific Agonist M3
SEQ ID NO. 16 Bispecific Agonist M4
SEQ ID NO. 17 Bispecific Agonist M5
SEQ ID NO. 18 Bispecific Agonist M6
SEQ ID NO. 19 Bispecific Agonist M7
SEQ ID NO. 20 Bispecific Agonist M8
SEQ ID NO. 21 Bispecific Agonist M9
SEQ ID NO. 22 Bispecific Agonist M10
SEQ ID NO. 22 Bispecific Agonist M11
Table 14
As various changes can be made in the above-described subject matter without departing from the scope and spirit of the present invention, it is intended that all subject matter contained in the above description, or defined in the appended claims, be interpreted as descriptive and illustrative of the present invention. Many modifications and variations of the present invention are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications, and variances which fall within the scope of the appended claims.
,CLAIMS:We claim:
1. A pharmaceutical composition for promoting healing of wounds, comprising:
at least one Bispecific Agonist (BA) protein consisting of an immune cell modulator including a cytokine and a growth factor, the Bispecific Agonist (BA) protein comprising a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
2. The pharmaceutical composition according to claim 1, wherein the cytokine is selected from IL-2, IL-7, IL-11IL-15, IL17 and IL-21.
3. The pharmaceutical composition according to claim 1, wherein the growth factor is selected from EGF, PDGF, VEGF and FGF.
4. The pharmaceutical composition according to claim 1, wherein at least one cytokine is linked with at least growth factor with a linker molecule.
5. The pharmaceutical composition according to claim 1, wherein Bispecific Agonist (BA) protein targets on binding domains or interacting domains of IL-7R and EGF-R receptors.
6. The pharmaceutical composition according to claim 1, wherein mutations in the Bispecific agonist protein, the Bispecific Agonist (BA) protein targets on binding domains or interacting domains of IL-7R and EGF-R receptors without altering functionality in terms of pathways being stimulated, further the Bispecific Agonist (BA) protein may retain the potency that could be either equivalent to its corresponding single unmodified protein, or the potency may be higher or lower but offers therapeutically acceptable agonistic or antagonistic properties
7. The pharmaceutical composition according to claim 1, wherein the Bispecific Agonist protein is a single chain polypeptide molecule retaining functionality of both the proteins, and can be administered therapeutically
8. The pharmaceutical composition according to claim 1, wherein the Bispecific Agonist (BA) protein is SEQ ID NO: 1 and a molecular weight is approximately 24852.33 Daltons.
9. The pharmaceutical composition according to claim 1, wherein the Bispecific Agonist (BA) protein is SEQ ID NO: 3 and a molecular weight is approximately 25615.45 Daltons.
10. The pharmaceutical composition according to claim 1, wherein the Bispecific Agonist (BA) protein is SEQ ID NO: 5 and a molecular weight is approximately 26450.51 Daltons.
11. The pharmaceutical composition according to claim 1, wherein the Bispecific Agonist (BA) protein is SEQ ID NO: 7 and a molecular weight is approximately 27881.98 Daltons.
12. A pharmaceutical composition for treatment of wounds, comprising:
at least one Bispecific Agonist (BA) protein comprising an Interleukin-7 (IL-7) and an Epidermal growth factor (EGF) combined together with a linker molecules , where the Bispecific Agonist (BA) protein comprising a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
13. The pharmaceutical composition according to claim 12, wherein at least one cytokine is linked with at least growth factor with a linker molecule.
14. The pharmaceutical composition according to claim 12, wherein the treatment of patients includes with acute wounds, chronic wounds, and immune suppressive conditions that may have originated with various comorbidities such Diabetes, Burns, transplantations, etc.,
15. A Bispecific Agonist (BA) protein for treatment of wounds, comprising:
an Interleukin-7 (IL-7) and an epidermal growth factor (EGF), wherein the Bispecific Agonist (BA) protein is a single chain polypeptide molecule selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
16. A formulation for treatment of wounds, comprising:
at least one Bispecific Agonist (BA) protein comprising an Interleukin-7 (IL-7) and an Epidermal growth factor (EGF),
the Bispecific Agonist (BA) is encapsulated in Gelma Hydrogel,
wherein the Bispecific Agonist (BA) protein comprising a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
17. The formulation according to claim 16, wherein the Gelma Hydrogel is composed of gelatin, a natural polymer derived from animals or by recombinant expression of collagen is modified with methacrylic anhydride to introduce cross linkable groups.
18. The formulation according to claim 16, wherein the Gelma Hydrogel is prepared by dissolving LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate) in PBS (phosphate-buffered saline) at three different concentrations such as 0.25% (w/v), 0.05% (w/v), and 0.01% (w/v), where a solution is heated at 55? until the LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate) is fully dissolved, and obtain Gelma powder of 2.5 % or 5.0 % of concentrations, then exposed to a UV-visible light (350 nm-400 nm) for one minute and incubated at 37? for 24 hours in a 96- well plate.
19. The formulation according to claim 16, wherein the Gelma hydrogel is prepared by dissolving LAP (Lithium phenyl-2, 4, 6 trimethylbenzoylphosphinate) and a photoinitiator in PBS (phosphate-buffered saline) at 0.25% (w/v) and heating at 55? until the LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate) is fully dissolved.
20. The formulation according to claim 16, wherein the Bispecific Agonist (BA) is encapsulated in Gelma Hydrogel to achieve slow, sustained release, enhancing the pharmacokinetic profiles and consequently improving therapeutic outcomes.
21. The formulation according to claim 16, wherein the Bispecific Agonist can be administered as an injectable with or without hydrogel, depending on the indication and condition of the patient being treated for modulation of pharmacokinetics and pharmacological outcomes, including the management of any potential toxicities.
22. The formulation according to claim 16, wherein the Bispecific Agonist can also be administered topically in various forms, such as a cream, nanoparticle suspension, or as an active pharmaceutical ingredient alongside compatible biomaterials or as a patch releasing BA in controlled manner.