Peptide hydrogel encapsulated protein formulations to improve protein stability at elevated temperatures

Abstract

Disclosed herein are peptide hydrogel-based formulations. In some embodiments, the peptide hydrogel-based formulations encapsulate antibodies. In some embodiments, the peptide hydrogel-based formulations prevent formation of high molecular weight protein species.

Description

PEPTIDE HYDROGEL ENCAPSULATED PROTEIN FORMULATIONS TO IMPROVE PROTEIN STABILITY AT ELEVATED TEMPERATURESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 724,050 filed November 22, 2024, the entire contents of which is incorporated by reference herein.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (26114-WO-PCT_SL.xml; Size: 10,454 bytes, created on January 20, 2025) are herein incorporated by reference in their entirety.FIELD

[0003] This disclosure relates generally to peptide hydrogel-based formulations and methods of making the same.BACKGROUND

[0004] Biologies are fragile molecules with susceptibility to various degradation pathways, which will result in the loss of efficacy. One important pathway is aggregation, which is most commonly induced by thermal stress. Therefore, biologic formulations are typically stored in refrigerated liquid, frozen, or lyophilized powder state. The manufacturing, handling, shipping and storage of these formulations also involve cold chain logistics. This is not only costly but also less accessible to less developed regions. To improve patient access, there is a need to develop formulations that can prevent protein aggregation, particularly under thermal stress conditions.

[0005] Hydrogels are useful in biological applications because they have high water content, which is suitable for the capsulation of various types of cargo molecules. With proper choice of gel-forming materials, for example peptide-based hydrogels, additional desired properties including biocompatibility and ease of manufacturing can be incorporated. Due to these advantages, hydrogels are currently being used in applications involving drug delivery of therapeutic proteins.

[0006] The physical structure of hydrogel can be described as water-containing, crosslinking networks. Therefore, when hydrogels are used to encapsulate biologic molecules, the encapsulated molecules can be retained within such network structures to inhibit theintermolecular interactions thus preventing the aggregation pathway. Together with the previously mentioned properties, a peptide hydrogel is an ideal system for the formulation of biologies to maintain stability under thermal stress conditions.SUMMARY

[0007] The present disclosure relates to peptide hydrogel-based formulations and methods of making peptide hydrogel-based formulations.

[0008] In some embodiments, the peptide hydrogel-based formulations encapsulate proteins, such as antibodies (e.g., pembrolizumab). In some embodiments, the peptide hydrogel-based formulations comprise a buffer. In some embodiments, the peptide hydrogel-based formulations comprise a buffer and an inorganic salt. In some embodiments, the buffer comprises L-histidine hydrochloride. In some embodiments, the inorganic salt comprises sodium chloride. In some embodiments, the inorganic salt (e.g., sodium chloride) functions to reduce repulsive electrostatic forces between chains of the peptides resulting in physical cross-linking of the peptides and formation of the peptide hydrogel-based formulations.

[0009] In an aspect, a formulation is provided comprising: between about 9.5 mg / ml and about 10.5 mg / ml of a peptide; between about 17.8 mg / ml and about 18.8 mg / ml sodium chloride; and between about 128 mg / ml and about 138 mg / ml of an antibody, wherein the antibody comprises a light chain comprising at least 90% sequence identity to SEQ ID NO: 2 and a heavy chain comprising at least 90% sequence identity to SEQ ID NO: 3, wherein the antibody binds to PD-1, and wherein the peptide comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1.

[0010] In some embodiments, the antibody comprises a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 4, an HCDR2 comprising SEQ ID NO: 5, and an HCDR3 comprising SEQ ID NO: 6; and a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 7, an LCDR2 comprising SEQ ID NO: 8, and an LCDR3 comprising SEQ ID NO: 9.

[0011] In an aspect, a peptide hydrogel-based formulation is provided, comprising: between about 9.5 mg / ml and about 10.5 mg / ml of a peptide; between about 17.8 mg / ml and about 18.8 mg / ml sodium chloride; and between about 128 mg / ml and about 138 mg / ml of an antibody, wherein the antibody comprises a light chain sequence that has at least 90% sequence identity to SEQ ID NO: 2 and a heavy chain sequence that has at least 90% identity to SEQ ID NO: 3, wherein the antibody binds to PD-1, wherein the peptide comprises a sequence having at least95% sequence identity to SEQ ID NO: 1, and wherein the addition of sodium chloride leads to physical cross-linking of the peptide and formation of a peptide hydrogel.

[0012] In some embodiments, the antibody comprises a heavy chain complementarity' determining region (HCDR) 1 comprising SEQ ID NO:4, an HCDR2 comprising SEQ ID NO:5. and an HCDR3 comprising SEQ ID NO: 6; and a light chain complementarity- determining region (LCDR) 1 comprising SEQ ID NO:7, an LCDR2 comprising SEQ ID NO:8, and an LCDR3 comprising SEQ ID NO:9.

[0013] In some embodiments, the peptide in the formulation or the peptide hydrogel-based formulation comprises SEQ ID NO: 1. In some embodiments, the formulation comprises about 10.0 mg / ml peptide. In some embodiments, the formulation comprises about 18.3 mg / ml sodium chloride. In some embodiments, the formulation comprises about 133 mg / ml antibody. In some embodiments, the antibody comprises SEQ ID NO: 2 and SEQ ID NO: 3.

[0014] In some embodiments, the peptide concentration is about 10.0 mg / ml. In some embodiments, the sodium chloride concentration is about 18.3 mg / ml. In some embodiments, the antibody concentration is about 133 mg / ml. In some embodiments, the peptide hydrogel-based formulation further comprises betyveen about 51 mg / ml and about 61 mg / ml of sucrose. In some embodiments, the peptide hydrogel -based formulation further comprises between about 0.06 mg / ml and 0.26 mg / ml of polysorbate 80. In some embodiments, the peptide hydrogel-based formulation further comprises about 56 mg / ml of sucrose and about 0.16 mg / ml of polysorbate 80.

[0015] In an aspect, a peptide hydrogel-based formulation is provided, comprising: betyveen about 9.5 mg / ml and about 10.5 mg / ml of a peptide; between about 17.8 mg / ml and about 18.8 mg / ml sodium chloride; and between about 128 mg / ml and about 138 mg / ml of an antibody, wherein the antibody comprises a light chain comprising at least 90% sequence identity to SEQ ID NO: 2 and a heavy chain comprising at least 90% sequence identity to SEQ ID NO: 3, wherein the antibody binds to PD-1; wherein the peptide comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1, and wherein physical cross-linking of the peptide leads to formation of a peptide hydrogel.

[0016] In some embodiments, the peptide comprises SEQ ID NO: 1. In some embodiments, the antibody comprises a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO:4, an HCDR2 comprising SEQ ID NO:5. and an HCDR3 comprising SEQ ID NO:6; and a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO:7, an LCDR2 comprising SEQ ID NO:8, and an LCDR3 comprising SEQ ID NO:9.

[0017] In some embodiments, the peptide concentration is about 10.0 mg / ml. In some embodiments, the sodium chloride concentration is about 18.3 mg / ml. In some embodiments, the antibody concentration is about 133 mg / ml.

[0018] In some embodiments, the antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 3. In some embodiments, the antibody is pembrolizumab.

[0019] In some embodiments, the peptide comprises SEQ ID NO: 1, the peptide concentration is about 10.0 mg / ml, the sodium chloride concentration is about 18.3 mg / ml, the antibody concentration is about 133 mg / ml, and the antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 3.

[0020] In some embodiments, the peptide hydrogel-based formulation further comprises between about 51 mg / ml and about 61 mg / ml of sucrose. In some embodiments, the peptide hydrogel-based formulation further comprises between about 0.06 mg / ml and about 0.26 mg / ml of polysorbate 80.

[0021] In an aspect, a method of making a peptide hydrogel-based formulation with an encapsulated antibody is provided, comprising: adding a peptide and an antibody to a solution containing water to make an antibody / peptide solution, wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1 and wherein the antibody comprises a sequence that is at least 90% identical to SEQ ID NO: 2 and a sequence that is at least 90% identical to SEQ ID NO: 3, wherein the antibody binds to PD-1 ; and adding sodium chloride to the antibody / peptide solution, wherein the presence of sodium chloride allows physical cross-linking of the peptide and formation of a peptide hydrogel and encapsulation of the antibody.

[0022] In some embodiments, the antibody / peptide solution is filtered prior to adding the sodium chloride. In some embodiments, the sodium chloride that is added is in solution. In some embodiments, the ratio of sodium chloride solution: antibody / peptide solution equals from about 0.8 to about 1.2: from about 14.1 to about 15.1.

[0023] In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is between about 0.8 and about 1.2: about 2.65 and about 2.85. In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is about 1.0: about 2.75. In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is between about 0.8 and about 1.2: about 6.2 and about 6.8. In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is about 1.0: about 6.5. In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is betweenabout 0.8 and about 1.2: between about 13.8 and about 14.2. In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (ii) is about 1.0: about 14.0.

[0024] In some embodiments, the antibody solution further comprises between about 1.99 mg / ml and about 2.19 mg / ml of L-histidine hydrocholoride. In some embodiments, the antibody solution comprises about 2.09 mg / ml of L-histidine hydrochloride.

[0025] In some embodiments, the antibody solution further comprises between about 65 mg / ml and about 75 mg / ml of sucrose. In some embodiments, the antibody solution comprises about 70 mg / ml of sucrose.

[0026] In some embodiments, the antibody solution further comprises from about 0. 15 mg / ml to about 0.25 mg / ml of polysorbate 80. In some embodiments, the antibody solution comprises about 0.20 mg / ml of polysorbate 80.

[0027] In some embodiments, the antibody solution comprises about 165 mg / ml of the antibody. In some embodiments, the antibody in the antibody solution comprises a light chain that comprises SEQ ID NO: 2 and a heavy chain that comprises SEQ ID NO: 3.

[0028] In some embodiments, the ratio of sodium chloride solution: antibody / peptide solution is about 1.0: about 14.6.

[0029] In an aspect, a method of making a hydrogel with an encapsulated antibody is provided, comprising: providing a solution comprising a peptide in water in a first container (first solution), wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1; mixing an inorganic salt and an antibody solution in a second container (second solution), wherein the antibody comprises a light chain comprising a sequence that is at least 90% identical to SEQ ID NO: 2 and a heavy chain comprising a sequence that is at least 90% identical to SEQ ID NO: 3, wherein the antibody comprises a concentration that is between about 160 mg / ml and about 170 mg / ml and wherein the antibody binds to PD-1: and mixing the first solution with the second solution to form a third solution, comprising the hydrogel with the encapsulated antibody.

[0030] In some embodiments, the first solution is mixed with the second solution in a syringe.

[0031] In some embodiments, the peptide comprises the sequence of SEQ ID NO: 1.

[0032] In some embodiments, the antibody comprises a light chain that comprises SEQ ID NO: 2 and a heavy chain that comprises SEQ ID NO: 3.

[0033] In some embodiments, the inorganic salt comprises sodium chloride. In some embodiments, the concentration of sodium chloride in the second solution is between about 4.8 M and about 5.2 M. In some embodiments, the concentration of sodium chloride in the second solution is about 5.0 M.

[0034] In some embodiments, the antibody concentration in the antibody solution is about 165 mg / ml.

[0035] In some embodiments, the concentration of the peptide in the first solution is between about 48 mg / ml and about 52 mg / ml. In some embodiments, the concentration of the peptide in the first solution is about 50 mg / ml.

[0036] In some embodiments, the method further comprises incubating the third solution. In some embodiments, the third solution is incubated for 10 minutes or less.

[0037] In an aspect, a peptide hydrogel-based formulation is provided, wherein the formulation comprises: about 8 mg / ml to about 12 mg / ml of a peptide, wherein the peptide comprises a sequence that is at least 90% identical to S EQ ID NO: 1; from about 118 mg / ml to about 126 mg / ml of an antibody, wherein the antibody comprises a light chain comprising a sequence that is at least 90% identical to SEQ ID NO: 2 and a heavy chain comprising a sequence that is at least 90% identical to SEQ ID NO: 3, wherein the antibody binds PD-1; and from about 15 mg / ml to about 19 mg / ml of an inorganic salt.

[0038] In some embodiments, the peptide comprises the sequence of SEQ ID NO: 1.

[0039] In some embodiments, the antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 3.

[0040] In some embodiments, the inorganic salt comprises sodium chloride.

[0041] In some embodiments, the formulation comprises about 122 mg / ml of the antibody.

[0042] In some embodiments, the formulation comprises about 17 mg / ml of the inorganic salt.

[0043] In an aspect, a method of making a peptide hydrogel-based formulation with an encapsulated antibody is provided, comprising:(i) adding lyophilized peptide to a first container containing water (peptide solution), and adding sodium chloride to a second container containing water (sodium chloride solution), wherein the final concentration of the lyophilized peptide powder in the first container is between about 70 mg / ml and about 80 mg / ml and the final concentration of the sodium chloride in the second container is between about 279 mg / ml and about 289 mg / ml, and wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1;(ii) adding an antibody solution and the peptide solution, to a third container (antibody / peptide solution), wherein the antibody solution comprises between about 160 and 170 mg / ml of antibody and wherein the antibody comprises a sequence that is at least 90% identical to SEQ ID NO: 2 and a sequence that is at least 90% identical to SEQ ID NO: 3; and(iii) filtering the sodium chloride solution and filtering the antibody / peptide solution;(iv) adding the filtered sodium chloride solution and the filtered antibody / peptide solution to a fourth container, wherein, after adding the filtered solutions to the fourth container, the fourth container comprises a ratio of filtered sodium chloride solution: filtered antibody / peptide solution equals between about 0.8 and 1.2: between about 14.1 and 15.1.

[0044] In some embodiments, the ratio of the peptide solution: antibody solution in step (ii) is between about 0.8 and about 1.2 (peptide solution): about 2.65 and 2.85 (antibody solution). In some embodiments, the ratio of the peptide solution: antibody solution in step (ii) is about 1.0 (peptide solution): about 2.75 (antibody solution).

[0045] In some embodiments, the ratio of the peptide solution: antibody solution in step (ii) is between about 0.8 and about 1.2 (peptide solution): about 6.4 and about 6.6 (antibody solution). In some embodiments, the ratio of the peptide solution: antibody solution in step (ii) is about 1.0 (peptide solution): about 6.5 (antibody solution).

[0046] In some embodiments, the ratio of the peptide solution: antibody solution in step (ii) is between about 0.8 and about 1.2 (peptide solution): between about 13.8 and about 14.2 (antibody solution). In some embodiments, the ratio of the peptide solution: antibody solution in step (ii) is about 1.0 (peptide solution): about 14.0 (antibody solution).

[0047] In some embodiments, the antibody solution further comprises between about 1.99 mg / ml and about 2.19 mg / ml of L-histidine hydrocholoride. In some embodiments, the antibody solution comprises about 2.09 mg / ml of L-histidine hydrochloride.

[0048] In some embodiments, the antibody solution further comprises between about 65 mg / ml and about 75 mg / ml of sucrose. In some embodiments, the antibody solution comprises about 70 mg / ml of sucrose.

[0049] In some embodiments, the antibody solution comprises between about 0.15 mg / ml and about 0.25 mg / ml of polysorbate 80. In some embodiments, the antibody solution comprises about 0.20 mg / ml of polysorbate 80.

[0050] In some embodiments, the final concentration of the lyophilized peptide in the first container and the second container is about 75 mg / ml.

[0051] In some embodiments, the final concentration of the sodium chloride in the first container and the second container is about 284 mg / ml.

[0052] In some embodiments, the antibody solution comprises about 165 mg / ml of antibody. In some embodiments, the antibody in the antibody solution comprises SEQ ID NO: 2 and SEQ ID NO: 3.26114

[0053] In some embodiments, in step (iv) the ratio of filtered sodium chloride solution: filtered antibody / peptide solution is about 1.0 (filtered sodium chloride solution): about 14.6 (filtered antibody / peptide solution).

[0054] In an aspect, a method of making a hydrogel with an encapsulated antibody is provided, comprising: adding a peptide to water in a first container (first solution), wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1; adding an inorganic salt to an antibody solution in a second container (second solution), wherein the antibody comprises a light chain comprising a sequence that is at least 90% identical to SEQ ID NO: 2 and a heavy chain comprising a sequence that is at least 90% identical to SEQ ID NO: 3, wherein the antibody compnses a concentration that is between about 160 mg / ml and about 170 mg / ml; and filtering and mixing the first solution with the second solution in a syringe to create a third solution.

[0055] In some embodiments, the first solution is mixed with the second solution in a syringe.

[0056] In some embodiments, the peptide comprises the sequence of SEQ ID NO: 1. In some embodiments, the concentration of the peptide is between about 48 mg / ml and about 52 mg / ml. In some embodiments, the concentration of the peptide is about 50 mg / ml.

[0057] In some embodiments, the antibody comprises a light chain that comprises SEQ ID NO: 2 and a heavy chain that comprises SEQ ID NO: 3. In some embodiments, the antibody concentration in the antibody solution is about 165 mg / ml.

[0058] In some embodiments, the inorganic salt comprises sodium chloride. In some embodiments, the concentration of sodium chloride in the second solution is between about 4.8 M and about 5.2 M. In some embodiments, the concentration of sodium chloride in the second solution is about 5.0 M.

[0059] In some embodiments, the method further comprises incubating the third solution. In some embodiments, the third solution is incubated for 10 minutes or less.

[0060] In an aspect, a peptide hydrogel-based formulation is provided. In some embodiments, the peptide hydrogel-based formulation comprises: between about 8 mg / ml and about 12 mg / ml of a peptide, wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1; between about 118 mg / ml and about 126 mg / ml of an antibody, wherein the antibody comprises a light chain comprising a sequence that is at least 90% identical to SEQ ID NO: 2 and a heavy chain comprising a sequence that is at least 90% identical to SEQ ID NO: 3, wherein the antibody binds to PD-1; and between about 15 mg / ml and about 19 mg / ml of an inorganic salt.

[0061] In some embodiments, the peptide comprises the sequence of SEQ ID NO: 1.26114

[0062] In some embodiments, the antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 3. In some embodiments, the antibody concentration comprises about 122 mg / ml.

[0063] In some embodiments, the inorganic salt comprises sodium chloride. In some embodiments, the inorganic salt concentration is about 17 mg / ml.

[0064] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 provides a representative chromatogram showing separation of the main peak (between about 2.1 minutes and 3.3 minutes) and the high molecular weight peak (between about 1.4 minutes and 2. 1 minutes), after separation of pembrolizumab using size exclusion chromatography, as described in Example 2.

[0066] FIGs. 2A-2C provide data summarizing the percent peak area for high molecular weight species at different temperatures, using Prototypes 1-6 of hydrogel formulations, as described in Example 2. FIG. 2A shows percent peak of high molecular weight species at 5°C, using each of Prototypes 1-6, as described in Example 2. FIG. 2B shows percent peak of high molecular weight species at 25°C, using each of Prototypes 1-6, as described in Example 2. FIG. 2C shows percent peak of high molecular weight species at 40°C, using each of Prototypes 1-6, as described in Example 2.

[0067] FIG. 3 provides data summarizing pembrolizumab stability at different time points, after expelling a hydrogel encapsulating pembrolizumab from a syringe, as described in Example 3.

[0068] FIG. 4 provides data summarizing various rheological measurements, after expelling a hydrogel encapsulating pembrolizumab from a syringe, as described in Example 3.

[0069] FIG. 5 provides a schematic of an exemplary embodiment expelling and recovering a hydrogel comprising a peptide (SEQ ID NO: 1) that encapsulates pembrolizumab, from a syringe, as described in Example 3.

[0070] FIG. 6 provides data showing inhibition of pembrolizumab monomer loss from nonreduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS) experiments.

[0071] FIG. 7 provides data showing inhibition of low molecular weight (LMW) species formation using CD-SDS experiments.26114

[0072] FIG. 8 provides visual observations of turbidity development of pembrolizumab samples at different dilutions and different incubation durations.

[0073] FIG. 9 provides data showing a plot of turbidity measurements of pembrolizumab samples at different dilutions and different incubation durations.

[0074] FIGs. 10A-10F provide data showing pembrolizumab binding potency using enzyme- linked immunosorbent assay (ELISA).

[0075] FIG. 11 provides data comparing immunogenicity between pembrolizumab and pembrolizumab encapsulated in a hydrogel.

[0076] FIG. 12 provides data comparing PK profiles in pembrolizumab in hydrogel and pembrolizumab in a histidine formulation.DETAILED DESCRIPTION

[0077] The present disclosure is directed to formulations and peptide hydrogel-based formulations that can be used in protein therapeutics.

[0078] In some embodiments, the peptide hydrogel-based formulations comprise an encapsulated protein (e.g., an antibody such as pembrolizumab). In some embodiments, the antibody comprises a light chain sequence that is at least 90% identical to SEQ ID NO: 2. In some embodiments, the antibody comprises a heavy chain sequence that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the antibody binds to PD-1. In some embodiments, the antibody comprises a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO:4, an HCDR2 comprising SEQ ID NO:5, and an HCDR3 comprising SEQ ID NO:6; and a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 7, an LCDR2 comprising SEQ ID NO:8, and an LCDR3 comprising SEQ ID NO:9.

[0079] In some embodiments, the peptide hydrogel-based formulations disclosed herein function as protection matrices to prevent the aggregation of encapsulated proteins (e.g., an antibody such as pembrolizumab) at various temperatures, for example, temperatures that are at least 25°C. at least 30°C. at least 35°C, at least 40°C, at least 45°C, at least 50°C, at least 60°C. at least 65°C, at least 70°C, or at least 75°C. In some embodiments, the peptide hydrogel-based formulations disclosed herein function as protection matrices to prevent the aggregation of the encapsulated proteins (e.g., an antibody such as pembrolizumab) when the proteins are incubated for various time periods. In some embodiments, the time-period for incubation is least 15 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0080] In some embodiments, the peptide hydrogel-based formulations disclosed herein prevent aggregation of proteins (e.g., an antibody such as pembrolizumab) at high temperatures through arresting protein molecular interaction kinetics, thereby preventing or reducing aggregation formation of the proteins. In some embodiments, any of the peptide hydrogel-based formulations described herein prevent the aggregation of proteins.

[0081] In some embodiments, the peptide hydrogel-based formulations function to inhibit monomer loss of encapsulated proteins (e.g., antibodies such as pembrolizumab) at various temperatures, for example, temperatures that are least 25°C, at least 30°C, at least 35°C, at least 40°C, at least 45°C, at least 50°C, at least 60°C, at least 65°C, at least 70°C. or at least 75°C. In some embodiments, the temperature is at least 25°C. In some embodiments, the temperature is at least 40°C. In some embodiments, the peptide hydrogel-based formulations disclosed herein function to inhibit monomer loss of encapsulated proteins (e.g., antibodies such as pembrolizumab). when the proteins are incubated at various time periods. In some embodiments, the time-period for incubation is least 15 days, at least I month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months. In some embodiments, any of the peptide hydrogel-based formulations described herein prevent monomer loss of the encapsulated proteins.

[0082] In some embodiments, the peptide hydrogel-based formulations function to inhibit formation of low molecular weight (LMW) species of encapsulated proteins (e.g., antibodies such as pembrolizumab) at various temperatures, for example, temperatures that are least 25°C, at least 30°C, at least 35°C. at least 40°C, at least 45°C, at least 50°C, at least 60°C, at least 65°C, at least 70°C, or at least 75°C. In some embodiments, the temperature is at least 25°C. In some embodiments, the temperature is at least 40°C. In some embodiments, the peptide hydrogelbased formulations disclosed herein function to inhibit formation of LMW species of encapsulated proteins (e.g., antibodies such as pembrolizumab), when the proteins are incubated at various time periods. In some embodiments, the time-period for incubation is least 15 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months. In some embodiments, any of the peptide hydrogel-based formulations described herein prevent formation of LMW species of the encapsulated proteins.

[0083] In some embodiments, the formulations and the peptide hydrogel-based formulations comprise a peptide that is at least 95% identical to SEQ ID NO: 1.

[0084] In some embodiments, the formulations and the peptide hydrogel-based formulations comprise an inorganic salt (e.g., sodium chloride). In some embodiments, the inorganic salt in the peptide hydrogel-based formulations functions to reduce repulsive electrostatic reactions between chains of the peptide, thereby allowing physical cross-linking of the peptide and formation of the peptide hydrogel-based formulation.Formulations

[0085] In certain aspects, the disclosure includes embodiments of peptide hydrogel-based formulations that include a peptide.

[0086] In some embodiments, the formulation further includes a salt, for example, an inorganic salt (e.g., sodium chloride). In some embodiments, the inorganic salt comprises one or more of the following: sodium chloride, potassium chloride, magnesium chloride, calcium chloride.

[0087] In some embodiments, the formulation further includes a buffer (e.g., L-Histidine, hydrochloride). In some embodiments, the peptide hydrogel-based formulations further include a sugar (e.g., sucrose). In some embodiments, the sugar comprises one or more of the following: sucrose, mannitol, dextrose, trehalose, lactose, and maltose.

[0088] In some embodiments, the formulation further includes polysorbate. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the polysorbate is polysorbate 80.

[0089] In some embodiments, the peptide hydrogel-based formulation includes one or more of (i) an inorganic salt (e.g., sodium chloride), (ii) a buffer (e.g., L-Histidine, hydrochloride), (iii) a sugar (e.g., sucrose), and (iv) polysorbate (e.g., polysorbate 20 or polysorbate 80).

[0090] In some embodiments, the concentration of the peptide in the formulation is between about 5 mg / ml and about 25 mg / ml, for example, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18, mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml. about 24 mg / ml, or about 25 mg / ml. In some embodiments, the concentration of the peptide is about 10 mg / ml.

[0091] In some embodiments, the concentration of the peptide in the formulation is between about 0.5 w / v % about 2.5 w / v %, for example, about 0.5 w / v %, about 0.6 w / v %, about 0.7 w / v %, about 0.8 w / v %, about 0.9 w / v %, about 1.0 w / v %, about 1.1 w / v %, about 1.2 w / v %, about 1.3 w / v %, about 1.4 w / v %, about 1.5 w / v %, about 1.6 w / v %, about 1.7 w / v %, about 1.8 w / v %, about 1.9 w / v %, about 2.0 w / v%, about 2.1 w / v %, about 2.2 w / v %, about 2.3 w / v %, about261142.4 w / v %, or about 2.5 w / v %. In some embodiments, the concentration of the peptide is about 1.0 w / v %.

[0092] In some embodiments, the peptide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1, for example, about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity7, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, or about 99% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide comprises SEQ ID NO: 1.

[0093] In some embodiments, the concentration of the inorganic salt in the formulation is between about 17.3 mg / ml and about 19.3 mg / ml, for example, about 17.3 mg / ml, about 17.4 mg / ml, about 17.5 mg / ml, about 17.6 mg / ml, about 17.7 mg / ml, about 17.8 mg / ml, about 17.9 mg / ml, about 18.0 mg / ml, about 18.1 mg / ml, about 18.2 mg / ml, about 18.3 mg / ml, about 18.4 mg / ml, about 18.5 mg / ml, about 18.6 mg / ml, about 18.7 mg / ml, about 18.8 mg / ml. about 18.9 mg / ml, about 19.0 mg / ml, about 19. 1 mg / ml, about 19.2 mg / ml, or about 19.3 mg / ml. In some embodiments, the inorganic salt is sodium chloride. In some embodiments, the concentration of the sodium chloride in the peptide hydrogel-based formulation is about 18.3 mg / ml.

[0094] In some embodiments, the concentration of the inorganic salt in the formulation is between about 1.73 w / v % and about 1.93 w / v %. for example, about 1.73 w / v %, about 1.74 w / v %, about 1.75 w / v %, about 1.76 w / v %, about 1.77 w / v % about 1.78 w / v %, about 1.79 w / v %, about 1.80 w / v %, about 1.81 w / v %, about 1.82 w / v %, about 1.83 w / v %, about 1.84 w / v %, about 1.85 w / v %, about 1.86 w / v %, about 1.87 w / v %, about 1.88 w / v %, about 1.89 w / v %, about 1.90 w / v %, about 1.91 w / v %, about 1.92 w / v %, or about 1.93 w / v %.

[0095] In some embodiments, the concentration of the sugar in the formulation is between about 46 mg / ml and about 66 mg / ml, for example, about 46 mg / ml, about 47 mg / ml, about 48 mg / ml, about 49 mg / ml, about 50 mg / ml, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, about 55 mg / ml, about 56 mg / ml, about 57 mg / ml, about 58 mg / ml, about 59 mg / ml. about 60 mg / ml, about 61 mg / ml, about 62 mg / ml, about 63 mg / ml, about 64 mg / ml. or about 65 mg / ml. In some embodiments, the concentration of the sugar in the formulation is about 56 mg / ml.

[0096] In some embodiments, the concentration of the sugar in the formulation is between about 4.6 w / v % and about 6.6 w / v %. for example, about 4.6 w / v %, about 4.7 w / v %. about 4.8 w / v %, about 4.9 w / v %, about 5.0 w / v %, about 5.1 w / v %, about 5.2 w / v %, about 5.3 w / v %, about 5.4 w / v %, about 5.5 w / v %, about 5.6 w / v %, about 5.7 w / v %, about 5.8 w / v %, about 5.9 w / v %, about 6.0 w / v %, about 6.1 w / v %, about 6.2 w / v %, about 6.3 w / v %, about 6.4 w / v %,or about 6.5 w / v %. In some embodiments, the concentration of the sugar in the peptide hydrogel -based formulation is about 5.6 w / v %.

[0097] In some embodiments, the concentration of the polysorbate in the formulation is between about 0.060 mg / ml and about 0.260 mg / ml, for example, about 0.06 mg / ml, about 0.07 mg / ml. about 0.08 mg / ml, about 0.09 mg / ml, about 0. 10 mg / ml, about 0. 11 mg / ml. about 0. 12 mg / ml, about 0.13 mg / ml, about 0.14 mg / ml, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.20 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, or about 0.26 mg / ml.

[0098] In some embodiments, the concentration of the polysorbate in the formulation is between about 0.006 w / v % and about 0.026 w / v %, for example, about 0.006 w / v %, about 0.007 w / v%, about 0.008 w / v%, about 0.009 w / v%, about 0.010 w / v %, about 0.011 w / v %, about 0.012 w / v %, about 0.013 w / v %, about 0.014 w / v %, about 0.015 w / v %, about 0.016 w / v %, about 0.017 w / v %, about 0.018 w / v %, about 0.019 w / v %, about 0.020 w / v %, about 0.021 w / v %, about 0.022 w / v %, about 0.023 w / v %, about 0.024 w / v %, about 0.025 w / v %, or about 0.026 w / v %.

[0099] In some embodiments, the concentration of the buffer in the formulation is between about 1.50 mg / ml and about 1.80 mg / ml, for example, about 1.50 mg / ml, about 1.51 mg / ml, about 1.52 mg / ml, about 1.53 mg / ml, about 1.54 mg / ml, about 1.55 mg / ml. about 1.56 mg / ml, about 1.57 mg / ml, about 1.58 mg / ml, about 1.59 mg / ml, about 1.60 mg / ml, about 1.61 mg / ml, about 1.62 mg / ml, about 1.63 mg / ml, about 1.64 mg / ml, about 1.65 mg / ml, about 1.66 mg / ml, about 1.67 mg / ml, about 1.68 mg / ml, about 1.69 mg / ml, about 1.70 mg / ml, about 1.71 mg / ml, about 1.72 mg / ml, about 1.73 mg / ml, about 1.74 mg / ml, about 1.75 mg / ml. about 1.76 mg / ml, about 1.77 mg / ml. about 1.78 mg / ml, about 1.79 mg / ml, or about 1.80 mg / ml.

[0100] In some embodiments, the concentration of the buffer in the peptide hydrogel -based formulation is between about 0.150 w / v %, about 0.151 w / v %, about 0.152 w / v %, about 0.153 w / v %, about 0.154 w / v %, about 0.155 w / v %, about 0.156 w / v %. about 0.157 w / v %, about 0. 158 w / v %. about 0. 159 w / v %, about 0. 160 w / v %. about 0. 161 w / v %, about 0. 162 w / v %, about 0. 163 w / v %, about 0. 164 w / v %, about 0. 165 w / v %, about 0. 166 w / v %, about 0. 167 w / v %, about 0.168 w / v %, about 0.169 w / v %, about 0.170 w / v %, about 0.171 w / v %, about 0.172 w / v %, about 0.173 w / v %, about 0.174 w / v %, about 0.175 w / v %, about 0.176 w / v %, about 0. 177 w / v %, about 0. 178 w / v %, about 0. 179 w / v %, or about 0. 180 w / v %.

[0101] In some embodiments, the formulation further includes an antibody. In some embodiments, the antibody comprises a light chain having at least 80% sequence identity with SEQ ID NO: 2 and a heavy chain having at least 80% sequence identity' with SEQ ID NO: 3,wherein the antibody binds to PD-1. For example, the light chain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 2. In some embodiments, the light chain comprises the sequence of SEQ ID NO: 2. For example, the heavy chain is at least 80% sequence identity with SEQ ID NO: 3, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 3. In some embodiments, the heavy chain comprises SEQ ID NO: 3.

[0102] In some embodiments, the antibody comprises a heavy chain having at least 80% identity with SEQ ID NO: 3 and a heavy chain complementarity' determining region (HCDR) 1 comprising SEQ ID NO: 4, an HCDR2 comprising SEQ ID NO: 5, and an HCDR3 comprising SEQ ID NO: 6.

[0103] In some embodiments, the antibody comprises a light chain having at least 80% identity with SEQ ID NO: 2 and a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 7, an LCDR2 comprising SEQ ID NO: 8, and an LCDR3 comprising SEQ ID NO: 9.

[0104] In some embodiments, the antibody comprises a heavy chain complementarity determining region (HCDR) 1 comprises SEQ ID NO: 4, an HCDR2 comprising SEQ ID NO: 5, and an HCDR3 comprising SEQ ID NO: 6; and a light chain complementarity7determining region (LCDR) 1 comprising SEQ ID NO: 7, an LCDR2 comprising SEQ ID NO: 8, and an LCDR3 comprising SEQ ID NO: 9.Peptide Hydrogel-Based Formulations

[0105] In certain aspects, the disclosure includes embodiments of peptide hydrogel-based formulations that include a peptide.

[0106] In some embodiments, the peptide hydrogel-based formulations further include a salt, for example, an inorganic salt (e g., sodium chloride). In some embodiments, the inorganic salt can comprise any one or more of the following: sodium chloride, potassium chloride, magnesium chloride, calcium chloride.

[0107] In some embodiments, the inorganic salt functions to reduce repulsive electrostatic interactions between chains of the peptide thereby allowing physical cross linking of the chains and formation of a peptide hydrogel.26114

[0108] In some embodiments, the peptide hydrogel-based formulations further include a buffer (e.g., L-Histidine, hydrochloride). In some embodiments, the peptide hydrogel-based formulations further include a sugar (e.g., sucrose). In some embodiments, the sugar comprises one or more of the following: sucrose, mannitol, dextrose, trehalose, lactose, maltose.

[0109] In some embodiments, the peptide hydrogel-based formulations further include polysorbate. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the polysorbate is polysorbate 80.

[0110] In some embodiments, the peptide hydrogel-based formulations include any one or more of (i) an inorganic salt (e.g., sodium chloride), (ii) a buffer (e.g., L-Histidine, hydrochloride), (iii) a sugar (e.g., sucrose), and (iv) polysorbate (e.g., polysorbate 20 or polysorbate 80).[OHl] In some embodiments, the concentration of the peptide in the peptide hydrogel-based formulation is between about 5 mg / ml and about 25 mg / ml, for example, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml. about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml. about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18, mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, or about 25 mg / ml. In some embodiments, the concentration of the peptide is about 10 mg / ml.

[0112] In some embodiments, the concentration of the peptide in the peptide hydrogel-based formulation is between about 0.5 w / v % about 2.5 w / v %, for example, about 0.5 w / v %, about 0.6 w / v %, about 0.7 w / v %, about 0.8 w / v %, about 0.9 w / v %, about 1.0 w / v %, about 1.1 w / v %, about 1.2 w / v %, about 1.3 w / v %, about 1.4 w / v %, about 1.5 w / v %, about 1.6 w / v %, about 1.7 w / v %, about 1.8 w / v %, about 1.9 w / v %. about 2.0 w / v%, about 2.1 w / v %, about 2.2 w / v %, about 2.3 w / v %, about 2.4 w / v %, or about 2.5 w / v %. In some embodiments, the concentration of the peptide is about 1.0 w / v %.

[0113] In some embodiments, the peptide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1, for example, about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity7, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, or about 99% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide comprises SEQ ID NO: 1.

[0114] In some embodiments, the concentration of the inorganic salt in the peptide hydrogelbased formulation is between about 17.3 mg / ml and about 19.3 mg / ml, for example, about 17.3 mg / ml, about 17.4 mg / ml, about 17.5 mg / ml, about 17.6 mg / ml, about 17.7 mg / ml, about 17.8 mg / ml, about 17.9 mg / ml, about 18.0 mg / ml, about 18.1 mg / ml, about 18.2 mg / ml, about 18.326114 mg / ml, about 18.4 mg / ml, about 18.5 mg / ml, about 18.6 mg / ml, about 18.7 mg / ml, about 18.8 mg / ml, about 18.9 mg / ml, about 19.0 mg / ml, about 19.1 mg / ml, about 19.2 mg / ml, or about 19.3 mg / ml. In some embodiments, the concentration of the sodium chloride in the peptide hydrogelbased formulation is about 18.3 mg / ml.

[0115] In some embodiments, the concentration of the inorganic salt in the peptide hydrogelbased formulation is between about 1.73 w / v % and about 1.93 w / v %, for example, about 1.73 w / v %, about 1.74 w / v %, about 1.75 w / v %, about 1.76 w / v %, about 1.77 w / v % about 1.78 w / v %, about 1.79 w / v %, about 1.80 w / v %, about 1.81 w / v %, about 1.82 w / v %, about 1.83 w / v %, about 1.84 w / v %, about 1.85 w / v %, about 1.86 w / v %, about 1.87 w / v %, about 1.88 w / v %, about 1.89 w / v %, about 1.90 w / v %, about 1.91 w / v %, about 1.92 w / v %, or about 1.93 w / v %.

[0116] In some embodiments, the peptide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1, for example, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%. about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide comprises SEQ ID NO: 1.

[0117] In some embodiments, the concentration of the sugar in the peptide hydrogel-based formulation is between about 46 mg / ml and about 66 mg / ml, for example, about 46 mg / ml, about 47 mg / ml, about 48 mg / ml, about 49 mg / ml, about 50 mg / ml, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, about 55 mg / ml, about 56 mg / ml, about 57 mg / ml. about 58 mg / ml, about 59 mg / ml, about 60 mg / ml, about 61 mg / ml, about 62 mg / ml, about 63 mg / ml, about 64 mg / ml, or about 65 mg / ml. In some embodiments, the concentration of the sugar in the peptide hydrogel-based formulation is about 56 mg / ml.

[0118] In some embodiments, the concentration of the sugar in the peptide hydrogel-based formulation is between about 4.6 w / v % and about 6.6 w / v %, for example, about 4.6 w / v %, about 4.7 w / v %, about 4.8 w / v %, about 4.9 w / v %, about 5.0 w / v %, about 5. 1 w / v %, about 5.2 w / v %, about 5.3 w / v %, about 5.4 w / v %, about 5.5 w / v %, about 5.6 w / v %, about 5.7 w / v %, about 5.8 w / v %, about 5.9 w / v %, about 6.0 w / v %, about 6. 1 w / v %, about 6.2 w / v %, about 6.3 w / v %. about 6.4 w / v %. or about 6.5 w / v %. In some embodiments, the concentration of the sugar in the peptide hydrogel-based formulation is about 5.6 w / v %.

[0119] In some embodiments, the concentration of the polysorbate in the peptide hydrogelbased formulation is between about 0.060 mg / ml and about 0.260 mg / ml, for example, about0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml. about 0.09 mg / ml, about 0.10 mg / ml, about0.11 mg / ml, about 0.12 mg / ml, about 0.13 mg / ml, about 0.14 mg / ml, about 0.15 mg / ml, about0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.20 mg / ml, about261140.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, or about 0.26 mg / ml.

[0120] In some embodiments, the concentration of the polysorbate in the peptide hydrogelbased formulation is between about 0.006 w / v % and about 0.026 w / v %, for example, about 0.006 w / v %, about 0.007 w / v%, about 0.008 w / v%, about 0.009 w / v%, about 0.010 w / v %, about 0.011 w / v %, about 0.012 w / v %, about 0.013 w / v %, about 0.014 w / v %, about 0.015 w / v %, about 0.016 w / v %, about 0.017 w / v %, about 0.018 w / v %, about 0.019 w / v %, about 0.020 w / v %, about 0.021 w / v %, about 0.022 w / v %, about 0.023 w / v %, about 0.024 w / v %, about 0.025 w / v %, or about 0.026 w / v %.

[0121] In some embodiments, the concentration of the buffer in the peptide hydrogel-based formulation is between about 1.50 mg / ml and about 1.80 mg / ml, for example, about 1.50 mg / ml, about 1.51 mg / ml, about 1.52 mg / ml, about 1.53 mg / ml, about 1.54 mg / ml, about 1.55 mg / ml, about 1.56 mg / ml, about 1.57 mg / ml, about 1.58 mg / ml, about 1.59 mg / ml. about 1.60 mg / ml, about 1.61 mg / ml, about 1.62 mg / ml, about 1.63 mg / ml, about 1.64 mg / ml, about 1.65 mg / ml, about 1.66 mg / ml, about 1.67 mg / ml, about 1.68 mg / ml, about 1.69 mg / ml, about 1.70 mg / ml, about 1.71 mg / ml, about 1.72 mg / ml, about 1.73 mg / ml, about 1.74 mg / ml, about 1.75 mg / ml, about 1.76 mg / ml, about 1.77 mg / ml, about 1.78 mg / ml, about 1.79 mg / ml, or about 1.80 mg / ml.

[0122] In some embodiments, the concentration of the buffer in the peptide hydrogel-based formulation is between about 0.150 w / v %, about 0.151 w / v %, about 0.152 w / v %, about 0.153 w / v %, about 0.154 w / v %, about 0.155 w / v %, about 0.156 w / v %, about 0.157 w / v %, about 0.158 w / v %, about 0.159 w / v %, about 0.160 w / v %, about 0.161 w / v %, about 0.162 w / v %, about 0. 163 w / v %, about 0. 164 w / v %, about 0. 165 w / v %. about 0. 166 w / v %, about 0. 167 w / v %, about 0.168 w / v %, about 0.169 w / v %, about 0.170 w / v %, about 0.171 w / v %, about 0.172 w / v %, about 0.173 w / v %, about 0.174 w / v %, about 0.175 w / v %, about 0.176 w / v %, about 0.177 w / v %, about 0.178 w / v %, about 0.179 w / v %, or about 0.180 w / v %.

[0123] In some embodiments, the various components in the peptide hydrogel-based formulations are present at a range of concentrations as shown in Table 1.Table 1. - Embodiments of the Concentrations of the Components in the Peptide Hydrogel- Based Formulations26114Peptide Hydrogel-Based Formulations with an Encapsulated Antibody (e.g., pembrolizumab)

[0124] In certain aspects, the disclosure includes embodiments of peptide hydrogel-based formulations that include a peptide and an encapsulated antibody (e.g., pembrolizumab (light chain: SEQ ID NO: 2; heavy’ chain: SEQ ID NO: 3)).

[0125] In some embodiments, peptide hydrogel-based formulations that include SEQ ID NO: 2 and SEQ ID NO: 3 described herein, are capable of preventing aggregation of proteins at temperatures that are at least 30°C, at least 35°C, at least 40°C, at least 45°C, at least 50°C, at least 60°C, at least 65°C, at least 70°C, or at least 75°C.

[0126] In some embodiments, the peptide hydrogel-based formulations that encapsulate an antibody further include a salt, for example, an inorganic salt (e.g., sodium chloride). In some embodiments, the inorganic salt includes any of the following: sodium chloride, potassium chloride, magnesium chloride, and calcium chloride.

[0127] In some embodiments, the presence of the inorganic salt allows physical cross linking of the peptide chains and formation of the peptide hydrogel-based formulation.

[0128] In some embodiments, the inorganic salt functions to reduce repulsive electrostatic interactions between chains of the peptide thereby allowing physical cross linking of the peptide chains and formation of the peptide hydrogel.

[0129] In some embodiments, the peptide hydrogel-based formulations include an antibody and further include a buffer (e.g., L-Histidine, hydrochloride). In some embodiments, the buffer includes any of the following: sodium citrate, potassium citrate, citric acid, sodium acetate, acetic acid, potassium acetate, glutamic acid, sodium phosphate monobasic, sodium phosphate dibasic, potassium phosphate monobasic, and potassium phosphate dibasic.26114

[0130] In some embodiments, the peptide hydrogel-based formulations include an antibody and further include a sugar (e.g., sucrose). In some embodiments, the sugar is any of the following: sucrose, mannitol, dextrose, trehalose, lactose, maltose.

[0131] In some embodiments, the peptide hydrogel-based formulations that encapsulate an antibody further include polysorbate 80. In some embodiments, the peptide hydrogel-based formulations that encapsulate an antibody further include polysorbate 20. In some embodiments, the peptide hydrogel-based formulations that encapsulate an antibody further include any one or more of (i) an inorganic salt (e.g., sodium chloride), (ii) a buffer (e.g., L-Histidine, hydrochloride), (iii) a sugar (e g., sucrose), and (iv) polysorbate 80.

[0132] In some embodiments, the encapsulated antibody comprises a light chain having at least 80% sequence identity with SEQ ID NO: 2 and a heavy chain having at least 80% sequence identity with SEQ ID NO: 3, wherein the antibody binds to PD-1. For example, the light chain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 2. In some embodiments, the light chain comprises the sequence of SEQ ID NO: 2. For example, the heavy chain is at least 80% sequence identity with SEQ ID NO: 3, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 3. In some embodiments, the heavy chain comprises SEQ ID NO: 3.

[0133] In some embodiments, the antibody comprises a heavy chain having at least 80% identity with SEQ ID NO: 3 and a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 4, an HCDR2 comprising SEQ ID NO: 5, and an HCDR3 comprising SEQ ID NO: 6.

[0134] In some embodiments, the antibody comprises a light chain having at least 80% identity with SEQ ID NO: 2 and a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 7, an LCDR2 comprising SEQ ID NO: 8, and an LCDR3 comprising SEQ ID NO: 9.

[0135] In some embodiments, the antibody comprises a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO:4, an HCDR2 comprising SEQ ID NO:5. and an HCDR3 comprising SEQ ID NO: 6; and a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO:7, an LCDR2 comprising SEQ ID NO:8, and an LCDR3 comprising SEQ ID NO:9.26114

[0136] In some embodiments, the concentration of the antibody (e.g., pembrolizumab) in the peptide hydrogel-based formulation is between about 100 mg / ml and about 160 mg / ml, for example, about 100 mg / ml, about 101 mg / ml, about 102 mg / ml, about 103 mg / ml, about 104 mg / ml, about 105 mg / ml, about 106 mg / ml, about 107 mg / ml, about 108 mg / ml, about 109 mg / ml. about 110 mg / ml, about 111 mg / ml. about 112 mg / ml, about 113 mg / ml, about 114 mg / ml, about 115 mg / ml, about 116 mg / ml, about 117 mg / ml, about 118 mg / ml, about 119 mg / ml, about 120 mg / ml, about 121 mg / ml, about 122 mg / ml, about 123 mg / ml, about 124 mg / ml, about 125 mg / ml, about 126 mg / ml, about 127 mg / ml, about 128 mg / ml, about 129 mg / ml, about 130 mg / ml, about 131, mg / ml, about 132 mg / ml, about 133 mg / ml. about 134 mg / ml, about 135 mg / ml, about 136 mg / ml, about 137 mg / ml, about 138 mg / ml, about 139 mg / ml, about 140 mg / ml, about 141 mg / ml, about 142 mg / ml, about 143 mg / ml, about 144 mg / ml, about 145 mg / ml, about 146 mg / ml, about 147 mg / ml, about 148 mg / ml, about 149 mg / ml, about 150 mg / ml, about 151 mg / ml, about 152 mg / ml, about 153 mg / ml, about 154 mg / ml, about 155 mg / ml, about 156 mg / ml, about 157 mg / ml, about 158 mg / ml, about 159 mg / ml, or about 160 mg / ml.

[0137] In some embodiments, the concentration of the antibody (e.g., pembrolizumab) in the peptide hydrogel-based formulation is between about 10.0 w / v %, and about 16.0 w / v %, for example about 10.0 w / v %, about 10.1 w / v %. about 10.2 w / v %, about 10.3 w / v %, about 10.4 w / v %, about 10.5 w / v %, about 10.6 w / v %, about 10.7 w / v %, about 10.8 w / v %, about 10.9 w / v %, about 11.0 w / v %, about 11.1 w / v %, about 11.2 w / v %, about 11.3 w / v %, about 11.4 w / v %, about 11.5 w / v %, about 11.6 w / v %, about 11.7 w / v %, about 11.8 w / v %, about 11.9 w / v %, about 12.0 w / v %, about 12. 1 w / v %, about 12.2 w / v %, about 12.3 w / v %, about 12.4 w / v %, about 12.5 w / v %, about 12.6 w / v %, about 12.7 w / v %, about 12.8 w / v %, about 12.9 w / v %, about 13.0 w / v %, about 13.1 w / v %, about 13.2 w / v %, about 13.3 w / v %, about 13.4 w / v %, about 13.5 w / v %, about 13.6 w / v %, about 13.7 w / v %, about 13.8 w / v %, about 13.9 w / v %, about 14.0 w / v %, about 14.1 w / v %, about 14.2 w / v %, about 14.3 w / v %, about 14.4 w / v %. about 14.5 w / v %, about 14.6 w / v %, about 14.7 w / v %, about 14.8 w / v %. about 14.9 w / v %, about 15.0 w / v %, about 15.1 w / v %, about 15.2 w / v %, about 15.3 w / v %, about 15.4 w / v %, about 15.5 w / v %, about 15.6 w / v %, about 15.7 w / v %, about 15.8 w / v %, about 15.9 w / v %, or about 16.0 w / v %.

[0138] In some embodiments, the concentration of the peptide in the peptide hydrogel-based formulation is between about 5 mg / ml and about 25 mg / ml, for example, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml,26114 about 18, mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, or about 25 mg / ml. In some embodiments, the concentration of the peptide is about 10 mg / ml.

[0139] In some embodiments, the concentration of the peptide in the peptide hydrogel-based formulation is between about 0.5 w / v % about 2.5 w / v %. for example, about 0.5 w / v %. about 0.6 w / v %, about 0.7 w / v %, about 0.8 w / v %, about 0.9 w / v %, about 1.0 w / v %, about 1.1 w / v %, about 1.2 w / v %, about 1.3 w / v %, about 1.4 w / v %, about 1.5 w / v %, about 1.6 w / v %, about 1.7 w / v %, about 1.8 w / v %, about 1.9 w / v %, about 2.0 w / v%, about 2.1 w / v %, about 2.2 w / v %, about 2.3 w / v %, about 2.4 w / v %, or about 2.5 w / v %. In some embodiments, the concentration of the peptide is about 1.0 w / v %.

[0140] In some embodiments, the peptide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1, for example, about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, or about 99% sequence identity to SEQ ID NO: 1 . In some embodiments, the peptide comprises SEQ ID NO: 1.

[0141] In some embodiments, the concentration of the inorganic salt in the peptide hydrogelbased formulation is between about 17.3 mg / ml and about 19.3 mg / ml, for example, about 17.3 mg / ml, about 17.4 mg / ml, about 17.5 mg / ml, about 17.6 mg / ml, about 17.7 mg / ml, about 17.8 mg / ml, about 17.9 mg / ml, about 18.0 mg / ml, about 18.1 mg / ml, about 18.2 mg / ml, about 18.3 mg / ml, about 18.4 mg / ml, about 18.5 mg / ml, about 18.6 mg / ml, about 18.7 mg / ml, about 18.8 mg / ml, about 18.9 mg / ml, about 19.0 mg / ml, about 19.1 mg / ml, about 19.2 mg / ml. or about 19.3 mg / ml. In some embodiments, the concentration of the sodium chloride in the peptide hydrogelbased formulation is about 18.3 mg / ml.

[0142] In some embodiments, the concentration of the inorganic salt in the peptide hydrogelbased formulation is between about 1.73 w / v % and about 1.93 w / v %, for example, about 1.73 w / v %. about 1.74 w / v %, about 1.75 w / v %, about 1.76 w / v %, about 1.77 w / v % about 1.78 w / v %, about 1.79 w / v %, about 1.80 w / v %, about 1.81 w / v %, about 1.82 w / v %, about 1.83 w / v %, about 1.84 w / v %, about 1.85 w / v %, about 1.86 w / v %, about 1.87 w / v %, about 1.88 w / v %, about 1.89 w / v %, about 1.90 w / v %, about 1.91 w / v %, about 1.92 w / v %, or about 1.93 w / v %.

[0143] In some embodiments, the peptide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1, for example, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide comprises SEQ ID NO: 1.26114

[0144] In some embodiments, the concentration of the sugar in the peptide hydrogel-based formulation is between about 46 mg / ml and about 66 mg / ml, for example, about 46 mg / ml, about 47 mg / ml, about 48 mg / ml, about 49 mg / ml, about 50 mg / ml, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, about 55 mg / ml, about 56 mg / ml, about 57 mg / ml, about 58 mg / ml. about 59 mg / ml, about 60 mg / ml, about 61 mg / ml, about 62 mg / ml, about 63 mg / ml. about 64 mg / ml, or about 65 mg / ml. In some embodiments, the concentration of the sugar in the peptide hydrogel-based formulation is about 56 mg / ml.

[0145] In some embodiments, the concentration of the sugar in the peptide hydrogel-based formulation is between about 4.6 w / v % and about 6.6 w / v %, for example, about 4.6 w / v %, about 4.7 w / v %, about 4.8 w / v %, about 4.9 w / v %, about 5.0 w / v %, about 5. 1 w / v %, about 5.2 w / v %, about 5.3 w / v %, about 5.4 w / v %, about 5.5 w / v %, about 5.6 w / v %, about 5.7 w / v %, about 5.8 w / v %, about 5.9 w / v %, about 6.0 w / v %, about 6.1 w / v %, about 6.2 w / v %, about 6.3 w / v %, about 6.4 w / v %, or about 6.5 w / v %. In some embodiments, the concentration of the sugar in the peptide hydrogel-based formulation is about 5.6 w / v %.

[0146] In some embodiments, the concentration of the polysorbate in the peptide hydrogelbased formulation is between about 0.060 mg / ml and about 0.260 mg / ml, for example, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.10 mg / ml, about0. 11 mg / ml, about 0. 12 mg / ml, about 0. 13 mg / ml. about 0. 14 mg / ml, about 0.15 mg / ml, about0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.20 mg / ml, about0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, or about0.26 mg / ml.

[0147] In some embodiments, the concentration of the polysorbate in the peptide hydrogelbased formulation is between about 0.006 w / v % and about 0.026 w / v %, for example, about 0.006 w / v %, about 0.007 w / v%, about 0.008 w / v %, about 0.009 w / v %, about 0.010 w / v %, about 0.011 w / v %, about 0.012 w / v %, about 0.013 w / v %, about 0.014 w / v %, about 0.015 w / v%, about 0.016 w / v %, about 0.017 w / v %, about 0.018 w / v %, about 0.019 w / v %, about 0.020 w / v %. about 0.021 w / v%, about 0.022 w / v%, about 0.023 w / v %, about 0.024 w / v %. about 0.025 w / v %, or about 0.026 w / v %.

[0148] In some embodiments, the concentration of the buffer in the peptide hydrogel-based formulation is between about 1.50 mg / ml and about 1.80 mg / ml, for example, about 1.50 mg / ml, about 1.51 mg / ml, about 1.52 mg / ml, about 1.53 mg / ml, about 1.54 mg / ml. about 1.55 mg / ml, about 1.56 mg / ml, about 1.57 mg / ml, about 1.58 mg / ml, about 1.59 mg / ml, about 1.60 mg / ml, about 1.61 mg / ml, about 1.62 mg / ml, about 1.63 mg / ml, about 1.64 mg / ml, about 1.65 mg / ml, about 1.66 mg / ml, about 1.67 mg / ml, about 1.68 mg / ml, about 1.69 mg / ml, about 1.70 mg / ml,26114 about 1.71 mg / ml, about 1.72 mg / ml, about 1.73 mg / ml, about 1.74 mg / ml, about 1.75 mg / ml, about 1.76 mg / ml, about 1.77 mg / ml, about 1.78 mg / ml, about 1.79 mg / ml, or about 1.80 mg / ml.

[0149] In some embodiments, the concentration of the buffer in the peptide hydrogel-based formulation is between about 0.150 w / v %, about 0.151 w / v %, about 0.152 w / v %, about 0.153 w / v %. about 0. 154 w / v %, about 0. 155 w / v %, about 0. 156 w / v %. about 0. 157 w / v %, about 0.158 w / v %, about 0.159 w / v %, about 0.160 w / v %, about 0.161 w / v %, about 0.162 w / v %, about 0.163 w / v %, about 0.164 w / v %, about 0.165 w / v %, about 0.166 w / v %, about 0.167 w / v %, about 0.168 w / v %, about 0.169 w / v %, about 0.170 w / v %, about 0.171 w / v %, about 0.172 w / v %, about 0.173 w / v %, about 0.174 w / v %, about 0.175 w / v %. about 0.176 w / v %, about 0. 177 w / v %, about 0. 178 w / v %, about 0. 179 w / v %, or about 0.180 w / v %.

[0150] In an aspect, a peptide hydrogel-based formulation is provided, comprising: between about 9.5 mg / ml and about 10.5 mg / ml of a peptide; between about 17.8 mg / ml and about 18.8 mg / ml sodium chloride; and between about 128 mg / ml and about 138 mg / ml of an antibody, wherein the antibody comprises a light chain comprising at least 90% sequence identity to SEQ ID NO: 2 and a heavy chain comprising at least 90% sequence identity to SEQ ID NO: 3, wherein the antibody binds to PD-1; wherein the peptide comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1, and wherein physical cross-linking of the peptide leads to formation of a peptide hydrogel.

[0151] In some embodiments, the concentration of the peptide is about 9.5 mg / ml, about 9.6 mg / ml, about 9.7 mg / ml, about 9.8 mg / ml, about 9.9 mg / ml, about 10.0 mg / ml, about 10.1 mg / ml, about 10.2 mg / ml, about 10.3 mg / ml, about 10.4 mg / ml, or about 10.5 mg / ml.

[0152] In some embodiments, the concentration of the sodium chloride is about 17.8 mg / ml, about 17.9 mg / ml. about 18.0 mg / ml, about 18.1 mg / ml, about 18.2 mg / ml, about 18.3 mg / ml, about 18.4 mg / ml, about 18.5 mg / ml, about 18.6 mg / ml, about 18.7 mg / ml, or about 18.8 mg / ml.

[0153] In some embodiments, the concentration of the antibody is about 128 mg / ml, about 129 mg / ml, about 130 mg / ml, about 131 mg / ml, about 132 mg / ml, about 133 mg / ml, about 134 mg / ml. about 135 mg / ml, about 136 mg / ml. about 137 mg / ml, or about 138 mg / ml.

[0154] In some embodiments, the peptide comprises a sequence having at least 95% sequence identity, having at least 96% sequence identity, having at least 97% sequence identity, having at least 98% sequence identity, or having at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide comprises SEQ ID NO: 1.

[0155] In some embodiments, the antibody light chain comprises a sequence having at least 90% sequence identity, having at least 91% sequence identity, having at least 92% sequence identity, having at least 93% sequence identity, having at least 94% sequence identity, having at26114 least 95% sequence identity, having at least 96% sequence identity, having at least 97% sequence identity, having at least 98% sequence identity, or having at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the antibody light chain comprises SEQ ID NO: 2.

[0156] In some embodiments, the antibody light chain comprises a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 7. an LCDR2 comprising SEQ ID NO: 8, and an LCDR3 comprising SEQ ID NO: 9.

[0157] In some embodiments, the antibody heavy chain comprises a sequence having at least 90% sequence identity, having at least 91% sequence identity, having at least 92% sequence identity, having at least 93% sequence identity, having at least 94% sequence identity, having at least 95% sequence identity, having at least 96% sequence identity, having at least 97% sequence identity7, having at least 98% sequence identity, or having at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the antibody heavy chain comprises SEQ ID NO: 3.

[0158] In some embodiments, the antibody heavy chain comprises a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 4, an HCDR2 comprising SEQ ID NO: 5, and an HCDR3 comprising SEQ ID NO: 6.

[0159] In some embodiments, the antibody is pembrolizumab.

[0160] In some embodiments, the peptide hydrogel-based formulation further comprises between about 51 mg / ml and about 61 mg / ml of sucrose, for example, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, about 55 mg / ml, about 56 mg / ml, about 57 mg / ml, about 58 mg / ml, about 59 mg / ml, about 60 mg / ml, or about 61 mg / ml.

[0161] In some embodiments, the peptide hydrogel-based formulation further comprises between about 0.06 mg / ml and about 0.26 mg / ml of polysorbate 80, for example, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.10 mg / ml, about 0.11 mg / ml, about 0.12 mg / ml, about 0.13 mg / ml, about 0.14 mg / ml, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.20 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, or about 0.26 mg / ml.

[0162] In some embodiments, the various components are present in the peptide hydrogelbased formulation at a range of concentrations as shown in Table 2.Table 2 - Embodiments of the Concentrations of the Components in the Peptide Hydrogel- Based Formulations that Include an AntibodyAssembly of the Peptide Hydrogel-Based Formulations

[0163] In some embodiments, the peptide hydrogel-based formulations described herein selfassemble when the peptide is contacted with an inorganic salt. In some embodiments, the inorganic salt comprises one or more of the following: sodium chloride, potassium chloride, magnesium chloride, and calcium chloride. In some embodiments, the peptide hydrogel-based formulations self-assemble when the peptide is contacted with an antibody (e.g., pembrolizumab) and inorganic salt. In some embodiments, the inorganic salt comprises any of the follow ing: sodium chloride, potassium chloride, magnesium chloride, and calcium chloride.

[0164] In some embodiments, the presence of the organic salt allows physical cross-linking of the chains of the peptide and formation of the peptide hydrogel. In some embodiments, the organic salt functions to reduce repulsive electrostatic interactions between chains of the peptide, thereby allowing physical cross-linking of the chains of the peptide and formation of the peptide hydrogel.

[0165] In some embodiments, assembly of peptide hydrogel-based formulations described herein results in encapsulation of the antibody. In some embodiments, encapsulation of the antibody in the peptide hydrogel-based formulation protects the antibody from external stimuli of the environment. In some embodiments, encapsulation of the antibody in the peptide hydrogelbased formulation prevents degradation of the antibody.

[0166] In some embodiments, the peptide hydrogel-based formulation comprises a synthetic peptide that comprises a sequence that is at least 90% identical to SEQ ID NO: 1, for example, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94%identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 1. In some embodiments, the synthetic peptide comprises SEQ ID NO: 1.

[0167] In some embodiments, the peptide is contacted with an antibody that comprises a light chain that is at least 90% identical to SEQ ID NO: 2, for example, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 2. In some embodiments, the light chain comprises SEQ ID NO: 2. In some embodiments, the peptide is contacted with an antibody that comprises a heavy chain that is at least 90% identical to SEQ ID NO: 3, for example, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 3. In some embodiments, the heavy chain comprises SEQ ID NO: 3.Methods of Self-Assembling the Peptide Hydrogel-Based Formulations

[0168] In an aspect, methods of self-assembling the peptide hydrogel -based formulations are provided. In some embodiments, the method includes mixing a solution of an antibody in water with a peptide solution comprising a peptide in water or other solvent and adding sodium chloride, wherein the presence of the sodium chloride allows physical cross-linking of the chains of the peptide and formation of the peptide hydrogel.

[0169] In some embodiments, the method includes the following steps 1-6, as outlined below:1. Adding water or a solvent to a first container and adding water or a solvent to a second container.2. Adding sodium chloride to the first container followed by mixing (sodium chloride solution).3. Adding a peptide to the second container followed by mixing (peptide solution).4. Adding an antibody solution (e.g., a solution containing pembrolizumab) and the peptide solution from step 3. to a third container, in a desired ratio, followed by mixing (antibody / peptide solution).

[0170] In some embodiments, the desired ratio of peptide solution: antibody solution is between about 0.8 and about 1.2 (peptide solution): between about 2.65 and about 2.85 (antibody solution). In some embodiments, the desired ratio of peptide solution: antibody solution is about 1 (peptide solution): about 2.75 (antibody solution).

[0171] In some embodiments, the desired ratio of peptide solution: antibody solution is between about 0.8 and about 1.2 (peptide solution): between about 13.8 and about 14.2 (antibody26114 solution). In some embodiments, the desired ratio of peptide solution: antibody solution is about 1 (peptide solution): about 14.0 (antibody solution).5. Filtering the antibody / peptide solution from step 4. and filtering the sodium chloride solution from step 2.6. In a fourth container, mixing the filtered antibody / peptide solution and the filtered sodium chloride solution from step 5, in a desired ratio.

[0172] In some embodiments, the desired ratio of the filtered sodium chloride solution: filtered antibody / peptide solution is between about 0.8 and about 1.2 (filtered sodium chloride solution): between about 14.1 and about 15.1 (filtered antibody / peptide solution). In some embodiments, the desired ratio of the filtered sodium chloride solution: filtered antibody / peptide solution is about 1 (filtered sodium chloride solution): about 14.6 (filtered antibody / peptide solution).

[0173] In embodiments, water is used as the solvent in the above method. Water can include inorganic salt (e.g., sodium chloride). The inorganic salt can include any of the following sodium chloride, potassium chloride, magnesium chloride, calcium chloride.

[0174] A buffer salt (e.g., L-Histidine, hydrochloride) can be added to the solvent. The buffer salt could include any of the following sodium citrate, potassium citrate, citric acid, sodium acetate, acetic acid, potassium acetate, glutamic acid, and sodium phosphate monobasic, sodium phosphate dibasic, potassium phosphate monobasic, and potassium phosphate dibasic.

[0175] A sugar (e.g., sucrose) can be added to the solvent. The sugar can be any of the following sucrose, mannitol, dextrose, trehalose, lactose, and maltose.

[0176] A surfactant can be added to the solvent. In some embodiments, the surfactant comprises polysorbate 80 or polysorbate 20.

[0177] In some embodiments, the antibody solution comprises between about 150 mg / ml and about 180 mg / ml of pembrolizumab (SEQ ID NO: 2 and SEQ ID NO: 3), for example, about 150 mg / ml, about 151 mg / ml, about 152 mg / ml, about 153 mg / ml, about 154 mg / ml, about 155 mg / ml, about 156 mg / ml, about 157 mg / ml, about 158 mg / ml, about 159 mg / ml, about 160 mg / ml. about 161 mg / ml, about 162 mg / ml. about 163 mg / ml, about 164 mg / ml, about 165 mg / ml, about 166 mg / ml, about 167 mg / ml, about 168 mg / ml, about 169 mg / ml, about 170 mg / ml, about 171 mg / ml, about 172 mg / ml, about 173 mg / ml, about 174 mg / ml, about 175 mg / ml, about 176 mg / ml, about 177 mg / ml, about 178 mg / ml, about 179 mg / ml, or about 180 mg / ml.

[0178] In some embodiments, the antibody solution comprises between about 15.0 w / v % and about 18.0 w / v % of pembrolizumab (SEQ ID NO: 2 and SEQ ID NO: 3), for example, about 15.0 w / v %, about 15.1 w / v %, about 15.2 w / v %, about 15.3 w / v %, about 15.4 w / v %, about2611415.5 w / v %, about 15.6 w / v %, about 15.7 w / v %, about 15.8 w / v %, about 15.9 w / v %, about16.0 w / v %, about 16.1 w / v %, about 16.2 w / v %, about 16.3 w / v %, about 16.4 w / v %, about16.5 w / v %, about 16.6 w / v %, about 16.7 w / v %, about 16.8 w / v %, about 16.9 w / v %, about17.0 w / v %, about 17.1 w / v %, about 17.2 w / v %, about 17.3 w / v %, about 17.4 w / v %, about17.5 w / v %, about 17.6 w / v %, about 17.7 w / v %, about 17.8 w / v %, about 17.9 w / v %, or about18.0 w / v %.

[0179] In some embodiments, the antibody solution comprises between about 1.95 mg / ml and about 2.25 mg / ml of L-histidine hydrochloride, for example, about 1.95 mg / ml, about 1.96 mg / ml, about 1.97 mg / ml, about 1.98 mg / ml, about 1.99 mg / ml, about 2.00 mg / ml. about 2.01 mg / ml, about 2.02 mg / ml, about 2.03 mg / ml, about 2.04 mg / ml, about 2.05 mg / ml, about 2.06 mg / ml, about 2.07 mg / ml, about 2.08 mg / ml, about 2.09 mg / ml, about 2.10 mg / ml, about 2.11 mg / ml, about 2.12 mg / ml, about 2.13 mg / ml, about 2.14 mg / ml, about 2.15 mg / ml, about 2.16 mg / ml, about 2.17 mg / ml, about 2.18 mg / ml, about 2.19 mg / ml, about 2.20 mg / ml. about 2.21 mg / ml, about 2.22 mg / ml, about 2.23 mg / ml, about 2.24 mg / ml, or about 2.25 mg / ml.

[0180] In some embodiments, the antibody solution comprises between about 0.195 w / v % and about 0.225 w / v % of L-histidine hydrochloride, for example, about 0.195 w / v %, about 0.196 w / v %, about 0.197 w / v %, about 0.198 w / v %, about 0.199 w / v %, about 0.200 w / v %, about 0.201 w / v %. about 0.202 w / v %, about 0.203 w / v %, about 0.204 w / v %, about 0.205 w / v %, about 0.206 w / v %, about 0.207 w / v %, about 0.208 w / v %, about 0.209 w / v %, about 0.210 w / v %, about 0.211 w / v %, about 0.212 w / v %, about 0.213 w / v %, about 0.214 w / v %, about 0.215 w / v %, about 0.216 w / v %, about 0.217 w / v %, about .0218 w / v %, about 0.219 w / v %, about 0.220 w / v %, about .0221 w / v %, about .0222 w / v %, about .0223 w / v %, about .0224 w / v %, or about .0225 w / v %.

[0181] In some embodiments, the antibody solution comprises between about 60 mg / ml, and about 80 mg / ml of sucrose, for example about 60 mg / ml, about 61 mg / ml, about 62 mg / ml, about 63 mg / ml, about 64 mg / ml, about 65 mg / ml, about 66 mg / ml, about 67 mg / ml, about 68 mg / ml, about 69 mg / ml, about 70 mg / ml, about 71 mg / ml, about 72 mg / ml, about 73 mg / ml. about 74 mg / ml, about 75 mg / ml, about 76 mg / ml, about 77 mg / ml, about 78 mg / ml, about 79 mg / ml, or about 80 mg / ml.

[0182] In some embodiments, the antibody solution comprises between about 6.0 w / v %, and about 8.0 w / v % of sucrose, for example about 6.0 w / v %, about 6. 1 w / v %, about 6.2 w / v %, about 6.3 w / v %, about 6.4 w / v %, about 6.5 w / v %, about 6.6 w / v %, about 6.7 w / v %, about 6.8 w / v %, about 6.9 w / v %, about 7.0 w / v %, about 7.1 w / v %, about 7.2 w / v %, about 7.3 w / v %,26114 about 7.4 w / v %, about 7.5 w / v %, about 7.6 w / v %, about 7.7 w / v %, about 7.8 w / v %, about 7.9 w / v %, or about 8.0 w / v %.

[0183] In some embodiments, the antibody solution comprises between about 0.05 mg / ml and about 0.35 mg / ml of polysorbate 80, for example, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml. about 0. 10 mg / ml. about 0. 11 mg / ml, about0.12 mg / ml, about 0.13 mg / ml, about 0.14 mg / ml, about 0.15 mg / ml, about 0.16 mg / ml, about0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.20 mg / ml, about 0.21 mg / ml, about0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.26 mg / ml, about0.27 mg / ml, about 0.28 mg / ml, about 0.29 mg / ml. about 0.30 mg / ml, about 0.31 mg / ml, about0.32 mg / ml, about 0.33 mg / ml, about 0.34 mg / ml, or about 0.35 mg / ml.

[0184] In some embodiments, the antibody solution comprises between about 0.005 w / v % and about 0.035 w / v % of polysorbate 80, for example, about 0.005 w / v %, about 0.006 w / v %, about 0.007 w / v %. about 0.008 w / v %, about 0.009 w / v %, about 0.010 w / v %, about 0.011 w / v %, about 0.012 w / v %, about 0.013 w / v %, about 0.014 w / v %. about 0.015 w / v %, about 0.016 w / v %, about 0.017 w / v %, about 0.018 w / v %, about 0.019 w / v %, about 0.020 w / v %, about 0.021 w / v %, about 0.022 w / v %, about 0.023 w / v %, about 0.024 w / v %, about 0.025 w / v %, about 0.026 w / v %. about 0.027 w / v %, about 0.028 w / v %, about 0.029 w / v %, about 0.030 w / v %, about 0.031 w / v %, about 0.032 w / v %, about 0.033 w / v %. about 0.034 w / v %, or about 0.035 w / v %.

[0185] In some embodiments, the antibody comprises a light chain sequence that is at least 90% identical to SEQ ID NO: 2 and comprises a heavy chain sequence that is at least 90% identical to heavy chain SEQ ID NO: 3, wherein the antibody binds to PD-1. In some embodiments, the light chain sequence is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 2. In some embodiments, the light chain sequence comprises SEQ ID NO: 2. In some embodiments, the heavy chain sequence is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 3. In some embodiments, the heavy chain sequence comprises SEQ ID NO: 3.

[0186] In some embodiments, the peptide used in the above protocol comprises a sequence that is at least 90% identical to SEQ ID NO: 1, for example, about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical,26114 about 96% identical about 97% identical, about 98% identical, or about 99% identical to SEQ ID NO: 1. In some embodiments, the peptide comprises SEQ ID NO: 1.

[0187] In some embodiments, the peptide that is added to the first container and second container in steps 2 and 3 of the above method comprises a lyophilized peptide. In some embodiments, the lyophilized peptide is prepared through any lyophilization technique described herein or otherwise known in the art.

[0188] In some embodiments, after mixing in steps 2 and 3 of the above method, the concentration of the peptide in the first container and the second container is between about 60 mg / ml and 90 mg / ml, for example, about 60 mg / ml. about 61 mg / ml, about 62 mg / ml, about 63 mg / ml, about 64 mg / ml, about 65 mg / ml, about 66 mg / ml, about 67 mg / ml, about 68 mg / ml, about 69 mg / ml, about 70 mg / ml, about 71 mg / ml, about 72 mg / ml, about 73 mg / ml, about 74 mg / ml, about 75 mg / ml, about 76 mg / ml, about 78 mg / ml, about 79 mg / ml, about 80 mg / ml, about 81 mg / ml, about 82 mg / ml, about 83 mg / ml, about 84 mg / ml, about 85 mg / ml, about 86 mg / ml, about 87 mg / ml, about 88 mg / ml, about 89 mg / ml, or about 90 mg / ml.

[0189] In some embodiments, after mixing in steps 2 and 3 of the above method, the concentration of the peptide in the first container and the second container is between about 6.0 w / v % and 9.0 w / v %, for example, about 6.0 w / v %, about 6.1 w / v %, about 6.2 w / v %, about6.3 w / v %, about 6.4 w / v %. about 6.5 w / v %. about 6.6 w / v %, about 6.7 w / v %, about 6.8 w / v %, about 6.9 w / v %, about 7.0 w / v %, about 7.1 w / v %, about 7.2 w / v %, about 7.3 w / v %, about7.4 w / v %, about 7.5 w / v %, about 7.6 w / v %, about 7.8 w / v %, about 7.9 w / v %, about 8.0 w / v %, about 8.1 w / v %, about 8.2 w / v %, about 8.3 w / v %, about 8.4 w / v %, about 8.5 w / v %, about 8.6 w / v %, about 8.7 w / v %, about 8.8 w / v %. about 8.9 w / v %, or about 9.0 w / v %.

[0190] In some embodiments, after mixing in steps 2 and 3 of the above method, the concentration of the sodium chloride is between about 270 mg / ml and about 300 mg / ml, for example, about 270 mg / ml, about 271 mg / ml, about 272 mg / ml, about 273 mg / ml, about 274 mg / ml, about 275 mg / ml, about 276 mg / ml, about 277 mg / ml, about 278 mg / ml, about 279 mg / ml. about 280 mg / ml, about 281 mg / ml. about 282 mg / ml, about 283 mg / ml, about 284 mg / ml, about 285 mg / ml, about 286 mg / ml, about 287 mg / ml, about 288 mg / ml, about 289 mg / ml, about 290 mg / ml, about 291 mg / ml, about 292 mg / ml, about 293 mg / ml, about 294 mg / ml, about 295 mg / ml, about 296 mg / ml, about 297 mg / ml, about 298 mg / ml, about 299 mg / ml, or about 300 mg / ml.

[0191] In some embodiments, after mixing in steps 2 and 3 of the above method, the concentration of the sodium chloride is between about 27.0 w / v % and about 30.0 w / v %, for example, about 27.0 w / v %, about 27. 1 w / v %, about 27.2 w / v %, about 27.3 w / v %, about 27.426114 w / v %, about 27.5 w / v %, about 27.6 w / v %, about 27.7 w / v %, about 27.8 w / v %, about 27.9 w / v %, about 28.0 w / v %, about 28.1 w / v %, about 28.2 w / v %, about 28.3 w / v %, about 28.4 w / v %, about 28.5 w / v %, about 28.6 w / v %, about 28.7 w / v %, about 28.8 w / v %, about 28.9 w / v %, about 29.0 w / v %, about 29. 1 w / v %, about 29.2 w / v %, about 29.3 w / v %, about 29.4 w / v %. about 29.5 w / v %, about 29.6 w / v %, about 29.7 w / v %, about 29.8 w / v %. about 29.9 w / v %, or about 30.0 w / v %.

[0192] In some embodiments, the desired ratio of the peptide solution: antibody solution in step 4 above is between about 0.7 and about 1.3 (peptide solution): between about 2.70 and about 2.80 (antibody solution). In some embodiments, the desired ratio of the peptide solution: antibody solution in step 4 above is between about 0.9 and about 1.1 (peptide solution): between about 2.73 and about 2.77 (antibody solution). In some embodiments, the desired ratio of the peptide solution: antibody solution in step 4 above is about 1.0 (peptide solution): about 2.75 (antibody solution).

[0193] In some embodiments, the desired ratio of the peptide solution: antibody solution in step 4 above is between about 0.7 and about 1.3 (peptide solution): between about 6.0 and about 7.0 (antibody solution). In some embodiments, the desired ratio of the peptide solution: antibody solution in step 4 above is between about 0.9 and about 1.1 (peptide solution): between about 6.3 and about 6.7 (antibody solution). In some embodiments, the desired ratio of the peptide solution: antibody solution in step 4 above is about 1.0 (peptide solution): 6.5 (antibody solution).

[0194] In some embodiments, the desired ratio of the peptide solution: antibody solution in step 4 above is between about 0.7 and about 1.3 (peptide solution): between about 13.5 and about 14.5 (antibody solution). In some embodiments, the desired ratio of the peptide solution: antibodysolution in step 4 above is between about 0.9 and about 1.1 (peptide solution): between about 13.8 and about 14.2 (antibody solution). In some embodiments, the desired ratio of peptide solution: antibody solution in step 4 above is about 1.0 (peptide solution: about 14.0 (antibodysolution).

[0195] In some embodiments, the desired ratio of the filtered sodium chloride solution: filtered antibody / peptide solution from step 6 above is between about 0.7 and about 1.3 (filtered sodium chloride solution): between about 14.1 and about 15.1 (filtered antibody / peptide solution). In some embodiments, the desired ratio of filtered sodium chloride solution: filtered antibody / peptide solution from step 6 above is between about 0.9 and about 1.1 (filtered sodium chloride solution): between about 14.4 and about 14.8 (filtered antibody / peptide solution). In some embodiments, the desired ration of the filtered sodium chloride solution: filtered26114 antibody / peptide solution is about 1.0 (filtered sodium chloride solution): about 14.6 (filtered antibody / peptide solution).

[0196] In an aspect, a method of making a peptide hydrogel-based formulation with an encapsulated antibody is provided, comprising the following steps:(i) mixing a solution of peptide in water with an antibody solution to create an antibody / peptide solution, wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1 and wherein the antibody comprises a light chain sequence comprising at least 90% identical to SEQ ID NO: 2 and heavy chain sequence comprising at least 90% identical to SEQ ID NO: 3, wherein the antibody binds to PD-1; and(ii) adding sodium chloride to the antibody / peptide solution, wherein the presence of sodium chloride allows physical cross-linking of the peptide and formation of a peptide hydrogel and encapsulation of the antibody.

[0197] In some embodiments, the antibody / peptide solution is filtered prior to adding the sodium chloride.

[0198] In some embodiments, the sodium chloride that is added is in solution. In some embodiments, the ratio of sodium chloride solution: antibody / peptide solution equals from about 0.8 to about 1.2 (sodium chloride solution): from about 14. 1 to about 15. 1 (antibody / peptide solution). In some embodiments, the ratio of sodium chloride solution: antibody / peptide solution is about 0.8, about 0.9, about 1.0, about 1.1, or about 1.2 (sodium chloride solution): about 14.1, about 14.2, about 14.3, about 14.4, about 14.5, about 14.6, about 14.7, about 14.8, about 14.9, about 15.0, or about 15.1 (antibody / peptide solution).

[0199] In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is between about 0.8 and about 1.2: about 2.65 and about 2.85.

[0200] In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is about 1.0: about 2.75.

[0201] In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is between about 0.8 and about 1.2: about 6.2 and about 6.8.

[0202] In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is about 1.0: about 6.5.

[0203] In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (i) is between about 0.8 and about 1.2: between about 13.8 and about 14.2.

[0204] In some embodiments, the ratio of the solution of peptide in water: antibody solution in step (ii) is about 1.0: about 14.0.26114

[0205] In some embodiments, the antibody solution further comprises between about 1.99 mg / ml and about 2.19 mg / ml of L-histidine hydrocholoride, for example, about 1.99 mg / ml, about 2.00 mg / ml, about 2.01 mg / ml, about 2.02 mg / ml, about 2.03 mg / ml, about 2.04 mg / ml, about 2.05 mg / ml, about 2.06 mg / ml, about 2.07 mg / ml, about 2.08 mg / ml. about 2.09 mg / ml, about 2. 10 mg / ml, about 2. 11 mg / ml, about 2. 12 mg / ml, about 2. 13 mg / ml. about 2. 14 mg / ml. about 2.15 mg / ml, about 2.16 / ml, about 2.17 mg / ml, about 2.18 mg / ml, or about 2.19 mg / ml of L-histidine hydrocholoride.

[0206] In some embodiments, the antibody solution further comprises between about 65 mg / ml and about 75 mg / ml of sucrose, for example, about 65 mg / ml, about 66 mg / ml. about 67 mg / ml, about 68 mg / ml, about 69 mg / ml, about 70 mg / ml, about 71 mg / ml, about 72 mg / ml, about 73 mg / ml, about 74 mg / ml, or about 75 mg / ml of sucrose.

[0207] In some embodiments, the antibody solution further comprises between about 0.15 mg / ml to about 0.25 mg / ml of polysorbate 80. for example, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.20 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, or about 0.25 mg / ml of polysorbate 80.

[0208] In some embodiments, the antibody solution comprises between about 160 mg / ml and about 170 mg / ml of the antibody , for example, about 160 mg / ml, about 161 mg / ml, about 162 mg / ml. about 163 mg / ml, about 164 mg / ml, about 165 mg / ml, about 166 mg / ml, about 167 mg / ml, about 168 mg / ml, about 169 mg / ml, or about 170 mg / ml.Methods of Formulating Hydrogels in Syringes

[0209] In an aspect, a method of formulating a hydrogel in a syringe is provided. In some embodiments, the hydrogel comprises an encapsulated antibody (e.g., pembrolizumab). In some embodiments, the method comprises (i) adding a peptide to a solution comprising an inorganic salt and an antibody, (ii) filtering and mixing the solution in a syringe, (iii) incubating the resulting solution in the syringe to allow hydrogel formation. In some embodiments, the peptide comprises SEQ ID NO: 1. In some embodiments, the antibody is pembrolizumab.

[0210] In some embodiments the method of formulating a hydrogel in a syringe comprises the following steps:1. Preparing a first solution containing a peptide.2. Preparing a second solution by adding an inorganic salt to an antibody solution.3. Filtering and mixing the first solution with the second solution in a syringe, to create a homogenous solution.261144. Incubating the first solution and the second solution in the syringe to allow hydrogel formation in the syringe.

[0211] In some embodiments, the first solution is added to the syringe before mixing the first solution with the second solution.

[0212] In some embodiments, the peptide in step 1 comprises a sequence that is at least 90% identical, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the peptide in step 1 comprises SEQ ID NO: 1.

[0213] In some embodiments, the concentration of the peptide in the first solution is between about 45 mg / ml and about 55 mg / ml, for example, about 45 mg / ml, about 46 mg / ml, about 47 mg / ml, about 48 mg / ml, about 49 mg / ml, about 50 mg / ml, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, or about 55 mg / ml.

[0214] In some embodiments, the concentration of the peptide in the first solution is between about 4.5 w / v % and 5.5 w / v % of SEQ ID NO: I, for example, about 4.5 w / v %. about 4.6 w / v %, about 4.7 w / v %, about 4.8 w / v %, about 4.9 w / v %, about 5.0 w / v %, about 5. 1 w / v %, about 5.2 w / v %, about 5.3 w / v %, about 5.4 w / v %, or about 5.5 w / v %.

[0215] In some embodiments, the syringe is a plastic syringe. In some embodiments, the syringe is a glass syringe.

[0216] In some embodiments, the inorganic salt in the second solution comprises one or more of: sodium chloride, potassium chloride, magnesium chloride, and calcium chloride.

[0217] In some embodiments, the concentration of the inorganic salt in the second solution is between about 4.5 M and 5.5 M. for example, about 4.5 M, about 4.6 M, about 4.7 M, about 4.8 M, about 4.9 M, about 5.0 M, about 5. 1 M, about 5.2 M, about 5.3 M, about 5.4 M, or about 5.5 M.

[0218] In some embodiments, the antibody in the second solution comprises a light chain having at least 80% sequence identity with SEQ ID NO: 2 and a heavy chain having at least 80% sequence identity with SEQ ID NO: 3, wherein the antibody binds to PD-1. For example, the light chain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 2. In some embodiments, the light chain comprises the sequence of SEQ ID NO: 2. For example, the heavy chain is at least 80% sequence identity with SEQ ID NO: 3, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at26114 least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 3. In some embodiments, the heavy chain comprises SEQ ID NO: 3.

[0219] In some embodiments, the concentration of the antibody in the antibody solution is between about 150 mg / ml and about 180 mg / ml, for example, about 150 mg / ml, about 151 mg / ml. about 152 mg / ml, about 153 mg / ml. about 154 mg / ml, about 155 mg / ml, about 156 mg / ml, about 157 mg / ml, about 158 mg / ml, about 159 mg / ml, about 160 mg / ml, about 161 mg / ml, about 162 mg / ml, about 163 mg / ml, about 164 mg / ml, about 165 mg / ml, about 166 mg / ml, about 167 mg / ml, about 168 mg / ml, about 169 mg / ml, about 170 mg / ml, about 171 mg / ml, about 172 mg / ml, about 173 mg / ml, about 174 mg / ml, about 175 mg / ml, about 176 mg / ml, about 177 mg / ml, about 178 mg / ml, about 179 mg / ml, or about 180 mg / ml.

[0220] In some embodiments, the concentration of the antibody in the antibody solution is between about 15.0 w / v % and about 18.0 w / v %, for example, about 15.0 w / v %, about 15.1 w / v %, about 15.2 w / v %, about 15.3 w / v %, about 15.4 w / v %, about 15.5 w / v %, about 15.6 w / v %, about 15.7 w / v %, about 15.8 w / v %, about 15.9 w / v %, about 16.0 w / v %, about 16.1 w / v %, about 16.2 w / v %, about 16.3 w / v %, about 16.4 w / v %, about 16.5 w / v %, about 16.6 w / v %, about 16.7 w / v %, about 16.8 w / v %, about 16.9 w / v %, about 17.0 w / v %, about 17.1 w / v %, about 17.2 w / v %, about 17.3 w / v %, about 17.4 w / v %, about 17.5 w / v %, about 17.6 w / v %, about 17.7 w / v %, about 17.8 w / v %, about 17.9 w / v %, or about 18.0 w / v %.

[0221] In some embodiments, the incubation period in step 4 is at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, or at least about 10 minutes. In some embodiments, the incubation period in step is greater than 10 minutes.

[0222] In an aspect, a method of making a hydrogel with an encapsulated antibody is provided, comprising the following steps:

[0223] (i) providing a solution comprising a peptide in water in a first container (first solution), wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1;

[0224] (ii) mixing an inorganic salt and an antibody solution in a second container (second solution), wherein the antibody comprises a light chain comprising a sequence that is at least 90% identical to SEQ ID NO: 2 and a heavy chain comprising a sequence that is at least 90% identical to SEQ ID NO: 3, wherein the antibody comprises a concentration that is between about 160 mg / ml and about 170 mg / ml and wherein the antibody binds to PD-1; and

[0225] (iii) mixing the first solution with the second solution to form a third solution, comprising the hydrogel with the encapsulated antibody.26114

[0226] In some embodiments, the first solution is mixed with the second solution in a syringe.

[0227] In some embodiments, the peptide comprises a sequence that has at least 90% sequence identity with SEQ ID NO: 1, for example, at about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity’, about 98% sequence identity, or about 99% sequence identity with SEQ ID NO: 1. In some embodiments, the peptide comprises SEQ ID NO: 1.

[0228] In some embodiments, the light chain comprise a sequence that has at least at least 90% sequence identity with SEQ ID NO: 2, for example, at about 90% sequence identity, about 91% sequence identity', about 92% sequence identity, about 93% sequence identity, about 94% sequence identity', about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, or about 99% sequence identity' with SEQ ID NO: 2. In some embodiments, the light chain sequence comprises SEQ ID NO: 2.

[0229] In some embodiments, the heavy chain comprise a sequence that has at least at least 90% sequence identity with SEQ ID NO: 3, for example, at about 90% sequence identity', about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, or about 99% sequence identity with SEQ ID NO: 3. In some embodiments, the heavy chain sequence comprises SEQ ID NO: 2.

[0230] In some embodiments, the inorganic salt comprises sodium chloride. In some embodiments, the concentration of the sodium chloride is between about 4.8 M and about 5.2 M, for example, about 4.8 M, about 4.9 M, about 5.0 M, about 5. 1 M, or about 5.2 M.

[0231] In some embodiments, the antibody concentration is between about 160 mg / ml and about 170 mg / ml, for example, about 160 mg / ml, about 161 mg / ml, about 162 mg / ml, about 163 mg / ml, about 164 mg / ml, about 165 mg / ml, about 166 mg / ml, about 167 mg / ml, about 168 mg / ml, about 169 mg / ml, or about 170 mg / ml.

[0232] In some embodiments, the concentration of the peptide in the first solution is between about 45 mg / ml and about 55 mg / ml, for example, about 45 mg / ml, about 46 mg / ml, about 47 mg / ml, about 48 mg / ml, about 49 mg / ml, about 50 mg / ml, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, or about 55 mg / ml.

[0233] In some embodiments, the method further comprises incubating the third solution. In some embodiments, the third solution is incubated for less than 10 minutes, for example, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes.26114Hydrogels in Syringes

[0234] In some embodiments, a hydrogel in a syringe is provided. In some embodiments, the hydrogel in the syringe is prepared by any of the methods of formulating a hydrogel in a syringe described herein. In some embodiments, the hydrogel in a glass syringe comprises the following components: a peptide, an antibody (e.g., pembrolizumab), and an inorganic salt.

[0235] In some embodiments, the peptide comprises a sequence that is at least 90% identical, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the peptide in step 1 comprises SEQ ID NO: 1.

[0236] In some embodiments, the concentration of the peptide in the hydrogel in the syringe is between about 9 mg / ml and about 11 mg / ml, for example, about 9.1 mg / ml, about 9.2 mg / ml, about 9.3 mg / ml, about 9.4 mg / ml, about 9.5 mg / ml, about 9.6 mg / ml, about 9.7 mg / ml, about 9.8 mg / ml, about 9.9 mg / ml, about 10.0 mg / ml, about 10.1 mg / ml, about 10.2 mg / ml, about 10.3 mg / ml, about 10.4 mg / ml, about 10.5 mg / ml, about 10.6 mg / ml, about 10.7 mg / ml, about 10.8 mg / ml, about 10.9 mg / ml, or about 11.0 mg / ml.

[0237] In some embodiments, the concentration of the peptide in the hydrogel in the syringe is between about 0.90 w / v % and about 1.10 w / v %, for example, about 0.90 w / v %, about 0.91 w / v %, about 0.92 w / v %, about 0.93 w / v %, about 0.94 w / v %, about 0.95 w / v %, about 0.96 w / v %, about 0.97 w / v %, about 0.98 w / v %, about 0.99 w / v %, about 1.00 w / v %, about 1.01 w / v %, about 1.02 w / v %, about 1.03 w / v %, about 1.04 w / v %, about 1.05 w / v %, about 1.06 w / v %, about 1.07 w / v %, about 1.08 w / v %, about 1.09 w / v %, or about 1.10 w / v %.

[0238] In some embodiments, the antibody in the hydrogel in the syringe comprises a light chain having at least 80% sequence identity with SEQ ID NO: 2 and a heavy chain having at least 80% sequence identity with SEQ ID NO: 3, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%. at least 98%. or at least 99% to SEQ ID NO: 2. In some embodiments, the light chain comprises the sequence of SEQ ID NO: 2. For example, the heavy chain is at least 80% sequence identity with SEQ ID NO: 3, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 3. In some embodiments, the heavy chain comprises SEQ ID NO: 326114

[0239] In some embodiments, the concentration of the antibody in the hydrogel in the syringe is between about 112 mg / ml and about 132 mg / ml, for example, about 112 mg / ml, about 113 mg / ml, about 114 mg / ml, about 115 mg / ml, about 116 mg / ml, about 117 mg / ml, about 118 mg / ml, about 119 mg / ml, about 120 mg / ml, about 121 mg / ml, about 122 mg / ml, about 123 mg / ml. about 124 mg / ml, about 125 mg / ml. about 126 mg / ml, about 127 mg / ml, about 128 mg / ml, about 129 mg / ml, about 130 mg / ml, about 131 mg / ml, or about 132 mg / ml.

[0240] In some embodiments, the inorganic salt comprises one or more of: sodium chloride, potassium chloride, magnesium chloride, and calcium chloride.

[0241] In some embodiments, the concentration of the inorganic salt is between about 7 mg / ml, and about 27 mg / ml, for example, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml. or about 27 mg / ml.Cross Linking the Synthetic Peptides in the Peptide Hydrogel-Based Formulations

[0242] In some embodiments, the synthetic peptides in the peptide hydrogel-based formulations disclosed herein are physically cross-linked. In some embodiments, physical cross-linking of the synthetic peptides occurs through non-covalent interactions of chains in the synthetic peptides, such as, for example, electrostatic interactions or hydrogen bonding. In some embodiments, phy sical cross-linking of the synthetic peptides occurs through reducing repulsive electrostatic forces within chains of the synthetic peptides.

[0243] In some embodiments, the peptide hydrogel-based formulation includes an inorganic salt. In some embodiments, the inorganic salt comprises sodium chloride. In some embodiments, the sodium chloride functions to reduce repulsive forces of chains in the synthetic peptides that allows for physical cross linking of chains in the synthetic peptides and formation of a peptide hydrogel-based formulation.Synthetic Peptide Preparation

[0244] Synthetic peptides can be prepared by any process known in the art.

[0245] In some embodiments, synthetic peptides are prepared on amide resin via Fmoc-based solid state peptide synthesis method (Methods and protocols of modem solid phase peptide synthesis. Muriel Amblard, Jean- Alain Fehrentz, Jean Martinez & Gilles Subra. Molecular Biotechnology, 2006, Vol. 33, Page 239-254.). Crude synthetic peptide is purified using reverse-26114 phase high-performance liquid chromatography. The pure peptide is collected in solvent mixture of water, acetonitrile and formic acid.Lyophilization Techniques

[0246] In some embodiments, the peptides disclosed herein are lyophilized. The peptides can be lyophilized through any process known in the art.

[0247] In some embodiments, the peptides are lyophilized using the following method: a polypropylene container with purified peptide solution is firstly flash frozen using liquid nitrogen. After the liquid is fully frozen, the container is replaced in a vessel which is connected to a benchtop lyophilizer (Labconco Freezone model) through a vacuum manifold. The lyophilization process can be controlled with collector temperature at -80°C and vacuum at O.OlmTor for two days or until it renders a dried lyophilized cake.

[0248] In some embodiments, lyophilization of the peptide produces a lyophilized powder that includes the peptides. In some embodiments, the lyophilized powder is used to assembling peptide hydrogel-based formulations using the methods described herein.Definitions

[0249] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0250] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0251] As used herein, the articles “a” and “an” refer to one or to more than one (i. e. , to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0252] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).

[0253] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values26114 disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0254] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of and “consisting essentially of the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0255] The terms “% identical”, “% identity” or similar terms are intended to refer, in particular to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared and introducing gaps, if necessary, to achieve maximum percent sequence identity. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing said sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. Two sequences can be optimally aligned for comparison even when the sequences are of different lengths, which includes optimally aligning sequences in which one sequence is truncated on either or both of its 5’ and 3’ ends relative to the sequence to which it is being aligned. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT. FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).EXAMPLES

[0256] The following examples are meant to be illustrative and should not be construed as further limiting.Example 1: Materials and Methods26114Materials Used in the Experiments

[0257] Standard materials used in experiments to prepare and test hydrogels that are described herein are as follows:- Lyophilized peptide the amino acid sequence of: VKVKVKVKV(d-P)PTKVEVKVKV (SEQ ID NO: 1)- Frozen drug substance of pembrolizumab includes a light chain of the pembrolizumab antibody (SEQ ID NO: 2) and a heavy chain of the pembrolizumab antibody (SEQ ID NO: 3).

[0258] Light chain of pembrolizumab: EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC (SEQ ID NO: 2).

[0259] Heavy’ chain of pembrolizumab: QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLG(-K) (SEQ ID NO: 3).

[0260] The loss of C-terminal lysine on the heavy chain of pembrolizumab is denoted by -K. The heavy chain N (position 297) is glycosylated as indicated in bold with formulation composition of 165 mg / mL protein, 2.09 mg / mL L-histidine hydrochloride, 70 mg / mL sucrose, 0.2 mg / mL polysorbate 80- Sodium chloride- Milli-Q waterFormulation Preparation Procedures

[0261] The compositions of the present disclosure can be prepared through techniques known in the art, such as, for example, by means of conventional dissolving, filtration, aseptic filling, and finishing. For example, the pharmaceutical compositions can be prepared by standard sterile26114 processing methods, which may include solution preparation, filtration, filling, and finishing. A standard formulation protocol is as follows:1. Frozen protein drug substance (165 mg / ml) (e.g., pembrolizumab (SEQ ID NO: 2 (light chain) and SEQ ID NO: 3(heavy chain)) is allowed to thaw in an appropriate container.2. In two separate containers, water or an aqueous solvent (e.g.. water containing any one or more of L-histidine, hydrogen chloride, sucrose and polysorbate 80) is added and stirring is initiated.3. 2 weight % - 8 weight % of lyophilized peptide powder as hydrogel forming peptide (e.g., SEQ ID NO: 1), and sodium chloride are added to the two containers separately, and visually confirming dissolution before proceeding to the next step.4. Thawed antibody solution and hydrogel precursor solution are filled into a same intermediate containers with desired ratio (weight ratio of peptide to antibody can be from 1:5 to 1:30); gentle stirring is applied to complete the mixing.5. Sodium chloride solution and mixed antibody solution are filtered and filled into same 2R vials with desired ratio (weight ratio of sodium chloride to peptide can be from about 7: 1 to about 0.8 : 1).6. The vials are stoppered, and allowed to incubate for 5 minutes, allowing the formulation to gel before being capped with aluminum seal.Peptide Synthesis

[0262] Peptides were prepared on amide resin via Fmoc-based solid state peptide synthesis method (Methods and protocols of modem solid phase peptide synthesis. Muriel Amblard. Jean- Alain Fehrentz, Jean Martinez & Gilles Subra. Molecular Biotechnology, 2006, Vol. 33, Page 239-254.). Crude synthetic peptide was purified using reverse-phase high-performance liquid chromatography. The pure peptide was collected in solvent mixture of water, acetonitrile and formic acid.Lyophilization Procedures

[0263] In a polypropylene container, peptide solution was added, then flash frozen using liquid nitrogen. After the liquid was fully frozen, the container was replaced in a vessel which was connected to a benchtop lyophilizer (Labconco Freezone model) through a vacuum manifold. The lyophilization process was controlled with collector temperature at -80C and vacuum at O.OlmTor for two days or until it rendered a lyophilized cake.26114Hydrogel Dissolution Analytical Procedure

[0264] The hydrogel was dissolved through the following protocol:1. To the 2R vial with hydrogel, 3x water was added, and vortexed to disintegrate the hydrogel.2. The suspension was further homogenized and transferred into an appropriate container.3. To the container, 4x water was added for further dilution, and homogenized appropriately.4. The solution was further diluted two-fold with water through pipetting and mixing.5. The final diluted solution was centrifuged at 5000 RPM for 5 minutes.6. After centrifugation, if solution formed phase separation, upper layer was withdrawn for size-exclusion chromatography analysis, which was monitored at UV 280 nm, with an appropriate column.Example 2: Encapsulating Pembrolizumab in Hydrogel Formulations

[0265] Previously frozen, formulated protein drug substance containing: 165 mg / mL monoclonal antibody (pembrolizumab (SEQ ID NO: 2 (light chain) and SEQ ID NO: 3 (heavy chain)), 2.09 mg / mL L-histidine hydrochloride, 70 mg / mL sucrose, 0.2 mg / mL polysorbate 80, was allowed to thaw in an appropriate container. In two separate containers, lyophilized peptide powder and sodium chloride were added, and charged with water to dissolve to afford the final concentration of 75 mg / mL peptide and 284 mg / mL sodium chloride, respectively. The thawed monoclonal antibody solution and peptide solution were filled into a same intermediate container with desired mixing ratios. Three ratios were prepared with ratio of peptide solution: monoclonal antibody solution = 1:2.75, 1 :6.5 and 1: 14. The sodium chloride solution and mixed monoclonal antibody / peptide solution were then filtered (0.22pm pore size, PVDF membrane filter) and filled into same 2R vials with a mixing ratio of sodium chloride solution: antibody / peptide solution = 1 : 14.6. The vials were stoppered and allowed to incubate for 5 minutes at room temperature, allowing the formulation to gel before being capped with aluminum seal.

[0266] Tables 3-5 show various hydrogel formulations that include the pembrolizumab antibody.Table 3 - An Embodiment of Components and Concentrations of a 0.5 w / v % Hydrogel26114Table 4 - An Embodiment of Components and Concentrations of a 1.0 w / v % HydrogelTable 5 - An Embodiment of Components and Concentrations of a 2.0 w / v % Hydrogel

[0267] To demonstrate the stability improvement of hydrogel formulations, control samples, which are non-gelling formulations were prepared and included in a stability study, as described in the prototype in Table 6. In Table 6, prototypes 1, 2 and 3 correspond to the hydrogel formulations described above. Prototypes 4. 5 and 6 are the non-gelling formulations as controls in the stability study. The stoppered and capped 2R vials were stored in stability chambers at the temperatures of 5°C, 25°C and 40°C, and redissolved at a time point, to analyze the protein26114 aggregation, i.e.. HMW (high molecular weight) species, as described in the hydrogel dissolution analytical procedure.Table 6 - Prototypes of the Hydrogel Formulations

[0268] HMW change on stability' was analyzed using size-exclusion chromatography (SEC).

[0269] Size exclusion chromatography (SEC) was performed on a UPLC system (Waters Acquity) with SEC column (Acquity HPLC Protein BEH SEC, 200 A, 1.7pm, 4.6mm x 150mm). 50mM phosphate buffer with additional 450mM L-Arg at pH7 was used as mobile phase.Samples were analyzed by isocratic elution and monitored by UV absorbance at 280nm. A reference protein sample was included in each set of analyses to serve as standard (STD) control.

[0270] A representative UV chromatogram is shown in FIG. 1. Drop-line method was used for peak area integration. In the chromatogram of STD, the main peak between about 2.1 min and 3.3 min is the monomer peak. The peak between 1.4 min and 2.1 min is the HMW peak. For the stability samples, all the peaks eluted earlier than monomer peak were grouped together as HMW species. The percentage peak area was analyzed using Empower.

[0271] The summarized percentage peak area of high molecular weight (HMW) species on stability- is shown in FIGs. 2A-2C. In each of FIGs. 2A-2C, the X-axis indicates time in the unit of month (M) and the temperatures in the unit of degrees Celsius. Tables 7-11 provide summaries of the percentage peak area for high molecular weight species.Table 7 - 5°C stability results (%HMW)26114*same set of TO sampleTable 8 - 25°C stability (%HMW)*same set of TO sampleTable 9 - 40°C stability results (%HMW)*same set of TO sampleTable 10 - HMW% peak area change compared with prototype 5 (pembrolizumab only)Table 11 - HMW% peak area change compared with prototype 6 (NaCl with pembrolizumab)Example 3: Preparation of pembrolizumab encapsulated in a peptide hydrogel in a glass syringe

[0272] To formulate pembrolizumab an encapsulated hydrogel in a syringe, 5 weight percentage (5 wt%) peptide stock solution was prepared in deionized water. Then, using a Becton26114Dickinson (BD) 1ml Syringe Luer-Lok syringe with an 18-gauge needle, 120.0 pL of 5 wt% of a peptide stock solution comprising SEQ ID NO: 1 was added to the glass syringe. Then in a 2mL glass vial, 37.2 pL of 5 M sodium chloride (NaCl) solution (Invitrogen) and 442.8 pL of a pembrolizumab 165 mg / mL liquid formulation solution, were mixed together. The pembrolizumab and NaCl mixture solution was then added to the glass syringe using a BD 1ml Syringe Luer-Lok syringe with an 18-gauge needle to mix all three components; pembrolizumab, NaCl and SEQ ID NO: 1 to achieve a homogenous solution in the glass syringe. Within 10 minutes, a hydrogel was formed inside the glass syringe. The final weight percentage of MAX8 peptide within the hydrogel is 1 wt%, and the final concentration of pembrolizumab and NaCl is -122 mg / mL and 17 mg / ml respectively.Table 12 - An Embodiment of Components and Concentrations of a 1.0 w / v % Hydrogel in a Syringe

[0273] Pembrolizumab thermal stability experiment: To determine the thermal stability of the pembrolizumab encapsulated in peptide (SEQ ID NO: 1) hydrogel, two additional control groups were prefilled in the glass syringe; 1) pembrolizumab (SEQ ID NO: 2 and SEQ ID NO: 3) liquid formulation composed of 442.8 pL of pembrolizumab 165 mg / mL liquid formulation solution and 157.2 pL of deionized water and 2)) pembrolizumab liquid formulation with 310 rnM NaCl composed of 442.8 pL of pembrolizumab 165 mg / mL liquid formulation solution, 120.0 pL of deionized water and 37.2 pL of 5 M sodium chloride (NaCl) solution. Then the pre-26114 filled syringes were staged for stability in three different temperature conditions (5°C, 25°C and 40°C).

[0274] Analysis of pembrolizumab thermal stability at different time points using size exclusion chromatography: At different timepoints (1 week, 2-week, 1 month, 2 month and 3 month), 50 pL of pembrolizumab was dispelled from the glass syringe. An aliquot of 10 pL. -122 mg / mL of pembrolizumab solution dispelled from the syringe was serially diluted with deionized water from -122 mg / mL 61 mg / mL, 20 mg / mL, 10 mg / mL to 2 mg / mL. Then 10 pL of 2 mg / mL pembrolizumab solution was subject to size exclusion chromatography analysis with ACQUITY UPLC Protein BEH SEC Column 200A, 1.7pm 4.6mm X 150 mm part# 186005225 using pH 7.0 50 mM phosphate with 450 mM arginine at a flow rate of 0.250 mL / minute. All samples were run in triplicates and the percentage of high molecular weight fraction in each sample were analyzed using empower software.

[0275] The results are shown in FIG. 3 summarizing the percent peak area for high molecular weight species at different temperatures, using formulations described above in Example 3. The left figure shows percent peak of high molecular weight species at 4°C, demonstrating no obvious change during the staging period. The middle figure shows percent peak of high molecular weight species at 25°C, demonstrating hydrogel formulation preventing aggregation of proteins. The right figure shows percent peak of high molecular weight species at 40°C, demonstrating hydrogel formulation preventing aggregation of proteins.

[0276] Rheological measurement: A stress-controlled HR-20 rheometer equipped with a 40- mm stainless-steel cone-and-plate geometry with 2° cone (TA Instruments) was used. All experiments were performed at 37°C. Loaded sample (hydrogel or pembrolizumab encapsulated hydrogel) was pre-sheared for 30s at 10 1 / s to remove loading stress history followed by shear rates from 0.1 - 1000 1 / s for steady state viscosity. Small amplitude oscillatory shear (SAGS) measurements were performed in the linear regime at 1% strain from 0.1 to 100 rad / s for frequency sweep and at 6 rad / s from 0.1 to 100% strain for amplitude sweep. For large-small strain cycles, the 1% and 20% strains were imposed alternatively at 6 rad / s for four cycles, and each strain for 60s.

[0277] FIG. 4 shows the rheological properties of peptide hydrogel encapsulating protein. The left figure shows the angular frequency sweep results of storage modulus G’ and loss modulus G”. Higher G’ than G” and a plateau of viscoelasticity show the gel -like behavior. The middle figure shows the decreased viscosity of peptide hydrogel with increasing shear rate demonstrating the shear-thinning behavior as well as the yield-stress feature suggesting a crosslinked microstructure. The right figure shows as strain increases exceeding about 10%, G’26114 decreases to the extent lower than G” demonstrating the liquid-like state of hydrogel enabling ease of injectability.

[0278] FIG. 5 shows the injectability and recovery of hydrogel encapsulating protein. The left figure shows the formation of self-supporting hydrogel in syringe, and the injectability of hydrogel from syringe, and the recovery of injected hydrogel from solution state to gel state. The middle scheme illustrates the injecting and recovery’ process. The right figure shows the measured rheological properties of storage modulus G’ and loss modulus G” of hydrogel encapsulating protein. When shearing at 1% strain, as shown by the white region, G’ is higher than G” demonstrating the gel state. When shearing at 20% strain, as shown by the colored region, G’ is lower than G” demonstrating the liquid state. Upon reducing strain from 20% to 1%, G’ can recover to the extent higher than G” less than one second demonstrating the fast recovery of hydrogel cross-linked microstructure.Example 4: Performing Non-Reduced Capillary Electrophoresis Sodium Dodecyl Sulfate (CE-SDS) to Analyze Monomer Loss

[0279] CE-SDS experiments w ere performed using a Maurice CE-SDS application kit from ProteinSimple (Bio-Techne, San Jose, CA, USA), containing CE-SDS cartridges, vials, caps, and 96-well plates, as well as CE-SDS separation matrix, sample buffer, wash and conditioning solutions and internal standards. For sample preparation, 200 pL of the protein samples at different stability condition were expelled from the syringe, and ran through Nab Protein A Plus Spin Kit (Thermo Fisher, Waltham, MA), following the manufacturer’s protocol. The purified protein was then buffer exchanged with deionized water using Amicon Ultra-0.5 mL 10 kDa MWCO centrifugal filters (Burlington, MA) to afford a final volume of 100 pL. The protein concentration w as then determined by absorption at 280 nm (extinction coefficient of 1.42 mL mg-1 cm-1) using NanoDrop 8000 (Thermo Fisher, Waltham, MA). Prior to the CE-SDS run, the protein solution was diluted with deionized water to 2 mg / mL at a volume of 25 pL. 2.5 pL of 200 mM freshly prepared N-ethylmaleimide (Thermo Fisher, Waltham, MA) water solution was then added to the protein solution and incubated in ice for 5 minutes. Then, 25 pL of Maurice 1 x sample buffer and 2 pL of Maurice CE-SDS 25 x Internal Standard (10 kDa recombinant protein) were added to the same solution mixture. The solution was then vortexed and heated at 70°C for 10 minutes. After cooling down on ice, the solution was vortexed again and centrifuged using a mini centrifuge to remove air bubbles. Then, 50 pL of the solution was loaded into the 96-well plate before the plate was loaded onto the Maurice (Bio-Techne, San Jose, CA). All samples were electrokinetically injected into the cartridge capillary' by applying 4600 V for 20 seconds.26114The sample separation was achieved by electrophoresis at 5750 V for 45 minutes. The electropherogram data was evaluated by Compass for iCE 4.0.0 (Bio-Techne, San Jose, CA, USA) to analyze the relative migration time (RMT) relative to the CE-SDS 25 x Internal Standard 10 kDa (recombinant protein), as well as the low molecular weight species (LMW) percentage and the monomer percentage for each sample.

[0280] FIG. 6 shows that at 25oC, pembrolizumab encapsulated by peptide gel showed an inhibition of monomer loss, which is most significant at the 12-month time point. At 40oC, pembrolizumab encapsulated by peptide gel showed an inhibition of monomer loss, which is obvious throughout the 12-month period. At 40oC at the 12-month time point, pembrolizumab and pembrolizumab with NaCl samples were visually observed to have significant aggregation with high turbidity and failed to yield any CE-SDS results.

[0281] FIG. 7 shows that at 25oC, pembrolizumab encapsulated by peptide gel showed an inhibition of LMW formation, which is obvious throughout the 12-month period. At 40oC, the three samples have comparable behavior regarding LMW formation. At 40oC at the 12-month time point, pembrolizumab and pembrolizumab with NaCl samples were visually observed to have significant aggregation with high turbidity7and failed to yield any CE-SDS results.Example 5: Analyzing Turbidity Development of Hydrogel Under Different Storage and Dilution Conditions

[0282] 200 pL of the protein samples at different stability conditions were expelled from the syringe, and diluted by deionized water to a nominal concentration of lOmg / mL. The mixture was vortexed to homogenize. For turbidity measurement, the solution was further diluted to 2.5mg / mL by deionized water and vortexed to homogenize. Then UV absorbance at 350nm and 500nm was measured to calculate turbidity as Absorbance350nm - Absorbance 500nm. The corresponding formulation matrix was used as a blank.

[0283] From left to right in FIG. 8: pembrolizumab control sample stored at -80oC then diluted to lOmg / mL by deionized water, pembrolizumab solution sample stored at 40oC for 12-months then diluted to lOmg / mL by deionized water, pembrolizumab with NaCl solution sample stored at 40C for 12-months then diluted to lOmg / mL by deionized water, and pembrolizumab encapsulated by 1% peptide (SEQ ID NO: 1) gel stored at 40oC for 12months then diluted to lOmg / mL and homogenized by deionized water. FIG. 8 shows that hydrogel encapsulation significantly inhibited the turbidity development after the 40oC and 12 months of storage.

[0284] FIG. 9 provides a plot showing turbidity (Absorbance 350nm-Absorbance500nm) results. Pembrolizumab encapsulated by hydrogel shows the lowest turbidity compared with26114 pembrolizumab solution and pembrolizumab solution with NaCl after 40oC and 12 months of storage.Example 6: Analyzing Pembrolizumab Binding Potency by Enzyme-Linked Immunosorbent Assay (ELISA)

[0285] Pembrolizumab samples staged at 25°C and 40 °C for 12 months and 6 months, respectively were analyzed using PD-1 [Biotinylated] : PD-L1 Inhibitor Screening enz me linked immunosorbent assay (ELISA) screening kit (ACRO Biosystems, Newark, DE) according to the manufacturer’s protocol. Fresh stock of pembrolizumab (stored at -80°C) was used a control group. The percentage activity was determined by the relative absorbance compared to the control wells that were not treated with PD-1 inhibitor. The half maximal inhibitory concentration (IC50) of each sample group was determined using GraphPad Prism 10.2.2’s (Graphpad Software LLC, San Diego. USA) built-in model: [Inhibitor] vs. response - variable slope (four parameters) analysis. All data points were collected in triplicates. For comparisons of the IC50 values, means were assessed using an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (sample size n=3). p values less than 0.05 were considered statistically significant.

[0286] The results are shown in FIGs. 10A-10F. The results indicate that pembrolizumab encapsulated by peptide gel at 4oC 12-month, 25oC 12-month and 40oC 6-month time points showed comparable binding activity comparing to -80oC control. At 40oC at the 12-month time point, pembrolizumab and pembrolizumab with NaCl samples were visually observed to have significant aggregation with high turbidity and failed to yield any ELISA results.Example 7; Assessing Formulation Immunogenicity by Dendritic Cell Maturation Assay

[0287] Healthy anonymized human donor CD 14+ monocytes were differentiated into monocyte-derived dendritic cells (MoDC) by 5 -day culture with 50 ng / ml each IL-4 and GM- CSF in RPMI-1640 culture medium supplemented with 1% penicillin / streptomycin and 10% fetal bovine serum. Immature MoDCs (50,000 per well) were harvested and pulsed with testing articles at 50 or 100 pg / mL protein dose in 200 pL per well in triplicate wells of a 96-well plate. The negative control was cells in medium and the positive controls for immunogenicity risk were Keyhole limpet hemocyanin (KLH) and Bococizumab (anti-PCSK9 lgG2). MoDCs were stained for dendritic cell phenotype markers (CDl lc, HLA-DR), maturation markers (CD40, CD86), and viability. At least 2000 events were collected on the SONY ID7000 flow cytometer and gated to determine the frequency of %CD40hlghCD86hlghmature MoDCs out of live Cdl lc+MoDCs.26114

[0288] The criteria for immunogenicity risk predicted by the DC maturation assay is a positive response in 30% or more of donors. The data is shown in FIG. 11. The data shows that there is no major difference in immunogenicity risk between pembrolizumab and pembrolizumab encapsulated by a peptide gel (both < 30% of responding donors).Example 8; Analyzing PK Profiles of Pembrolizumab in Hydrogel or Histidine Formulation Following a Single Subcutaneous Dose of 50 mg / kg

[0289] Wistar Han rat was chosen as the study species. Two study groups were designed. In group 1, the animals were dosed with pembrolizumab encapsulated with 1% peptide (SEQ ID NO: 1) gel, n=5. In group 2, the animals w ere dosed with pembrolizumab solution, n=3. In both groups, the animals w ere given a single subcutaneous dose at 50mg / kg.

[0290] The concentrations of pembrolizumab in rat plasma samples w ere tested using an electrochemiluminescence (ECL) based immunoassay. A total antibody or pembrolizumab functional assay was used.

[0291] In total antibody assay, biotinylated mouse anti-human IgG Fc (JDC10) (Southern Biotech, Cat# 9040-08) was used as a capture reagent and SulfoTAG-mouse anti IgG (CH2 domain) (Thermo Scientific. Cat # MA5- 16929. tagged by Radix) as a detection reagent. The minimum required dilution (MRD) was 20. The LLOQ of the assay was determined to be 0.00748 mg / mL in rat plasma. For pembrolizumab total antibody assay, MSD multi-array streptavidin 96-well plate (MesoScale Discovery cat# L15SA-1) was blocked with 5% bovine serum albumin (BSA) and incubated at room temperature for 1 hour. After incubation, plate was coated with capture reagent in assay buffer (MED buffer) (0.5% BSA [wt / v], 0.05% Tween 20 [v / v], 0.25% CHAPS [wt / v], 5 mM EDTA, in PBS, pH 7.4) for 1 hour.Standards / controls / diluted samples w ere transferred onto the washed MSD plate and incubated at room temperature for 1-1.5 hours. After incubation, detection reagent was transferred onto the washed MSD plate and incubated at room temperature for 1 hour. After a final wash step, MSD Read Buffer was added, and plates were read on MSD SECTOR Imager S 6000. The produced ECL signal w as proportional to the concentration of analyte, pembrolizumab, in samples that w as quantitatively measured by comparison with a standard curve. Total antibody assay was used for analysis of both sample and dose retainers.

[0292] In pembrolizumab functional assays, recombinant human PD1 / PDCD1 protein (Sino Biological, Cat# 10377-H08H) was used as a capture reagent and SulfoTAG-mouse anti IgG (CH2 domain) (Thermo Scientific, Cat # MA5- 16929, tagged by Radix) as a detection reagent. The minimum required dilution (MRD) was 20. The LLOQ of the assay was determined to be261140.0674 mg / mL in rat plasma. MSD multi-array High Binding 96-well plate (MesoScale Discovery cat# L15XB-3) was coated with capture reagent in DPBS and incubated at room temperature for 2-3 hours. The plate was washed then blocked with 5% bovine serum albumin (BSA) and incubated at room temperature for 1 hour. After incubation, Standards / controls / diluted samples were transferred onto the washed MSD plate and incubated at room temperature for 1-1.5 hours. After incubation, detection reagent in assay buffer (MED buffer) was transferred onto the washed MSD plate and incubated at room temperature for 1 hour. After a final wash step, MSD Read Buffer was added, and plates were read on MSD SECTOR Imager S 6000. The produced ECL signal was proportional to the concentration of analyte, pembrohzumab, in samples that were quantitatively measured by comparison with a standard curve.

[0293] The results are show n in FIG. 12 and Table 13.Table 13 - Data for Hydrogel and Histidine Formulations for Selected PK Parameters

[0294] Results show that there was no apparent difference betw een Cmax, Tmax, and volume of distribution. In addition, the absorption phase w as similar for both formulations (Ka of about 0039 hr'1). Also, a trend of increased clearance and reduced AUC was observed in animals treated with hydrogel formulation.

[0295] The following sequences are disclosed in the application:Table 14 - Sequences26114

[0296] The disclosed subject mater is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0297] All references (e.g.. publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

WHAT IS CLAIMED IS:

1. A formulation, comprising:(i) between about 9.5 mg / ml and about 10.5 mg / ml of a peptide;(ii) between about 17.8 mg / ml and about 18.8 mg / ml sodium chloride; and(iii) between about 128 mg / ml and about 138 mg / ml of an antibody, wherein the antibody comprises a light chain comprising at least 90% sequence identity to SEQ ID NO: 2 and a heavy chain comprising at least 90% sequence identity to SEQ ID NO: 3 and wherein the antibody binds to PD-1, and wherein the peptide comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1.

2. The formulation of claim 1, wherein the antibody comprises a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 4, an HCDR2 comprising SEQ ID NO: 5, and an HCDR3 comprising SEQ ID NO: 6; and a light chain complementarity' determining region (LCDR) 1 comprising SEQ ID NO: 7, an LCDR2 comprising SEQ ID NO: 8, and an LCDR3 comprising SEQ ID NO: 9.

3. The formulation of claim 1 or claim 2, wherein the antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 3.

4. The formulation of claim 1, wherein the peptide comprises SEQ ID NO: 1.

5. The formulation of any one of claims 1-4, wherein the peptide comprises a concentration of about 10.0 mg / ml.

6. The formulation of any one of claims 1-5, wherein the sodium chloride comprises a concentration of about 18.3 mg / ml.

7. The formulation of any one of claims 1-6, wherein the antibody concentration is about 133 mg / ml.

8. The formulation of any one of claims 1-7, wherein the antibody is pembrolizumab.

9. The formulation of claim 1, wherein the peptide comprises SEQ ID NO: 1, wherein the peptide concentration is about 10.0 mg / ml, wherein the sodium chloride concentration is about 18.3 mg / ml, wherein the antibody concentration is about 133 mg / ml. and wherein the antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 3.

10. The formulation of any of claims 1-9, further comprising between about 51 mg / ml and about 61 mg / ml of sucrose.

11. The formulation of any of claims 1-10, further comprising between about 0.06 mg / ml and 0.26 mg / ml of polysorbate 80.

12. The formulation of claim 9, further comprising about 56 mg / ml of sucrose and about 0.16 mg / ml of polysorbate 80.

13. A peptide hydrogel-based formulation, comprising:(i) between about 9.5 mg / ml and about 10.5 mg / ml of a peptide:(ii) between about 17.8 mg / ml and about 18.8 mg / ml sodium chloride; and(iii) between about 128 mg / ml and about 138 mg / ml of an antibody, wherein the antibody comprises a light chain comprising at least 90% sequence identity to SEQ ID NO: 2 and a heavy chain comprising at least 90% sequence identity to SEQ ID NO: 3, wherein the antibody binds to PD- 1 ; wherein the peptide comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1, and wherein physical cross-linking of the peptide leads to formation of a peptide hydrogel.

14. The formulation of claim 13, wherein the peptide comprises SEQ ID NO: 1.

15. The formulation of claim 13 or claim 14, wherein the antibody comprises a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 4, an HCDR2 comprising SEQ ID NO: 5, and an HCDR3 comprising SEQ ID NO: 6; and a light chaincomplementarity determining region (LCDR) 1 comprising SEQ ID NO: 7, an LCDR2 comprising SEQ ID NO: 8, and an LCDR3 comprising SEQ ID NO: 9.

16. The formulation of any one of claims 13-15, wherein the peptide concentration is about 10.0 mg / ml.

17. The formulation of any one of claims 13-16, wherein the sodium chloride concentration is about 18.3 mg / ml.

18. The formulation of any one of claims 13-17, wherein the antibody concentration is about 133 mg / ml.

19. The formulation of any one of claims 13-18. wherein the antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 3.

20. The formulation of any one of claims 13-19, wherein the antibody is pembrolizumab.

21. The formulation of claim 13. wherein the peptide comprises SEQ ID NO: 1, wherein the peptide concentration is about 10.0 mg / ml, wherein the sodium chloride concentration is about 18.3 mg / ml, wherein the antibody concentration is about 133 mg / ml. and wherein the antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 3.

22. The formulation of any of claims 13-21, further comprising between about 51 mg / ml and about 61 mg / ml of sucrose.

23. The formulation of any of claims 13-22 further comprising between about 0.06 mg / ml and about 0.26 mg / ml of polysorbate 80.

24. A method of making a peptide hydrogel-based formulation with an encapsulated antibody, comprising:(i) mixing a solution of a peptide in water with an antibody solution to create an antibody / peptide solution, wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1 and the antibody comprises a light chain comprising at least 90% sequence identity to SEQ ID NO: 2 and a heavy chain comprising at least 90% sequence identity to SEQ ID NO: 3, wherein the antibody binds to PD-1;(ii) adding sodium chloride to the antibody / peptide solution, wherein the presence of sodium chloride allows physical cross-linking of the peptide and formation of a peptide hydrogel and encapsulation of the antibody.

25. The method of claim 24, wherein the antibody / peptide solution is filtered prior to adding the sodium chloride.

26. The method of claim 24 or claim 25. wherein the sodium chloride that is added is in solution.

27. The method of claim 26, wherein the ratio of sodium chloride solution: antibody / peptide solution equals from about 0.8 to about 1.2: from about 14.1 to about 15.1.

28. The method of claim 24, wherein the ratio of the solution of peptide in water: antibody solution in step (i) is between about 0.8 and about 1.2: about 2.65 and about 2.85.

29. The method of claim 24, wherein the ratio of the solution of peptide in water: antibody solution in step (i) is about 1.0: about 2.75.

30. The method of claim 24, wherein the ratio of the solution of peptide in water: antibody solution in step (i) is between about 0.8 and about 1.2: about 6.2 and about 6.8.

31. The method of claim 24. wherein the ratio of the solution of peptide in water: antibody solution in step (i) is about 1.0: about 6.5.

32. The method of claim 24, wherein the ratio of the solution of peptide in water: antibody solution in step (i) is between about 0.8 and about 1.2: between about 13.8 and about 14.2.

33. The method of claim 25, wherein the ratio of the solution of peptide in water: antibody solution in step (ii) is about 1.0: about 14.0.

34. The method of any one of claims 24-33, wherein the antibody solution further comprises between about 1.99 mg / ml and about 2.19 mg / ml of L-histidine hydrocholoride.

35. The method of claim 34. wherein the antibody solution comprises about 2.09 mg / ml of L- histidine hydrochloride.

36. The method of any one of claims 24-35, wherein the antibody solution further comprises between about 65 mg / ml and about 75 mg / ml of sucrose.

37. The method of claim 36, wherein the antibody solution comprises about 70 mg / ml of sucrose.

38. The method of any one of claims 24-37, wherein the antibody solution further comprises from about 0.15 mg / ml to about 0.25 mg / ml of polysorbate 80.

39. The method of claim 38, wherein the antibody solution comprises about 0.20 mg / ml of polysorbate 80.

40. The method of claim 39, wherein the antibody solution comprises about 165 mg / ml of the antibody.

41. The method of any one of claims 24-40, wherein the antibody in the antibody solution comprises a light chain that comprises SEQ ID NO: 2 and a heavy chain that comprises SEQ ID NO: 3.

42. The method of any one of claims 27-41, wherein the ratio of sodium chloride solution: antibody / peptide solution is about 1.0: about 14.6.

43. A method of making a hydrogel with an encapsulated antibody, comprising:(i) providing a solution comprising a peptide in water in a first container (first solution), wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1;(ii) mixing an inorganic salt and an antibody solution in a second container (second solution), wherein the antibody comprises a light chain comprising a sequence that is at least 90% identical to SEQ ID NO: 2 and a heavy chain comprising a sequence that is at least 90% identical to SEQ ID NO: 3, wherein the antibody comprises a concentration that is between about 160 mg / ml and about 170 mg / ml and wherein the antibody binds to PD-1; and(iii) mixing the first solution with the second solution to form a third solution, comprising the hydrogel with the encapsulated antibody.

44. The method of claim 43, wherein the first solution is mixed with the second solution in a syringe.

45. The method of claim 43 or claim 44, wherein the peptide comprises the sequence of SEQ ID NO: 1.

46. The method of any one of claims 43-45, wherein the antibody comprises a light chain that comprises SEQ ID NO: 2 and a heavy chain that comprises SEQ ID NO: 3.

47. The method of any one of claims 43-46, wherein the inorganic salt comprises sodium chloride.

48. The method of claim 47, wherein the concentration of sodium chloride in the second solution is between about 4.8 M and about 5.2 M.

49. The method of claim 48, wherein the concentration of sodium chloride in the second solution is about 5.0 M.

50. The method of any one of claims 43-49, wherein the antibody concentration in the antibody solution is about 165 mg / ml.

51. The method of any one of claims 43-50, wherein the concentration of the peptide in the first solution is between about 48 mg / ml and about 52 mg / ml.

52. The method of claim 51. wherein the concentration of the peptide in the first solution is about 50 mg / ml.

53. The method of any one of claims 43-52, further comprising incubating the third solution.

54. The method of claim 53, wherein the third solution is incubated for 10 minutes or less.

55. A peptide hydrogel-based formulation that is produced by the method of any of claims 43-54.

56. The peptide hydrogel-based formulation of claim 55, wherein the formulation comprises:(i) about 8 mg / ml to about 12 mg / ml of a peptide, wherein the peptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1;(ii) from about 118 mg / ml to about 126 mg / ml of an antibody, wherein the antibody comprises a light chain comprising a sequence that is at least 90% identical to SEQ ID NO: 2 and a heavy chain comprising a sequence that is at least 90% identical to SEQ ID NO: 3, wherein the antibody binds PD-1; and(iii) from about 15 mg / ml to about 19 mg / ml of an inorganic salt.

57. The peptide hydrogel-based formulation of claim 56, wherein the peptide comprises the sequence of SEQ ID NO: 1.

58. The peptide hydrogel-based formulation of claim 56 or claim 57, wherein the antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 3.

59. The peptide hydrogel-based formulation of any one of claims 56-58, wherein the inorganic salt comprises sodium chloride.

60. The peptide hydrogel-based formulation of any one of claims 56-59, wherein the formulation comprises about 122 mg / ml of the antibody.

61. The peptide hydrogel-based formulation of any one of claims 56-60, wherein the formulation comprises about 17 mg / ml of the inorganic salt.

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