Peptide libraries having improved functional pharmacokinetic parameters and methods of synthesis and use thereof

Abstract

The present disclosure relates to the development of a synthetic peptide library. This synthetic peptide library comprises at least about 1×10^3, 1×10^4, 1×10^5, 1×10^6, or 1×10^7 peptide sequences derived from a single reference sequence, wherein a subset of the derived peptide sequences comprises peptide sequences comprising a functional pharmacokinetic parameter (FPKP) delectably improved relative to the single reference sequence.

Description

341812000140PEPTIDE LIBRARIES HAVING IMPROVED FUNCTIONAL PHARMACOKINETIC PARAMETERS AND METHODS OF SYNTHESIS AND USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application 63 / 694,778 filed, September 13, 2024, and U.S. Provisional Application 63 / 850,424 filed July 24, 2025, the disclosure of each of which are incorporated herein in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (341812000140SEQLIST.xml; Size: 5,421 bytes; and Date of Creation: September 11, 2025) is herein incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] The present invention concerns the design and construction of diverse peptide and polypeptide libraries. In particular, the invention concerns methods of analytical database design for creating datasets using multiple relevant parameters as filters, and methods for generating sequence diversity by directed chemical peptide and polypeptide synthesis, utilizing non-canonical amino acids, and chemical linkers.BACKGROUND OF THE INVENTION

[0004] Proteins can have many beneficial uses within organisms. In particular situations, proteins can be used to treat diseases and other biological conditions that can detrimentally impact the health of humans and other mammals. In various scenarios, proteins can participate in reactions that are beneficial to subjects and that can counteract one or more biological conditions being experienced by the subjects. In some examples, proteins can also bind to target molecules within an organism that may be detrimental to the health of a subject. For these reasons, many individuals and organizations have sought to develop proteins that may have therapeutic benefits.MOFO-360864280 1341812000140

[0005] The development of proteins can be a time-consuming and resource-intensive process. Often, candidate proteins for development can be identified as potentially having desired biophysical properties, three-dimensional (3D) structures, and / or behavior within an organism. To determine whether the candidate proteins have the desired characteristics, the proteins can be synthesized and then tested to determine whether the actual characteristics of the synthesized proteins correspond to the desired characteristics. Due to the number of resources needed to synthesize and test proteins for specified biophysical properties, 3D structures, and / or behaviors, the number of candidate proteins synthesized for therapeutic purposes is limited. In some situations, the number of proteins synthesized for therapeutic purposes can be limited by the loss of resources that takes place when candidate proteins are synthesized and do not have the desired characteristics.

[0006] The use of computer-implemented techniques to identify candidate proteins that have particular characteristics has increased. These conventional techniques, however, can be limited in their scope and accuracy. In various situations, conventional computer- implemented techniques to generate protein sequences can be limited by the amount of data available and / or the types of data available that may be needed by those conventional techniques to accurately generate protein sequences with specified characteristics. Additionally, the techniques utilized to produce models that can generate protein sequences with particular characteristics can be complex and the know-how needed to produce models that are accurate and efficient can be complex. In certain scenarios, the length of the protein sequences produced by conventional models can also be limited because the accuracy of conventional techniques can decrease as the lengths of the proteins increases. Thus, the number of proteins generated by conventional techniques is limited.

[0007] The development of peptide- or polypeptide-based drug candidates often starts with the screening of libraries of related peptide or polypeptide sequences, generated from nucleic acid sequences. For example, polypeptide populations are generated from nucleic acid libraries to create a diverse library of antibody sequences, which may be derived from a small number of lineages. Alternatively, polypeptide populations are generated from nucleic acid libraries to create a diverse library of peptide sequences having biological activity, and such peptide sequences may be selected for pharmacodynamic properties, which may be modeled in vitro or in vivo. Despite these and other technologies, there is a great need for new, efficient methods for the design, synthesis, and screening of highly diverse (poly)peptide libraries.MOFO-360864280 2341812000140SUMMARY OF THE INVENTION

[0008] In various aspects, the present invention concerns the design and construction of diverse peptide and polypeptide libraries. The techniques and systems described herein can be used to generate amino acid sequences of proteins accurately and efficiently.

[0009] In certain embodiments, provided herein are methods of manufacturing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject, comprising: (a) selecting a region of at least 10 amino acids of the therapeutic peptide; (b) selecting a plurality of sites within the region;(c) generating at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non- canonical amino acid, wherein each of the unique peptide variants comprise a C-terminal and / or N-terminal modification; and (d) manufacturing a peptide library comprising the unique peptide variants.

[0010] In certain embodiments, provided herein are methods of designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject, comprising:(a) selecting a region of at least 10 amino acids of the therapeutic peptide; (b) selecting a plurality of sites within the region;(c) generating at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein one or more of the unique peptide variants comprise a C-terminal and / or N- terminal modification.

[0011] In certain embodiments, provided herein are methods of determining a functional pharmacokinetic parameter (FPKP) of a plurality of unique peptide variants of a therapeutic peptide, comprising the steps of: (a) introducing into a non-human mammalian subject a peptide library comprising the unique peptide variants, the peptide library comprising at least IxlO3unique peptide variants, each peptide variant comprising at least one modification at one or more of a plurality of sites in a region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein each of the one or more of the unique peptide variants comprise a C-terminal and / or N-terminal modification, wherein less than 75 pg / kg of each unique peptide variant is introduced into the non-human mammalian subject; (b) detecting thereafter a subset of the unique peptide variants present in the pluralityMOFO-360864280 3341812000140 of unique peptide variants, thereby determining the FPPK of the detected unique peptide variants.

[0012] In certain embodiments, the disclosed are synthetic peptide libraries comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences derived from a single reference sequence or a set of reference sequences, wherein a subset of the derived peptide sequences comprises peptide sequences having a pharmacokinetic parameter detectably improved relative to the single reference sequence or set of reference sequences.

[0013] In additional embodiments, disclosed are synthetic peptide libraries comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences derived from a single reference sequence or a set of reference sequences, wherein a subset of the derived peptide sequences comprises peptide sequences having a pharmacokinetic parameter detectably improved relative to the single reference sequence or set of reference sequences, or having a dissociation constant value lower than the reference sequence or set of reference sequences.

[0014] In further embodiments, disclosed are synthetic peptide libraries comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences independently having at least one non-canonical amino acid. In some embodiments, disclosed are one or more synthetic peptides comprising at least one non-naturally occurring amino acid, wherein the synthetic peptide is capable of binding to a cellular or circulating target present in a human subject, and wherein the synthetic peptide is isolated from a derived synthetic peptide library comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences present in a mammalian subject. In preferred embodiments, disclosed are one or more synthetic peptides capable of binding to a cellular or circulating target present in a human subject, isolated from a derived peptide library comprising at least about lxlOA6 peptides present in a mammalian subject.

[0015] In additional embodiments, disclosed are one or more synthetic peptide libraries comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences independently having at least one amino acid difference from a naturally occurring human peptide sequence.

[0016] In certain aspects, disclosed are methods of determining a pharmacokinetic (PK) parameter of a plurality of synthetic peptides, comprising the steps of i) introducing into a mammalian subject the plurality of synthetic peptides; ii) detecting thereafter a subset of synthetic peptides present in the plurality, thereby determining the PK parameter of the detected synthetic peptides.MOFO-360864280 4341812000140

[0017] In certain embodiments, disclosed are methods of identifying a synthetic peptide having an increased pharmacokinetic (PK) parameter relevant to a reference synthetic peptide, comprising the steps of:

[0018] a) determining a pharmacokinetic (PK) parameter of a plurality of synthetic peptides, comprising the steps of i) introducing into a mammalian subject the plurality of synthetic peptides; ii) detecting thereafter a subset of synthetic peptides present in the plurality, thereby determining the PK parameter of the detected synthetic peptides; and iii) selecting a reference synthetic peptide from the detected synthetic peptides;

[0019] b) synthesizing chemical variants of the selected reference synthetic peptide;

[0020] c) introducing the synthesized chemical variants into a mammalian subject and determining the PK parameter of a plurality of the introduced synthesized chemical variants, comprising the step of i) introducing into a mammalian subject the plurality of synthetic peptides; and

[0021] d) identifying thereafter one or more synthetic peptides having the increased PK parameter.

[0022] Additionally disclosed are methods of identifying a synthetic peptide comprising an acceptable pharmacokinetic (PK) parameter, comprising the steps of

[0023] i) introducing into a mammalian subject a peptide library comprising at least about lxlOA4 synthetic peptide sequences derived from a single reference sequence comprising an unacceptable PK parameter; and

[0024] ii) detecting thereafter a subset of the synthetic peptide sequences present in the mammalian subject, thereby identifying the synthetic peptide comprising an acceptable PK parameter.

[0025] Disclosed further are methods of identifying a synthetic peptide comprising an acceptable pharmacokinetic (PK) parameter, comprising the steps of

[0026] i) introducing into a mammalian subject a peptide library comprising at least about lxlOA4 synthetic peptide sequences; and

[0027] ii) detecting at a desired time point a subset of the synthetic peptide sequences present in the mammalian subject, thereby identifying the synthetic peptide comprising an acceptable PK parameter.

[0028] In certain embodiments, disclosed are methods of identifying a target-binding compound having an acceptable pharmacokinetic (PK) parameter, comprising the steps of:MOFO-360864280 5341812000140

[0029] a) providing a first peptide library comprising a plurality of first peptide library members, the first peptide library members derived from a target-binding compound having an unacceptable PK parameter;

[0030] b) administering the first peptide library to a mammalian subject;

[0031] c) identifying one or more first peptide library members present in the mammalian subject having an acceptable PK parameter; and optionally the steps of

[0032] d) providing a second peptide library comprising a plurality of second peptide library members, the second peptide library members derived from the identified target-binding compound having an acceptable PK parameter;

[0033] e) administering the second peptide library to a mammalian subject; and

[0034] f) identifying one or more second peptide library members present in the mammalian subject having an acceptable PK parameter and having acceptable binding to the target compound.

[0035] In certain preferred embodiments, disclosed herein are methods of identifying a target-binding compound having an acceptable pharmacokinetic (PK) parameter, comprising the steps of:

[0036] a) providing a first peptide library comprising a plurality of first peptide library members, the first peptide library members derived from a target-binding compound having an unacceptable PK parameter;

[0037] b) administering the first peptide library to a mammalian subject;

[0038] c) identifying one or more first peptide library members present in the mammalian subject having an acceptable PK parameter;

[0039] d) building a machine learning model using one or more machine learning algorithms, and training the model using training data, wherein the training data comprise the chemical composition and acceptable PK parameter of the one or more first peptide library members;

[0040] e) using the trained model to predict the sequences of a plurality of second peptide library members.

[0041] In particular implementations, generative adversarial networks can be implemented to determine models that can produce amino acid sequences of proteins. The generative adversarial networks can be trained using a number of different training datasets to produce amino acid sequences for proteins having specified characteristics. For example, sequences of amino acids of proteins having particular biophysical properties and sequences of amino acids having a particular structure can be generated herein. Additionally, the techniques and systems described herein can utilize computer-implemented processes that analyze the aminoMOFO-360864280 6341812000140 acid sequences generated to determine a desired functional pharmacokinetic parameter (FPKP). The analysis of the amino acid sequences can determine whether the amino acid sequences produced by the system correspond to a desired set of characteristics. In certain implementations, the computer-implemented processes can filter amino acid sequences produced to identify amino acid sequences that correspond to a specified set of characteristics.

[0042] In certain aspects, the improved pharmacokinetic properties of the peptide is selected from one or more of the following properties: (1) is less susceptible to biotransformation in the patient, (2) is excreted from the body of the patient at a slower rate, (3) has increased stability of its tertiary structure, (4) has a longer plasma half-life, (5) has increased oral bioavailability, (6) has higher penetrance across the blood-brain barrier, and (7) has higher accumulation in a target tissue.

[0043] Also provided herein are methods of manufacturing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject, comprising: (a) selecting a region of at least 10 amino acids of the therapeutic peptide; (b) selecting a plurality of sites within the region; (c) generating at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein each of the unique peptide variants comprise a C-terminal and / or N-terminal modification; and (d) manufacturing a peptide library comprising the unique peptide variants.

[0044] Also provided herein are methods of designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject, comprising: (a) selecting a region of at least 10 amino acids of the therapeutic peptide; (b) selecting a plurality of sites within the region; (c) generating at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein one or more of the unique peptide variants comprise a C-terminal and / or N-terminal modification.

[0045] Also provided herein are methods for determining a functional pharmacokinetic parameter (FPKP) of a plurality of unique peptide variants of a therapeutic peptide, comprising the steps of: (a) introducing into a non-human mammalian subject a peptide library comprising the unique peptide variants, the peptide library comprising at least IxlO3MOFO-360864280 7341812000140 unique peptide variants, each peptide variant comprising at least one modification at one or more of a plurality of sites in a region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein each of the one or more of the unique peptide variants comprise a C-terminal and / or N-terminal modification, wherein less than 75 pg / kg of each unique peptide variant is introduced into the non-human mammalian subject; (b) detecting thereafter a subset of the unique peptide variants present in the plurality of unique peptide variants, thereby determining the FPPK of the detected unique peptide variants.

[0046] Also provided herein are kits comprising a peptide library manufactured according to the method described herein.

[0047] Also provided herein are peptide libraries manufactured according to the methods described herein. Also provided herein are peptide libraries designed according to the methods described herein.

[0048] Also provided herein are arrays comprising the peptide libraries described herein and a solid surface, wherein the derived peptide sequence is individually arrayed upon the solid surface.

[0049] Also provided herein are synthetic peptide librarys comprising at least about IxlO3, about IxlO4, about IxlO5, about IxlO6, or about IxlO7peptide sequences independently having at least one amino acid difference from a naturally occurring human peptide sequence.

[0050] Also provided herein are methods identifying a synthetic peptide having an increased pharmacokinetic (PK) parameter relevant to a reference synthetic peptide, comprising the steps of: determining a pharmacokinetic (PK) parameter of a plurality of synthetic peptides, comprising the steps of: introducing into a mammalian subject the plurality of synthetic peptides; detecting thereafter a subset of synthetic peptides present in the plurality of synthetic peptides, thereby determining the PK parameter of the detected synthetic peptides; and selecting a reference synthetic peptide from the detected synthetic peptides; synthesizing chemical variants of the selected reference synthetic peptide; introducing the synthesized chemical variants into a mammalian subject and determining the PK parameter of a plurality of the introduced synthesized chemical variants, and identifying thereafter one or more synthetic peptides having the increased PK parameter.

[0051] Also provided herein are methods of identifying a synthetic peptide comprising an acceptable pharmacokinetic (PK) parameter, comprising the steps of: introducing into a mammalian subject a peptide library comprising at least about IxlO3synthetic peptide sequences derived from a single reference sequence comprising an unacceptable PKMOFO-360864280 8341812000140 parameter; and detecting thereafter a subset of the synthetic peptide sequences present in the mammalian subject, thereby identifying the synthetic peptide comprising an acceptable PK parameter.

[0052] Also provided herein are methods of identifying a synthetic peptide comprising an acceptable pharmacokinetic (PK) parameter, comprising the steps of: introducing into a mammalian subject a peptide library comprising at least about IxlO3synthetic peptide sequences; and detecting at a desired time point a subset of the synthetic peptide sequences present in the mammalian subject, thereby identifying the synthetic peptide comprising an acceptable PK parameter.

[0053] Also provided herein are methods of identifying a target-binding compound having an acceptable pharmacokinetic (PK) parameter, comprising the steps of: providing a first peptide library comprising a plurality of first peptide library members, the first peptide library members derived from a target binding compound having an unacceptable PK parameter; introducing into the mammalian subject the first peptide library; identifying one or more first peptide library members present in the mammalian subject having an acceptable PK parameter; and optionally the steps of providing a second peptide library comprising a plurality of second peptide library members, the second peptide library members derived from the identified target binding compound having an acceptable PK parameter; introducing into the mammalian subject the second peptide library; andidentifying one or more second peptide library members present in the mammalian subject having an acceptable PK parameter and having acceptable binding to the target compound.

[0054] Also provided herein are methods of identifying a target binding compound having an acceptable pharmacokinetic (PK) parameter, comprising the steps of providing a first peptide library comprising a plurality of first peptide library members, the first peptide library members derived from a target-binding compound having an unacceptable PK parameter; introducing into a mammalian subject the first peptide library; identifying one or more first peptide library members present in the mammalian subject having an acceptable PK parameter; building a machine learning model using one or more machine learning algorithms, and training the model using training data, wherein the training data comprise the chemical composition and acceptable PK parameter of the one or more first peptide library members; and using the trained model to predict the sequences of a plurality of second peptide library members.

[0055] Also provided herein are systems for designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-humanMOFO-360864280 9341812000140 mammalian subject, comprising: one or more processors, and a non-transitory memory coupled to the one or more processors comprising instructions that, when executed by the one or more processors, cause the one or more processors to: receive a therapeutic peptide; select a region of at least 10 amino acids of the therapeutic peptide; select a plurality of sites within the region; and generate at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein one or more of the unique peptide variants comprise a C- terminal and / or N-terminal modification.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0057] FIG. 1 is a summary of the representative steps of the design and synthesis of a diverse peptide library that includes non-canonical amino acids using rapid flow-based peptide synthesis.

[0058] FIG. 2 is a summary of the datasets used to model functional pharmacokinetic parameters in the diverse peptide library.

[0059] FIG. 3 shows results of a principal component analysis of chemical properties for canonical amino acids compared to amino acids used for designing the diverse peptide library of some embodiments described herein.

[0060] FIG. 4A and 4B shows result of a principal component analysis of chemical properties for native peptides and peptide designed according to embodiments of the disclosure. FIG.4A shows Atrial Natriuretic Peptide (ANP) and the corresponding peptide variants. FIG. 4B is the same as FIG 4A but includes the native B-type Natriuretic Peptide (BNP) in PCA space as a comparison.

[0061] FIG. 5 shows mass spectrometry intensities for peptides as a percentage of the peptide intensity a timepoint 0 for capped and uncapped versions of 3 peptides recovered from rate plasma at various timepoints.

[0062] FIG. 6 provides an exemplary computer system according to some embodiments described herein.MOFO-360864280 10341812000140DETAILED DESCRIPTIONS OF THE INVENTION

[0063] The methods and systems described herein for designing a peptide library, manufacturing a peptide library, and determining functional pharmacokinetics parameters (FPKP) improve on methods for creating and screening large peptide- polypeptide-based drug candidates. The methods and systems described herein are allow for efficient design, synthesis, and screening of highly diverse (poly)peptide libraries.

[0064] The methods and systems described herein can be used to design, manufacture, and analyze a large diverse polypeptide library derived from a therapeutic peptide. The libraries designed, manufactured, and analyzed, according to the methods and systems described herein comprise non-canonical amino acids and C-terminal and / or N-terminal modifications. It was discovered that non-canonical amino acids increase chemical diversity and resulting peptide property diversity beyond those previously appreciated.

[0065] Also described herein are method wherein the highly diverse peptide libraries can be administered to non-human mammalian subjects for in-vivo analysis. Peptide instability in vivo is a core challenge in therapeutic development. In comparison to other drug modalities, stability and degradation of the peptide needs to be accounted for. The instability of peptides in vivo may come from a variety of factors including but limited to proteolytic degradation by proteases, renal clearance, hydrolysis and immunogenicity. Optimization of a therapeutic peptide by testing a high volume of peptide variants individually is often impractical due to time and resource limitations. One way to predict stability without individually testing peptide variants in vivo is through computationally estimating performance for the peptide variants. Due to the complexity of factors associated with pharmacokinetics for peptide in vivo, computational methods may not recreate in vivo dynamics effectively and with high accuracy. The disclosure demonstrates for the first time that the peptides from the highly diverse libraries can be recovered from an animal in a single or small number of experiments and detected using mass spectrometry which can lead to valuable insights about peptide variants performance in vivo and as potential drug candidates. Using these methods, the properties of peptides with non-canonical amino acids and terminal modifications can be measured and compared in vivo The methods and systems, thus, eliminate the need for expensive and time consuming experiments for therapeutic peptide optimization and allow for testing of more diverse peptides than was previously possible.MOFO-360864280 11341812000140DEFINITIONS

[0066] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), provides one skilled in the art with a general guide to many of the terms used in the present application.

[0067] For purposes of the present invention, the following terms are defined below.

[0068] As used herein, the term “pharmacokinetic property” or “PK” refers to a parameter that describes the disposition of an active agent or drug in an organism or host. Representative pharmacokinetic properties include plasma half-life, hepatic first-pass metabolism, solubility, lipophilicity, size, structure, ability to form hydrogen bonds, polarity, chemical stability, susceptibility to enzymatic reactions, absorption rate, clearance rate, volume of distribution, or the degree of blood serum protein, e.g. albumin, blood brain barrier crossing, oral bioavailability, cell penetration, biodistribution, binding, etc.

[0069] As used herein, the term “plasma half-life” refers to the time for one-half of an administered drug to be eliminated from the plasma of the patient through biological processes, e.g., biometabolism, excretion, etc.

[0070] As used herein, the term “volume of distribution” refers to the distribution and degree of retention of a drug throughout the various compartments of organisms, e.g. intracellular and extracellular spaces, tissues and organs, etc. This factor is expressed as the “apparent volume of distribution,” or Vd, which is the estimated volume of the body into which the drug has been distributed. A large Vd suggests that the drug has distributed more broadly throughout the body and may be associated with the longer half-life because a lesser portion of the drug will be in the plasma and thus delivered to the elimination points, the kidney and the liver.

[0071] The phrases “isolated,” “purified” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. An “isolated” region refers to a region that does not include the whole sequence of the polypeptide from which the region was derived. An “isolated” nucleic acid, protein, or respective fragment thereof has been substantially removed from its in vivo environment so that it may be manipulated by the skilled artisan, such as but not limited to nucleotideMOFO-360864280 12341812000140 sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in substantially pure quantities.

[0072] The term “reference sequence” or “reference peptide” as used herein generally refers to a collection of sequences available in various databases (also called the “subject sequence” or wild-type sequence) that can be used as a template to generate new sequences (also called “derived sequences” or “derived peptides”). Reference sequences can be used to identify, select and modify (e.g., substitute) one or more amino acids. Both reference sequences and the derived sequences can be aligned for comparison. The percent sequence identity is defined as a derived sequence’s percent identity to a reference sequence. For example, when stated “Sequence A having a sequence identity of 50% to Sequence B,” Sequence A is the derived sequence and Sequence B is the reference sequence. When using a sequence comparison algorithm, derived and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary. The percent sequence identity for the derived sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence. The equation for percent sequence identity from the aligned sequence is as follows: [(Number of Identical Positions) / (Total Number of Positions in the Derived Sequence)] x 100%.

[0073] The term “variable region” as used herein refers to a region of a peptide sequence that is not biologically active and is not necessary for the peptide to perform a function or induce physiological change (ie. binding).

[0074] The term “biologically active” or “binding region” can be used interchangeably to refer to a portion of a compound’s sequence (eg., a biologically active portion of the sequence) that interacts with a target. Non-limiting examples of targets include receptors, binders, cytokines, or another portion that induces a physiological change such as a signaling cascade or some other function.

[0075] The term “derivative”, “derived”, “engineered” or “variant” as used herein concerning a peptide refers to a peptide that is derived from the reference peptide. These terms refer to amino acid sequence variants that may have amino acids replaced, deleted, or inserted with naturally occurring or non-canonical amino acids in the variable or binding region, as compared to the wild-type polypeptide. A derivation can optionally include chemicalMOFO-360864280 13341812000140 modifications of the peptide such that the peptide still retains some of its fundamental pharmacologic activities. Chemical modifications of interest include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation, the substitution of non-canonical amino acids, or glycosylation of the peptide at any position. In addition, a derivative peptide may be a fusion of a polypeptide to a chemical compound, such as, but not limited to, another peptide, antibody, drug molecule or other therapeutic or pharmaceutical agent or a detectable probe. A “deletion” is the removal of one or more amino acids from within the wild-type protein, while a “truncation” is the removal of one or more amino acids from one or more ends of the wild-type protein. Thus, a variant peptide may be made by manipulation of genes encoding the polypeptide. A variant may be made by altering the basic composition or characteristics of the polypeptide, but not at least some of its fundamental pharmacologic activities.

[0076] The term “amino acid,” as used herein, means an amino acid moiety that comprises any naturally occurring or non-naturally occurring or synthetic amino acid residue, i.e., any moiety comprising at least one carboxyl and at least one amino residue directly linked by one, two, three or more carbon atoms, typically one (a) carbon atom. An amino acid may be an L- isomer or a D-isomer of an amino acid.

[0077] ‘ ‘Non-naturally encoded amino acid” or “non-canonical amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term “non-naturally encoded amino acid” include “non-natural amino acid,” “non-natural amino acid,” “non-naturally occurring amino acid,” and various hyphenated and non-hyphenated versions thereof, am. The term “non-naturally encoded amino acid” also refers to modifications (e.g., post-translational modifications) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but are not themselves naturally incorporated into the growing polypeptide chain by the translation complex. Examples of such non-naturally encoded amino acids include, but are not limited to, N- acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0078] The term “percent (%) amino acid sequence identity” as used herein, refers to the percentage of amino acid residues in a derived polypeptide, or the percent homology, that are identical to amino acid residues in a reference sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum % sequence identity. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art. OftenMOFO-360864280 14341812000140 publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align peptide sequences. For BLAST sequences under 30 amino acids in length, blast parameters are set to word size 2, gap opening penalty 9, gap extension penalty 9, scoring matrix PAM30, lookup table addition score threshold 16 (equivalent to NCBI BLAST’S “blastp -task blastp-short”). For sequences longer than 30 amino acids, the parameters are set to NCBI BLAST+ 2.16.0 blastp default parameters.

[0079] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” can be used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. The term “detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0080] The term “encode,” as used herein, refers to the ability of a polynucleotide to provide information or instructions sequence sufficient to produce a corresponding gene expression product. In a non-limiting example, mRNA can encode for a polypeptide during translation, whereas DNA can encode for an mRNA molecule during transcription.

[0081] The term “ex vivo” refers to an event that takes place outside of a subject’s body. An ex vivo assay may not be performed on a subject. Rather, it can be performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample can be an “in vitro” assay.

[0082] The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0083] The term “protein”, “peptide”, and “polypeptide” can be used interchangeably and in their broadest sense can refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits can be linked by peptide bonds. Alternatively, the subunits can be linked by other bonds, e.g., ester, ether, etc. A protein or peptide can contain at least two amino acids and no limitation can be placed on the maximum number of aminoMOFO-360864280 15341812000140 acids which can comprise a protein’s or peptide’s sequence. As used herein the term “amino acid” can refer to either natural amino acids, unnatural amino acids, or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetic s .

[0084] The term “in vitro” refers to an event that takes place contained in a container for holding laboratory reagents, such that it can be separated from the biological source from which the material can be obtained. In vitro assays can encompass cell-based assays in which living or dead cells can be employed. In vitro assays can also encompass a cell-free assays in which no intact cells can be employed.

[0085] The term “in vivo” refers to an event that takes place in a subject’s body.

[0086] The term “half-life extending moiety” refers to an increase in half-life (serum half-life and / or therapeutic half-life) and / or increased absorption, compared to a comparator such as an unconjugated form of a peptide or a wild-type reference peptide. Modifications described herein that improve or alter biological or biophysical properties, optionally via non-naturally encoded amino acids, directly or via linkers (e.g. peptide components or PEG), for example as PK extender components refers to a pharmaceutically acceptable moiety, domain, or molecule that is covalently linked (“conjugated” or “fused”) to a derived peptide. The term half-life extending moiety refers to a non-proteinaceous, half-life extending moiety such as a fatty acid or derivative thereof, a water-soluble polymer such as polyethylene glycol (PEG) or discontinuous PEG, hydroxyethyl starch (HES), lipid, branched or non-branched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups, and branched or unbranched C8-C30 alkyl groups; and proteinaceous half-life extending moieties such as serum albumin, transferrin, Adnectin (e.g., albumin-binding or pharmacokinetic extension (PKE) Adnectin), Fc domain, and unstructured polypeptides such as XTEN and PAS polypeptides (e.g. For example, a conformationally disordered polypeptide sequence consisting of the amino acids Pro, Ala, and / or Ser), and fragments of any of the foregoing.

[0087] The term “linker” or “spacer” may be any component that connects the half-life extending moiety to the derived peptide. Exemplary linkers include small organic compounds, water-soluble polymers of various lengths such as poly(ethylene glycol) or polydextran, and linkers of up to 50, 40, 30, 25, 20, 15, 10 or up to 6 amino acids in length, for example, including, but not limited to peptides or polypeptides.

[0088] The term “deamidation” refers to the tendency of an amino acid residue in a polypeptide to spontaneously undergo a deamidation reaction, thereby changing the chemical structure of the amino acid and potentially affecting the function of the polypeptide.MOFO-360864280 16341812000140Exemplary methods for measuring deamidation include imaging capillary isoelectric focusing (icIEF). The relative amount of deamidation can be determined relative to a comparative compound, for example, to identify polypeptides with reduced deamidation.

[0089] The term “in vivo proteolysis” refers to the cleavage of a polypeptide that can occur by proteases occurring in an organism when introduced into a living system (e.g., when injected into an organism). Proteolysis can potentially affect the biological activity or halflife of the polypeptide. For example, wild-type peptides can undergo cleavage, producing a truncated, inactive polypeptide. An exemplary method for measuring in vivo proteolysis of peptides is the Meso Scale Discovery (MSD)-based electrochemiluminescence immunosorbent assay (ECLIA). The relative amount of in vivo proteolysis can be determined relative to a comparative compound, for example, to identify polypeptides with reduced in vivo proteolysis.

[0090] The term “solubility” refers to the amount of a substance that can be dissolved in another substance, for example, the amount of an unmodified or derived peptide that can be dissolved in an aqueous solution. An exemplary method for measuring the solubility of unmodified or derived peptides is the plug flow solubility test. Relative solubility can be determined in relation to a comparison compound, for example, to identify polypeptides with increased solubility.

[0091] The term “biological activity” or “bioactivity” refers to a biological system, pathway, molecule or interaction with organisms including, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals and humans. Function refers to the ability of a molecule to affect any physical or biochemical property. For example, concerning the unmodified or derived peptide, the biological activity includes any of the functions performed by the peptide.

[0092] The term “bioavailability” as described herein refers to the extent a substance or drug becomes completely available in systemic circulation to its intended biological destination. Non-limiting examples of bioavailability include oral bioavailability, subcutaneous bioavailability, or penetration across the blood-brain barrier.

[0093] As used herein, the terms “adjusted serum half-life” or “adjusted in vivo half-life” and similar terms refer to a comparator, such as the positive or negative circulation half-life of a modified peptide compared to its unmodified form or wild-type peptide. Serum half-life can be measured by collecting blood samples at various time points following administration of the derived peptide and determining the concentration of the molecule in each sample. For example, peptides can be isolated from such blood samples taken over time and quantified onMOFO-360864280 17341812000140 a mass spectrometer. The resulting dataset comprising peptide counts over time can be fit to an exponential decay curve to estimate the time at which half of a given species is cleared. The correlation between serum concentration and time allows the calculation of serum halflife. The increased serum (in vivo) half-life may preferably be at least about two-fold, although smaller increases may be useful, for example, to enable a satisfactory dosing regimen or to avoid toxic effects. In some embodiments, the increase can be at least about 3- fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 2000- fold, or at least about 3000-fold.

[0094] As used herein, the term “unacceptable PK parameter”, or “unacceptable PK” relates to the PK parameter that is deemed undesirable or unimproved based on tested experimental parameters.DETAILED EMBODIMENTS

[0095] Techniques for performing the methods of the present invention are well known in the art and described in standard laboratory textbooks, including, for example, Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997); Molecular Cloning: A Laboratory Manual, Third Edition, J. Sambrook and D. W. Russell, eds., Cold Spring Harbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; O’Brian et al., Antibody Phage Display, Methods and Protocols, Humana Press, 2001; Phage Display: A Laboratory Manual, C. F. Barbas III et al. eds., Cold Spring Harbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; and Antibodies, G. Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can, for example, be performed using site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)). PCR amplification methods are described in U.S. Pat. Nos. 4,683,192, 4,683,202, 4,800,159, and 4,965,188, and in several textbooks including “PCR Technology: Principles and Applications for DNA Amplification”, H. Erlich, ed., Stockton Press, New York (1989); and “PCR Protocols: A Guide to Methods and Applications”, Innis et al., eds., Academic Press, San Diego, Calif. (1990).

[0096] Information concerning antibody sequence analysis using the Kabat database and Kabat conventions may be found, e.g., in Johnson et al., The Kabat database and a bioinformatics example, Methods Mol. Biol. 2004; 248: 11-25; and Johnson et al., Preferred CDRH3 lengths for antibodies with defined specificities, Int Immunol. 1998, December; 10(12): 1801-5.MOFO-360864280 18341812000140

[0097] Information regarding antibody sequence analysis using Chothia conventions may be found, e.g., in Chothia et al., Structural determinants in the sequences of immunoglobulin variable domain, J Mol. Biol. 1998 May 1; 278(2):457-79; Morea et al., Antibody structure, prediction and redesign, Biophys Chem. 1997; 68(l-3):9-16; Morea et al., Conformations of the third hypervariable region in the VH domain of immunoglobulins; J Mol. Biol. 1998, 275(2):269-94; Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins, J Mol. Biol. 1997, 273(4):927-48. Barre et al., Structural conservation of hypervariable regions in immunoglobulins evolution, Nat Struct Biol. 1994, l(12):915-20; Chothia et al., Structural repertoire of the human VH segments, J Mol. Biol. 1992, 227(3):799-817 Conformations of immunoglobulin hypervariable regions, Nature. 1989, 342(6252): 877-83; and Chothia et al., Review Canonical structures for the hypervariable regions of immunoglobulins, J Mol. Biol. 1987, 196(4):901-17).In silico Design of Diverse (Poly)peptide Libraries

[0098] The present disclosure relates to the generation of large, complex annotated databases of related peptides or unrelated peptides, based upon relevant single or multiple key parameters that can be individually directly defined computationally. The methods further enable the in vivo screening of diverse libraries. The large, complex, and diverse peptide libraries designed and generated herein can be used for large scale screening and optimization of therapeutic peptides.

[0099] Previously designed and manufactured libraries for screening rely on random amino acid substitutions with naturally occurring amino acids in a therapeutic peptide. These libraries are limited because a) randomized libraries are extremely inefficient, in that the vast majority of members will be non-performing; b) peptides comprised entirely of natural amino acids are too unstable for in vivo screening purposes; c) non-natural chemistries are often critical for drug pharmacokinetics and pharmacodynamics. In comparison, the libraries described herein can be designed based on biochemical insight and incorporate non-canonical amino acids that enable in vivo screening while improving pharmacokinetics and pharmacodynamics. By rationally, selectively diversifying peptide libraries position-by- position, incorporating any non-natural amino acid and scaling library diversity by design, the disclosed inventions enable in vivo screening of high-success-rate molecules at massively high-throughput and with tunable pharmacokinetics & pharmacodynamics.

[0100] As described herein, large scale in vivo pharmacokinetic screening of the described peptide libraries is possible because the libraries are designed and generated for administration to non-human mammalian subjects. After administration to the non-humanMOFO-360864280 19341812000140 mammal, methods such as mass spectrometry can be used to analyze pharmacokinetic properties of the peptide variants in vivo at a high throughput scale.

[0101] The present invention relates to modifications of known or pre-existing peptides that are variants or derivatives, and in specific embodiments, maintain one or more pharmacologic activities, and / or that improve the pharmacokinetic properties of the peptide. These modifications include, but are not limited to, variants and derivatives of the peptides that may increase their stability, specific activity, plasma half-life, increase protease resistance, and / or decrease the immunogenicity of the derived peptide, while retaining the ability of peptide binding profiles. Such variants include, but are not limited to, those which decrease the hydrolysis of the peptide, decrease the deamidation of the peptide, decrease the oxidation, decrease protease degradation, decrease the immunogenicity and / or increase the structural stability of the peptide. It is contemplated that two or more of the modifications described herein may be combined in one modified derived peptide, as well as combinations of one or more modifications described herein with other modifications to improve pharmacokinetic properties that are well known to those in the art.

[0102] Other pharmacokinetic parameters include, but are not limited to, reducing toxicity, improving solubility, reducing aggregation, increasing biological activity and / or target selectivity of the derived peptide, increasing manufacturability, and / or reducing immunogenicity of the derived peptide.

[0103] As discussed in US20200267608, one approach to improving the pharmacokinetic properties of the peptides is to create variants and derivatives of the peptides that are less susceptible to biotransformation. Biotransformation may decrease the pharmacologic activity of the peptide as well as decrease the rate at which it is eliminated from the patient’s body. One way of achieving this is to determine the amino acids and / or amino acid sequences that are most likely to be biotransformed and to replace these amino acids with ones that are not susceptible to that particular transformative process.

[0104] Hydrolysis is generally a problem in peptides containing aspartate. Aspartate is susceptible to dehydration to form a cyclic imide intermediate, causing the aspartate to be converted to the potentially inactive iso-aspartate analog, and ultimately cleaving the peptide chain. For example, in the presence of aspartic acid-proline in the peptide sequence, the acid- catalyzed formation of cyclic imide intermediate can result in cleavage of the peptide chain. As a non-limiting example, aspartate residues can be substituted for any non-canonical amino acid.MOFO-360864280 20341812000140

[0105] Hydrolysis is generally a problem in peptides containing aspartate. Aspartate is susceptible to dehydration to form a cyclic imide intermediate, causing the aspartate to be converted to the potentially inactive iso-aspartate analog, and ultimately cleaving the peptide chain. For example, in the presence of aspartic acid-proline in the peptide sequence, the acid- catalyzed formation of cyclic imide intermediate can result in cleavage of the peptide chain. As a non-limiting example, aspartate residues can be substituted for any naturally occurring or non-canonical amino acid.

[0106] It is contemplated that substituting other amino acids for asparagine and / or serine in the sequence of the derived peptide may result in a peptide with improved pharmacokinetic properties such as a longer plasma half-life and increased specific activity of a pharmacologic activity of the peptide. In one non-limiting instance of a variant, at least one or more asparagine residues of the derived peptide may be replaced with another amino acid residue, specifically a non-canonical residue. In another contemplated variant, one or more serine residues of the derived peptide may be replaced with another amino acid residue, specifically a non-canonical residue. In some variants of the derived peptide, one or more asparagine residues and one or more serine residues are substituted. In some embodiments, conservative substitutions are made. In other embodiments, non-conservative substitutions are made.

[0107] It is contemplated that substituting other amino acids for asparagine and / or serine in the sequence of the derived peptide may result in a peptide with improved pharmacokinetic properties such as a longer plasma half-life and increased specific activity of a pharmacologic activity of the peptide. In one non-limiting instance of a variant, at least one or more asparagine residues of the derived peptide may be replaced with another amino acid residue, specifically a naturally occurring amino acid residue, or a non-canonical amino acid residue. In another contemplated variant, one or more serine residues of the derived peptide may be replaced with another amino acid residue, specifically a naturally occurring amino acid residue, or a non-canonical amino acid residue. In some variants of the derived peptide, one or more asparagine residues and one or more serine residues are substituted. In some embodiments, conservative substitutions are made. In other embodiments, non-conservative substitutions are made.

[0108] Deamidation of amino acid residues is a particular problem in biotransformation. This base-catalyzed reaction frequently occurs in sequences containing asp aragine-gly cine or glutamine-glycine and follows a mechanism analogous to the aspartic acid-glycine sequence above. The deamidation of the asparagine-glycine sequence forms a cyclic imide intermediate that is subsequently hydrolyzed to form the aspartate or isoasparate analog of asparagine. InMOFO-360864280 21341812000140 addition, the cyclic imide intermediate can lead to racemization into D-aspartic acid or D- isoaspartic acid analogs of asparagine, all of which can potentially lead to inactive forms of the peptide.

[0109] It is contemplated that deamidation in the peptides may be prevented by replacing a glycine, asparagine and / or glutamine of the asparagine-glycine or glutamine-glycine sequences of the peptide with another amino acid and may result in a peptide with improved pharmacokinetic properties, such as a longer plasma half-life and increased specific activity of the pharmacologic activity of the peptide. In some embodiments, the one or more glycine residues of the derived peptide are replaced by another amino acid residue. In specific embodiments, one or more glycine residues of the derived peptide are replaced with a non- canonical amino acid residue. In some embodiments, the one or more asparagine or glutamine residues of the derived peptide are replaced by another amino acid residue. In specific embodiments, one or more asparagine or glutamine residues of the derived peptide are replaced with a non-canonical amino acid residue.

[0110] It is contemplated that deamidation in the peptides may be prevented by replacing a glycine, asparagine and / or glutamine of the asparagine-glycine or glutamine-glycine sequences of the peptide with another amino acid and may result in a peptide with improved pharmacokinetic properties, such as a longer plasma half-life and increased specific activity of the pharmacologic activity of the peptide. In some embodiments, the one or more glycine residues of the derived peptide are replaced by another amino acid residue. In specific embodiments, one or more glycine residues of the derived peptide are replaced with a non- canonical amino acid residue. In some embodiments, the one or more asparagine or glutamine residues of the derived peptide are replaced by another amino acid residue. In specific embodiments, one or more asparagine or glutamine residues of the derived peptide are replaced with a naturally occurring amino acid residue, or a non-canonical amino acid residue.

[0111] Reversible and irreversible oxidation of amino acids are other biotransformative processes that may also pose a problem that may reduce the pharmacologic activity, and / or plasma half-life of derived peptides. The cysteine and methionine residues are the predominant residues that undergo reversible oxidation. Oxidation of cysteine is accelerated at higher pH, where the thiol is more easily deprotonated and readily forms intra-chain or inter-chain disulfide bonds. These disulfide bonds can be readily reversed in vitro by treatment with dithiothreitol (DTT) or tris(2-carboxyethylphosphine) hydrochloride (TCEP).MOFO-360864280 22341812000140Methionine oxidizes by both chemical and photochemical pathways to form methionine sulfoxide and further into methionine sulfone, both of which are almost impossible to reverse.

[0112] It is contemplated that oxidation in the derived peptides may be prevented by replacing methionine and / or cysteine residues with other residues. In some embodiments, one or more methionine and / or cysteine residues of the derived peptide are replaced by another amino acid residue. In specific embodiments, a methionine residue is replaced with a non- canonical amino acid residue.

[0113] It is contemplated that oxidation in the derived peptides may be prevented by replacing methionine and / or cysteine residues with other residues. In some embodiments, one or more methionine and / or cysteine residues of the derived peptide are replaced by another amino acid residue. In specific embodiments, a methionine residue is replaced with a naturally occurring amino acid residue, or a non-canonical amino acid residue.

[0114] As described herein, a non-exhaustive list of non-canonical amino acids are available for purchase at the WuXi TIDES Product Portfolio, and can be selected for incorporation into the derived peptide sequences as described herein.

[0115] An additional non-exhaustive list of non-canonical amino acids can be seen as described in Castro et al., and can be selected for incorporation into the derived peptide sequences as described herein (Castro TG, et al., (2023). Biomolecules, 13(6):981). Additional non-canonical amino acids are listed in Chem Impex, Sigma, and Bachem, and can be selected for incorporation into the derived peptide.

[0116] In some embodiments, the peptide libraries described herein comprise at least about IxlO3unique peptide variants. In some embodiments, the peptide libraries described herein comprise at least about IxlO3, about IxlO4, about IxlO5, about IxlO6, or about IxlO7unique peptide variants. In some embodiments, the peptide libraries described herein comprise between about IxlO3and about IxlO7, between about IxlO3and about IxlO6, between about IxlO3and about IxlO5, or between about IxlO3and about IxlO4unique peptide variants. In some embodiments, the peptide libraries described herein comprise between about IxlO4and about IxlO7, between about IxlO5and about IxlO7, or between about IxlO6and about IxlO7unique peptide variants.

[0117] In some embodiments, each peptide variant in the peptide library comprises at least one modification at one or more of a plurality of sites in the therapeutic peptide from which the variants were designed. In some embodiments, each peptide variant in the peptide library comprises at least one modification at one or more of a plurality of sites in a region of the therapeutic peptide from which the variants were designed. In some embodiments, the regionMOFO-360864280 23341812000140 comprises at least about 10 amino acids. In some embodiments, the region comprises at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, or at least about 100 amino acids. In some embodiments, the region comprises between about 10 and about 100 amino acids, between about 10 and about 90 amino acids, between about 10 and about 80 amino acids, between about 10 and about 70 amino acids, between about 10 and about 60 amino acids, between about 10 and about 50 amino acids, between about 10 and about 40 amino acids, between about 10 and about 30 amino acids, or between about 10 and about 20 amino acids. In some embodiments, the region comprises between about 20 and about 100 amino acids, between about 30 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 60 and about 100 amino acids, between about 70 and about 100 amino acids, between about 80 and about 100 amino acids, or between about 90 and about 100 amino acids.

[0118] In some embodiments, the region is selected based on a binding property of the therapeutic peptide. In some embodiments, the region is selected based on amino acids predicted to contribute to binding of the therapeutic peptides to target. In some embodiments, the region is selected based on amino acids predicted not to contribute to binding of the therapeutic peptides to target. In some embodiments, the region of the peptide is an N- terminus of the therapeutic peptide. In some embodiments, the region of the peptide is a C- terminus of the therapeutic peptide. Selection of the region based on binding or non-binding amino acids may be based on the intended design of the therapeutic peptide variants. In a non-limiting example, the methods are for designing a peptide variation of the therapeutic peptide with increased stability but with no change in binding efficacy and the region comprises amino acids not expected to impact binding. One skilled in the art would appreciate the relationship between a region of interest for modification and an intended improvement in the therapeutic peptide.

[0119] In some embodiments, wherein at least one modification in peptide variants described herein comprises a non-canonical amino acid, such as a non-canonical amino acid described herein.

[0120] In some embodiments, each of the unique peptide variants comprise a C-terminal and / or N-terminal modification. In some embodiments, the one or more unique peptide variants comprise an amidated C-terminus and / or acetylated N-terminus. In some embodiments, the C-terminal modification for a peptide of the unique peptide variants isMOFO-360864280 24341812000140 selected from a group consisting of C-terminal carboxylic acids, C-terminal esterification, and C-terminal amidation. In some embodiments, the N-terminal modification for a peptide of the unique peptide variants is selected from a group consisting of trifluoroacetylation of the N-terminus, N-terminal propionylation, N-terminal monomethylation, N-terminal dimethylation, N-terminal trimethylation and N-terminal acetylation.

[0121] In some embodiments, one or more peptides in the unique peptide variants comprise a backbone modification. In some embodiments, the modification comprises a beta-amino acid, a n-methylated amino acid, an alpha-substituted amino acid, a gamma-substituted amino acid, and / or a peptoid.

[0122] The methods described herein can be used to design, manufacture, and determine FPKP for linear and or cyclic peptides. In some embodiments, the therapeutic peptide and the unique peptide variants thereof comprises a linear peptide sequence.

[0123] In some embodiments, the therapeutic peptides and the unique peptide variants thereof comprise cyclic peptide sequences. The cyclic peptide sequences may be used to generate cyclic polypeptides configured with loops and / or rings. In some embodiments, cyclic peptide sequences may comprise head-to-tail cyclization, wherein the N-terminus amino group and C-terminus (carboxyl group) of the peptide are covalently linked to form a circular backbone. In some embodiments, cyclic peptide sequences may comprise side chain cyclization wherein cyclization occurs between side chains of amino acids, such as through disulfide bonds (e.g., cysteine residues) or other covalent linkages. In some embodiments, cyclic peptide sequences may comprise backbone-to-side chain cyclization wherein a peptide backbone is linked to a side chain, forming a loop structure.

[0124] In some embodiments, disulfide cyclization between amino acids may be used to form a cyclic peptide sequence. In some embodiments, disulfide cyclization comprises formation of a covalent disulfide bond between the thiol groups of two cysteine residues. Cyclization of a generated peptide sequence with disulfide cyclization results in a loop structure in the generated polypeptide.Tertiary Structure Stabilization

[0125] The stability of the tertiary structure of the derived peptide will affect most aspects of the pharmacokinetics, including the pharmacologic activity, plasma half-life, and / or immunogenicity among others. See Kanovsky et al., Cancer Chemother. Pharmacol. 52:202- 208 (2003); Kanovsky et al., PNAS 23:12438-12443 (2001). Peptide helices often fall apart into random coils, becoming more susceptible to protease attack and may not penetrate the cell membrane well. Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000).MOFO-360864280 25341812000140Therefore, one way to stabilize the overall structure of the peptide is to stabilize the a-helix structure of the peptide. The intra-molecular hydrogen bonding associated with helix formation reduces the exposure of the polar amide backbone, thereby reducing the barrier to membrane penetration in a transport peptide, and thus increasing related pharmacologic activities and increasing the resistance of the peptide to protease cleavage.

[0126] One method to stabilize an a-helix is to replace the a-helix breaking amino acid residues such as glycine, proline, serine and aspartic acid, or helix neutral amino acid residues such as alanine, threonine, valine, glutamine, asparagine, cysteine, histidine, lysine or arginine, with helix forming residues, such as leucine, isoleucine, phenylalanine, glutamic acid, tyrosine, tryptophan and methionine. It is contemplated that the a-helix of derived peptides may be stabilized by replacing one or more glycine, proline, serine and / or aspartic acid residues with other amino acids. In specific embodiments, the glycine, proline, serine, aspartic acid, alanine, threonine, valine, glutamine, asparagine, cysteine, histidine, lysine and / or arginine residues are replaced by non-canonical amino acid residues. In other specific embodiments, one or more serine or glutamine residues in the a-helices of a derived peptide may be substituted.

[0127] One method to stabilize an a-helix is to replace the a-helix breaking amino acid residues such as glycine, proline, serine and aspartic acid, or helix neutral amino acid residues such as alanine, threonine, valine, glutamine, asparagine, cysteine, histidine, lysine or arginine, with helix forming residues, such as leucine, isoleucine, phenylalanine, glutamic acid, tyrosine, tryptophan and methionine. It is contemplated that the a-helix of derived peptides may be stabilized by replacing one or more glycine, proline, serine and / or aspartic acid residues with other amino acids. In specific embodiments, the glycine, proline, serine, aspartic acid, alanine, threonine, valine, glutamine, asparagine, cysteine, histidine, lysine and / or arginine residues are replaced by naturally occurring amino acid residues or non- canonical amino acid residues. In other specific embodiments, one or more serine or glutamine residues in the a-helices of a derived peptide may be substituted. Additionally, the Aib non-canonical amino acid may be substituted.

[0128] Another method to stabilize an a-helix tertiary structure involves using unnatural amino acid residues capable of 7t-stacking. For example, in Andrews and Tabor (Tetrahedron 55:11711-11743 (1999)), pairs of s-(3,5-dinitrobenzoyl)-Lys residues were substituted into the a-helix region of a peptide at different spacings. The overall results showed that the i,(i+4) spacing was the most effective stabilizing arrangement. Increasing the percentage of water, up to 90%, increased the helical content of the peptide. Pairs of s-acyl-Lys residues inMOFO-360864280 26341812000140 the same i,(i+4) spacing had no stabilizing effect, indicating that the majority of the stabilization arises from 7t-7t interactions. In one embodiment, the derived peptide may be modified so that the lysine residues are substituted by s-(3,5-dinitrobenzoyl)-Lys residues. In a specific embodiment, the lysine residues may be substituted by s-(3,5-dinitrobenzoyl)-Lys in a i,(i+4) spacing.

[0129] Another method to stabilize an a-helix tertiary structure involves disulfide bond formation between side-chains of the a-helix. It is also possible to stabilize helical structures by means of formal covalent bonds between residues separated in the peptide sequence. The commonly employed natural method is to use disulfide bonds (Pierret et al., (1995). Inti. J. Pept. Prot. Res., 46:471-479). In some embodiments, one or more non-canonical residue pairs are substituted into the a-helices of the derived peptide.

[0130] Another method to stabilize an a-helix tertiary structure involves disulfide bond formation between side-chains of the a-helix. It is also possible to stabilize helical structures by means of formal covalent bonds between residues separated in the peptide sequence. The commonly employed natural method is to use disulfide bonds (Pierret et al., (1995). Inti. J. Pept. Prot. Res., 46:471-479). In some embodiments, one or more naturally occurring residue pairs, or non-canonical residue pairs are substituted into the a-helices of the derived peptide. Peptide Library

[0131] Disclosed herein is a method for generating a synthetic peptide library comprising: creating a pooled peptide library comprising one or more peptide variants having at least one non-naturally occurring AA at selected positions on the peptide; screening the library against one or more pharmacokinetic (PK) parameters, optionally screening of the library against a target molecule; and optionally isolating the one or more variants.

[0132] In some embodiments, a derived peptide of the present disclosure can comprise a portion of a naturally occurring peptide. For example, an engineered peptide of the present disclosure can comprise a portion of an alpha-helical domain or a binding portion of a naturally occurring protein. As described herein, such portions can be further engineered in the development of the derived peptides to deviate from the portion of the naturally occurring peptide.

[0133] As disclosed herein, are methods for generating a peptide library comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences are derived from a single reference sequence, wherein a subset of the derived peptide sequences comprises peptide sequences comprising a functional pharmacokinetic parameter (FPKP) detectably improved relative to the single reference sequence.MOFO-360864280 27341812000140

[0134] In one embodiment, the peptide library is formed from the deviation of a known naturally occurring protein.

[0135] As disclosed herein, a peptide library can be generated starting from a naturally occurring peptide sequence. The binding or biologically active portion of the naturally occurring peptide sequence is conserved, while the non-binding or non-biologically active amino acids are substituted in one or more positions for non-canonical amino acids, resulting in the possibility of 103,104, 105, 106, or 107possible peptide sequences derived from a single reference sequence.

[0136] In another embodiment, the peptide library is generated using machine learning methods to generate non-canonical amino acid sequences for the derived peptide.

[0137] In another embodiment, the peptide library is formed from the experimental analysis of ten thousand proteins’ PK properties in blood circulation after 4 hours.

[0138] A peptide library can be formed in accordance with the methods disclosed in the Loftis et al. scientific literature (Loftis AR, et al., (2021). Proc Natl Acad Sci USA, 118(34): e2101596118).Table 1.MOFO-360864280 28341812000140

[0139] A non-exhaustive list of non-canonical amino acids are listed in Table 1, and are further disclosed in reference and are available for purchase at the WuXi TIDES Product Portfolio (https: / / tides.wuxiapptec.com / catalog-product / products), and can be selected for incorporation into the derived peptide sequences as described herein.

[0140] The non-conical amino acids may be selected from (R)-Fmoc-2-amino-4,4,4- trifluoro-butyric acid, (S)-2-Fmoc-4,4-difluoropentanoic Acid, Fmoc-Aib-OH, N-Fmoc-3- lodo-E- Alanine tert-butyl ester, Fmoc-Homoarginine, Fmoc-E-norArg(Pbf)-OH, Fmoc-Cit- OH, Fmoc-E-Arg(NO2)-OH, Fmoc-E-Arg(Me2)-OH.HCl, Dde-E-Orn(Fmoc)-OH, Fmoc- Homoser(Trt)-OH, Fmoc-E-Glu(OMe)-OH, Fmoc-N,N-dimethyl-E-Asparagine, Fmoc-Cit- OH, FMOC-O-PHOSPHO-E-SERINE, Fmoc-L-Tyr(PO(OMe)2)-OH, Fmoc-Tyr(SO3N / A)- 0HA H20, Fmoc-L-I2-Homo-Asp(OtBu)-OH, Fmoc-N,N-dimethyl-L-Glutamine, Fmoc-L- Asn(Me)-OH, Dde-L-Orn(Fmoc)-OH, Fmoc-Pen(Trt)-OH, Fmoc-L-Sec(Mob)-OH, Fmoc- Homoser(Trt)-OH, Fmoc-L-Glu(OMe)-OH, Fmoc-N,N-dimethyl-L-Asparagine, Fmoc-Cit- OH, FMOC-O-PHOSPHO-L-SERINE, Fmoc-L-Tyr(PO(OMe)2)-OH, Fmoc-Tyr(SO3N / A)- 0HA H20, Fmoc-L-I2-Homo-Asp(OtBu)-OH, Fmoc-N,N-dimethyl-L-Glutamine, Fmoc-L- Asn(Me)-OH, Dde-L-Orn(Fmoc)-OH, d-Ala, Gamma-Aminobutyric Acid, Fmoc-L-His(l- Me)-0H, Fmoc-L-(4-thiazolyl)-Alanine, Fmoc-L-4-pyrazole(3-CF3)-OH, 2Furyl- Alanine, (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-(lH-tetrazol-5-yl)butanoic acid, Fmoc-i2-cyclopropyl-L- Alanine, Fmoc-L-Cpg-OH, Fmoc-Deg-OH, Fmoc-L-IsoValine-OH, Fmoc-L-Thr(Me)-OH, Fmoc-L-tBuAla-OH, Fmoc-hArg(Pbf)-OH, Dde-L-Om(Fmoc)-OH, Fmoc-L-Lys(Pya)-OH, Fmoc-Lys(Me)2-OHA HCl, Fmoc-Lys(Me)3-OH Chloride, Dde-L- Dapa(Fmoc)-OH, Fmoc-L-Cys(Et)-OH, Fmoc-L-Selenomethionine, Fmoc-Hse(Me)-OH, Fmoc-L-Met(O)-OH, FM0C-D-MET(02)-0H, Fmoc-D-Phe(4-Me)-OH, Fmoc-L-Tyr(3,5- C12)-OH, Fmoc-L-3-(l-Naphthyl)-alanine, Fmoc-p-nitro-D-Phe-OH, Fmoc-L-3-Pal-OH, Fmoc-L- 5-Pyrimidine(2-OMe)-OH, Fmoc-L-3-thiophene(2,5-2Me)-OH, Fmoc-D-beta-(2- furyl)-alanine, Fmoc-(S)-3-Amino-3-phenylpropionic acid, Fmoc-L-Phe(4-F)-OH, Fmoc-L- Phe(4-Cl)-0H, Fmoc-L-Phe(4-Br)-OH, Fmoc-L-Phe(4-OiPr)-OH, Fmoc-L-Phe(4-SMe)-OH, Fmoc-3,4-dehydro-L-Proline, Fmoc-L-Hyp(Bzl)-OH, Fmoc-L-Azetidine-2-carboxylic acid, Fmoc-L-4,4-difluoroproline, (2S,4R)-l-(((9H-fluoren-9-yl)methoxy)carbonyl)-4- fluoropyrrolidine-2-carboxylic acid, (S)-4-(((9H-fluoren-9- yl)methoxy)carbonyl)thiomorpholine-3-carboxylic acid, (6S)-5-Fmoc-5- azaspiro[2.4]heptane-6-carboxylic acid, Fmoc-L-I±-Me-Pro-OH, Fmoc-2-Abz-OH, Fmoc-L- Phe-L-ThrPsi(Me,Me)Pro-OH, (R)-Fmoc-2-amino-4,4,4-trifluoro-butyric acid, Fmoc-L- Ser(O-Ethyl)-OH, S-Methyl Cysteine, Fmoc-beta-N,N-Dimethylamino-L-Ala, Fmoc-L-MOFO-360864280 29341812000140Ser(HP03Bzl)-0H, (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- ethoxybutanoic acid, , (R)-Fmoc-2-amino-4,4,4-trifluoro-butyric acid, Fmoc-L-Ser(O-Ethyl)- OH, S-Methyl Cysteine, Fmoc-beta-N,N-Dimethylamino-L-Ala, Fmoc-L-Ser(HPO3Bzl)-OH, (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-ethoxybutanoic acid, Fmoc-L-5- fluoroTryptophan, Fmoc-L-5-ChloroTryptophan, Fmoc-L-Trp(5-Br)-OH, Fmoc-L-3- Benzothienylalanine, Fmoc-5-Methoxy-L-Tryptophan, Fmoc-L-7-AzaTrp-OH, Fmoc-L-Oia- OH, Fmoc-L-3-naphthyridine-OH, Fmoc-L-5-Indazole(2-Me)-OH, Fmoc-L-I2-Homo-Trp- OH, FMOC-Beta-l-Trp-OH (n-Boc), FMOC-3-Tertbutyltyrosine, Fmoc-2,6-dimethyl-L- tyrosine, Fmoc-L-Phe(4-F)-OH, Fmoc-L-Dopa(acetonide)-OH, Fmoc-Tyrosine(OMe), Fmoc- L-Tyr(3-F, 4-Me)-0H, Fmoc-L-Tyr(3,5-C12)-OH, Fmoc-3,5-Dibromo-L-Tyr-OH, Fmoc-L- Tyr(3,5-DiI)-OH, Fmoc-Tyr(SO3N / A)-OHA H2O, Fmoc-L-Tyr(PO(OMe)2)-OH, (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2,3-dihydrobenzofuran-5-yl)propanoic acid, Fmoc-I2-cyclopropyl-L- Alanine, Fmoc-L-Cpg-OH, Fmoc-Deg-OH, Fmoc-L-IsoValine- OH, Fmoc-L-Thr(Me)-OH, or Fmoc-L-tBuAla-OH.

[0141] In some embodiments, the non-canonical amino acids may be selected from the non- canonical amino acids in Table 2.Table 2: Exemplary Non-Canonical amino acidsMOFO-360864280 30341812000140MOFO-360864280 31341812000140MOFO-360864280 32341812000140MOFO-360864280 33341812000140Synthesis of Diverse (Poly)peptide Libraries

[0142] In some cases, a derived peptide can comprise, or be derived from a portion of a naturally occurring protein that is from about 12 amino acids to about 100 amino acids, fromMOFO-360864280 34341812000140 about 13 amino acids to about 100 amino acids, from about 14 amino acids to about 100 amino acids, from about 15 amino acids to about 100 amino acids, from about 16 amino acids to about 100 amino acids, from about 17 amino acids to about 100 amino acids, from about 18 amino acids to about 100 amino acids, from about 19 amino acids to about 100 ammo acids, from about 20 amino acids to about 100 amino acids, from about 21 amino acids to about 100 amino acids, from about 22 amino acids to about 100 amino acids, from about 23 amino acids to about 100 amino acids, from about 24 amino acids to about 100 amino acids, from about 25 amino acids to about 100 amino acids, from about 26 ammo acids to about 100 ammo acids, from about 27 ammo acids to about 100 amino acids, from about 28 amino acids to about 100 amino acids, from about 29 amino acids to about 100 amino acids, from about 30 amino acids to about 100 amino acids, from about 31 amino acids to about 100 amino acids, from about 32 amino acids to about 100 amino acids, from about 33 amino acids to about 100 amino acids, from about 34 amino acids to about 100 amino acids, from about 35 amino acids to about 100 amino acids, from about 36 amino acids to about 100 amino acids, from about 37 amino acids to about 100 amino acids, from about 38 amino acids to about 100 amino acids, from about 39 amino acids to about 100 amino acids, from about 40 amino acids to about 100 amino acids, from about 41 amino acids to about 100 amino acids, from about 42 amino acids to about 100 amino acids, from about 43 amino acids to about 100 amino acids, from about 44 amino acids to about 100 amino acids, from about 45 amino acids to about 100 amino acids, from about 46 amino acids to about 100 amino acids, from about 47 amino acids to about 100 amino acids, from about 48 amino acids to about 100 amino acids, from about 49 amino acids to about 100 amino acids, from about 50 amino acids to about 100 amino acids, from about 51 amino acids to about 100 amino acids, from about 52 amino acids to about 100 amino acids, from about 53 amino acids to about 100 amino acids, from about 54 amino acids to about 100 amino acids, from about 55 amino acids to about 100 amino acids, from about 56 amino acids to about 100 amino acids, from about 57 amino acids to about 100 amino acids, from about 58 amino acids to about 100 amino acids, from about 59 amino acids to about 100 amino acids, from about 60 amino acids to about 100 amino acids, from about 61 amino acids to about 100 amino acids, from about 62 amino acids to about 100 amino acids, from about 63 amino acids to about 100 amino acids, from about 64 amino acids to about 100 amino acids, from about 65 amino acids to about 100 amino acids, from about 66 amino acids to about 100 amino acids, from about 67 amino acids to about 100 amino acids, from about 68 amino acids to about 100 amino acids, from about 69 amino acids to about 100 amino acids, from about 70 amino acids to about 100 amino acids, from about 71MOFO-360864280 35341812000140 amino acids to about 100 amino acids, from about 72 amino acids to about 100 amino acids, from about 73 amino acids to about 100 amino acids, from about 74 amino acids to about 100 amino acids, from about 75 amino acids to about 100 amino acids, from about 76 amino acids to about 100 amino acids, from about 77 amino acids to about 100 amino acids, from about 78 amino acids to about 100 amino acids, from about 79 amino acids to about 100 amino acids, from about 80 amino acids to about 100 amino acids, from about 81 amino acids to about 100 amino acids, from about 82 amino acids to about 100 amino acids, from about 83 amino acids to about 100 amino acids, from about 84 amino acids to about 100 amino acids, from about 85 amino acids to about 100 amino acids, from about 86 amino acids to about 100 amino acids, from about 87 amino acids to about 100 amino acids, from about 88 amino acids to about 100 amino acids, from about 89 amino acids to about 100 amino acids, from about 90 amino acids to about 100 amino acids, from about 91 amino acids to about 100 amino acids, from about 92 amino acids to about 100 amino acids, from about 93 amino acids to about 100 amino acids, from about 94 amino acids to about 100 amino acids, from about 95 amino acids to about 100 amino acids, from about 96 amino acids to about 100 amino acids, from about 97 amino acids to about 100 amino acids, from about 98 amino acids to about 100 amino acids, or from about 99 amino acids to about 100 amino acids.

[0143] In some embodiments, a derived peptide can comprise, or be derived from, a portion of a naturally occurring protein that is from 12 amino acids to 100 amino acids, from 13 amino acids to 100 amino acids, from 14 amino acids to 100 amino acids, from 15 amino acids to 100 amino acids, from 16 amino acids to 100 amino acids, from 17 amino acids to 100 amino acids, from 18 amino acids to 100 amino acids, from 19 amino acids to 100 ammo acids, from 20 amino acids to 100 amino acids, from 21 amino acids to 100 amino acids, from 22 amino acids to 100 amino acids, from 23 amino acids to 100 amino acids, from 24 amino acids to 100 amino acids, from 25 amino acids to 100 amino acids, from 26 ammo acids to 100 ammo acids, from 27 ammo acids to 100 amino acids, from 28 amino acids to 100 amino acids, from 29 amino acids to 100 amino acids, from 30 amino acids to 100 amino acids, from 31 amino acids to 100 amino acids, from 32 amino acids to 100 amino acids, from 33 amino acids to 100 amino acids, from 34 amino acids to 100 amino acids, from 35 amino acids to 100 amino acids, from 36 amino acids to 100 amino acids, from 37 amino acids to 100 amino acids, from 38 amino acids to 100 amino acids, from 39 amino acids to 100 amino acids, from 40 amino acids to 100 amino acids, from 41 amino acids to 100 amino acids, from 42 amino acids to 100 amino acids, from 43 amino acids to 100 amino acids, from 44 amino acids to 100 amino acids, from 45 amino acids to 100 amino acids, from 46MOFO-360864280 36341812000140 amino acids to 100 amino acids, from 47 amino acids to 100 amino acids, from 48 amino acids to 100 amino acids, from 49 amino acids to 100 amino acids, from 50 amino acids to 100 amino acids, from 51 amino acids to 100 amino acids, from 52 amino acids to 100 amino acids, from 53 amino acids to 100 amino acids, from 54 amino acids to 100 amino acids, from 55 amino acids to 100 amino acids, from 56 amino acids to 100 amino acids, from 57 amino acids to 100 amino acids, from 58 amino acids to 100 amino acids, from 59 amino acids to 100 amino acids, from 60 amino acids to 100 amino acids, from 61 amino acids to 100 amino acids, from 62 amino acids to 100 amino acids, from 63 amino acids to 100 amino acids, from 64 amino acids to 100 amino acids, from 65 amino acids to 100 amino acids, from 66 amino acids to 100 amino acids, from 67 amino acids to 100 amino acids, from 68 amino acids to 100 amino acids, from 69 amino acids to 100 amino acids, from 70 amino acids to 100 amino acids, from 71 amino acids to 100 amino acids, from 72 amino acids to 100 amino acids, from 73 amino acids to 100 amino acids, from 74 amino acids to 100 amino acids, from 75 amino acids to 100 amino acids, from 76 amino acids to 100 amino acids, from 77 amino acids to 100 amino acids, from 78 amino acids to 100 amino acids, from 79 amino acids to 100 amino acids, from 80 amino acids to 100 amino acids, from 81 amino acids to 100 amino acids, from 82 amino acids to 100 amino acids, from 83 amino acids to 100 amino acids, from 84 amino acids to 100 amino acids, from 85 amino acids to 100 amino acids, from 86 amino acids to 100 amino acids, from 87 amino acids to 100 amino acids, from 88 amino acids to 100 amino acids, from 89 amino acids to 100 amino acids, from 90 amino acids to 100 amino acids, from 91 amino acids to 100 amino acids, from 92 amino acids to 100 amino acids, from 93 amino acids to 100 amino acids, from 94 amino acids to 100 amino acids, from 95 amino acids to 100 amino acids, from 96 amino acids to 100 amino acids, from 97 amino acids to 100 amino acids, from 98 amino acids to 100 amino acids, or from 99 amino acids to 100 amino acids

[0144] In some embodiments, an engineered peptide can comprise, or be derived from, a portion of a naturally occurring protein that is at most 100 amino acids, at most 99 amino acids, at most 98 amino acids, at most 97 amino acids, at most 96 amino acids, at most 95 amino acids, at most 94 amino acids, at most 93 amino acids, at most 92 amino acids, at most 91 amino acids, at most 90 amino acids, at most 89 amino acids, at most 88 amino acids, at most 87 amino acids, at most 86 amino acids, at most 85 amino acids, at most 84 amino acids, at most 83 amino acids, at most 82 amino acids, at most 81 amino acids, at most 80 amino acids, at most 79 amino acids, at most 78 amino acids, at most 77 amino acids, at most 76 amino acids, at most 75 amino acids, at most 74 amino acids, at most 73 amino acids, atMOFO-360864280 37341812000140 most 72 amino acids, at most 71 amino acids, at most 70 ammo acids, at most 69 ammo acids, at most 68 ammo acids, at most 67 amino acids, at most 66 amino acids, at most 65 amino acids, at most 64 amino acids, at most 63 amino acids, at most 62 amino acids, at most 61 amino acids, at most 60 amino acids, at most 59 amino acids, at most 58 amino acids, at most 57 amino acids, at most 56 amino acids, at most 55 amino acids, at most 54 amino acids, at most 53 amino acids, at most 52 amino acids, at most 51 amino acids, at most 50 ammo acids, at most 49 ammo acids, at most 48 ammo acids, at most 47 amino acids, at most 46 amino acids, at most 45 amino acids, at most 44 amino acids, at most 43 amino acids, at most 42 amino acids, at most 41 amino acids, at most 40 amino acids, at most 39 amino acids, at most 38 amino acids, at most 37 amino acids, at most 36 amino acids, at most 35 amino acids, at most 34 amino acids, at most 33 amino acids, at most 32 amino acids, at most 31 amino acids, at most 30 amino acids, at most 29 amino acids, at most 28 amino acids, at most 27 amino acids, at most 26 amino acids, at most 25 amino acids, at most 24 amino acids, at most 23 amino acids, at most 22 amino acids, at most 21 amino acids, at most 20 ammo acids, at most 19 ammo acids, at most 18 ammo acids, at most 17 amino acids, at most 16 amino acids, at most 15 amino acids, at most 14 amino acids, at most 13 amino acids, or at most 12 amino acids.

[0145] In some embodiments, an engineered peptide can comprise, or be derived from, a portion of a naturally occurring protein that is 100 amino acids, 99 amino acids, 98 amino acids, 97 amino acids, 96 amino acids, 95 amino acids, 94 amino acids, 93 amino acids, 92 amino acids, 91 amino acids, 90 amino acids, 89 amino acids, 88 amino acids, 87 amino acids, 86 amino acids, 85 amino acids, 84 amino acids, 83 amino acids, 82 amino acids, 81 amino acids, 80 amino acids, 79 amino acids, 78 amino acids, 77 amino acids, 76 amino acids, 75 amino acids, 74 amino acids, 73 amino acids, 72 amino acids, 71 amino acids, 70 amino acids, 69 amino acids, 68 amino acids, 67 amino acids, 66 amino acids, 65 amino acids, 64 amino acids, 63 amino acids, 62 amino acids, 61 amino acids, 60 amino acids, 59 amino acids, 58 amino acids, 57 amino acids, 56 amino acids, 55 amino acids, 54 amino acids, 53 amino acids, 52 amino acids, 51 amino acids, 50 amino acids, 49 amino acids, 48 amino acids, 47 amino acids, 46 amino acids, 45 amino acids, 44 amino acids, 43 amino acids, 42 amino acids, 41 amino acids, 40 amino acids, 39 amino acids, 38 amino acids, 37 amino acids, 36 amino acids, 35 amino acids, 34 amino acids, 33 amino acids, 32 amino acids, 31 amino acids, 30 amino acids, 29 amino acids, 28 amino acids, 27 amino acids, 26 amino acids, 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 aminoMOFO-360864280 38341812000140 acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, or 12 amino acids.

[0146] In another preferred embodiment, the derived peptide will be less than 95%, or less than 90%, or less than 85%, or less than 80%, or less than 75%, or less than 70%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10% identical in its amino acid sequence to the wild-type reference sequence.

[0147] In the present disclosure, synthetic peptide libraries are synthesized without utilizing biological systems such as phage or in vitro translation. There are at least five subtypes of synthetic peptide libraries that differ from each other in the design of the library and the method used for the synthesis of the library.

[0148] Overlapping peptide libraries include the entirety of a larger protein used to produce a library of 8-20 amino acid peptides which overlap. These libraries can be used to identify the specific regions of a larger protein which participate in a given interaction or to provide predigested versions of a larger protein for binding.

[0149] Truncation peptide libraries refer to a given peptide that is produced with various or all N or C terminal truncations. These smaller fragments can be used to identify the minimal required region of a peptide for a given interaction being studied.

[0150] Random libraries include randomly generated peptides of a set length, or range of lengths, which can be used to identify novel binding partners of a target of interest.

[0151] In some embodiments, a target of interest includes receptors, binders, circulating molecules such as cytokines, or another portion that induces a physiological change such as a signaling cascade or some other function.

[0152] Alanine scanning libraries are libraries where each amino acid of a given protein or peptide is replaced with an alanine sequentially such that each peptide contains only one alanine mutation but all possible mutations to alanine are present. This method can be used to identify critical residues for binding.

[0153] Scrambled or positional peptide libraries highlight specific positions in the peptide that are substituted for many or all other amino acids. This emphasizes the effect of each amino acid at that position in the peptide, as the binding or other activity of the peptide can be tested. Scrambled libraries are often mutated with random peptides and are used as negative controls.MOFO-360864280 39341812000140

[0154] Solid phase peptide synthesis is defined as a process by which a peptide is anchored by its C-terminus to an insoluble polymer, and is assembled by successive addition of the protected amino acids in its sequence. This version of peptide synthesis is usually limited to a chain length of approximately 100 amino acids, with multiple possibilities of amino acid combinations.

[0155] Introducing a combinatorial peptide library can be done through chemical and biological synthesis. For use in the present disclosure, the peptide library is produced chemically via organic synthesis. Chemical peptide libraries begin with solid-phase peptide synthesis (SPSS). The general approach of SPS begins with attaching the first amino acid to a solid support through its carboxyl group, and each N-terminal protected amino acid is added in turns. During coupling, the carboxyl group of the incoming amino acid (or pool of mixed amino acids in the case of combinatorial library synthesis) must be activated, which is commonly achieved by using carbodiimides, amino acid halides, uronium (guanidinium N- oxides), or phosphonium salts (Wang YC, et al., (2014). Curr Top Pept Protein Res, 15:1-23; Carpino LA, et al., (1996). Acc Chem Res, 29:268-274). After each addition, the N- protecting group must be removed before the next amino acid (or pool of amino acids) can be added. A common N-protecting group is the fluorenylmethyloxycarbonyl (Fmoc) group that can be removed in basic conditions (Behrendt R, et al., (2016). J Pept Sci, 22:4-27).Removing waste products of synthesis can be accomplished by washing since the growing peptide is attached to a surface (Bozovicar K, et al., (2019). Int J Mol Sci, 21 ( 1 ):215) .

[0156] A more detailed method of SPPS includes a cycle of each amino acid being added consisting of the steps: (1) Cleavage of the N-protecting group, (2) washing steps, (3) coupling of a protected amino acid (or protected pool of amino acids), (4) washing steps. As the growing chain is bound to an insoluble support, the excess reagents and soluble byproducts can be removed by simple filtration. Washing steps with appropriate solvents ensure the complete removal of cleavage agents after the deprotection step in addition to the elimination of excesses of reagents and by-products resulting from the coupling step. Once the sequence has been completed, the peptide(s) must be cleaved off the resin. In general, acidolytic cleavage from the resin is the method of choice to release the peptide at the end of the synthesis, but a broad range of resins are susceptible to be cleaved by nucleophiles such as the Kaiser oxime resin, the p-carboxybenzyl alcohol linker, or by photolysis (Collins JM, et al., (2023). Nat Commun, 14( 10):8168).

[0157] Other SPPS combinations include using BOC or FMOC, manual synthesis, continuous flow synthesis, or fully automated SPPS.MOFO-360864280 40341812000140

[0158] Solid-phase peptide synthesis involves attaching a protected amino acid to a polymer bead and then carrying out deprotection and coupling steps to add amino acids one at a time to the end of the growing peptide chain. Solid-phase peptide synthesis is a multistep process that, even when automated, is extremely time-consuming for the relatively small amount of peptides generated. The time-consuming aspects of solid-phase synthesis include slow wash times, and the recirculation of low-concentration reagents rather than continuously replenishing high-concentration reagents, resulting in slow amide bond formation (Simon MD, et al., (2014). Chembiochem, 15(5):713-720). Rather than carrying out discrete steps as automated peptide synthesizers do, rapid flow synthesis can construct peptides as reagents that continuously flow across the polymer beads in the reactor.

[0159] Control of solid-phase peptide synthesis involves the use of feedback from one or more reactions and / or processes (e.g., reagent removal) taking place in the solidphase peptide synthesis system. As disclosed in US11584776B2, in some embodiments, a detector may detect one or more fluids flowing across a detection zone of a solid phase peptide synthesis system and one or more pooled signals may be generated corresponding to the fluid(s), in order to generate a non-canonical amino acid incorporated derived peptide, or pooled peptides. For instance, an electromagnetic radiation detector positioned downstream of a reactor may detect a fluid exiting the reactor after a deprotection reactor and produce a signal(s). In some embodiments, based at least in part on information derived from the signal(s), a parameter of the system may be modulated prior to and / or during one or more subsequent reactions and / or processes taking place in the solid phase peptide synthesis system. In some embodiments, the methods and systems, described herein, can be used to conduct quality control to determine and correct problems (e.g., aggregation, truncation, deletion) in reactions (e.g., coupling reactions) taking place in the solid phase peptide synthesis system.

[0160] In another embodiment, a method of operating a peptide synthesis system comprises producing a first and a second signal at a detection zone positioned downstream of a peptide synthesis reactor, comparing the first signal to the second signal and / or to a reference signal, and modulating a parameter of the system prior to and / or during a reaction in the reactor based at least in part on information derived from the comparing step, wherein the parameter is selected from the group consisting of a flow rate, protease resistance, confirmational flexibility or rigidity, target interaction, renal clearance, bioavailability, a reaction time, a temperature, a reactant type, a reactant concentration, blood-brain barrierMOFO-360864280 41341812000140 penetrance, a ratio of reactants, an addition of an additive, and combinations thereof as disclosed in US11584776B2.

[0161] Rapid flow-based peptide synthesis is a method that can incorporate an amino acid residue every 1.8 minutes under automatic control, or every 3 minutes under manual control, as described in Simon et al. (Simon MD, et al., (2014). Chembiochem, 15(5):713-720). This method begins with the analysis of existing kinetic data to optimize temperature parameters when breaking and forming bonds to improve the quality of sequence synthesis and purity. An HPLC pump is used to deliver DMF or 50% piperidine in DMF for common washing and deblocking steps, and a syringe pump is used to deliver coupling reagents. HPLC and variant characterization can be seen in Example 4. For the coupling step, quick connect is moved to a syringe pump to deliver an activated amino acid solution. The effluent is passed through a UV detector to continually monitor the absorbance at a specific wavelength, such as 304nm, where the Fmoc amino acids and the dibenzofulvene-piperidine deprotection adduct absorb strongly. As described in Simon et al., they chose to start with a 2 minute DMF wash at 10 mL / min, a 2 minute Fmoc deprotection at 6 mL / min, another 2 minute DMF wash, and a 6 minute room temperature coupling with 2 mmol of activated amino acid delivered at 1 mL / min. This procedure yielded highly pure material, enabling peptide synthesis in 12 minutes per residue. To achieve a maximal concentration of activated amino acid and rate of amide bond formation, coupling solutions were prepared by dissolving amino acids in one equivalent of 0.4 mHBTU in DMF. The activating base was added immediately before use, giving a final concentration of activated amino acid of about 0.3 M Simon MD, et al., (2014). Chembiochem, 15(5):713-720). To efficiently create a high-diversity combinatorial library, weighted pools of activated amino acids are added at selected coupling steps rather than a pure solution of one activated amino acid. As seen in Example 2, flow peptide synthesis is included herein.

[0162] In some embodiments, as disclosed in US9169287B2, the process of adding amino acid residues to immobilized peptides comprises exposing a deprotection reagent to the immobilized peptides to remove at least a portion of the protection groups from at least a portion of the immobilized peptides. The deprotection reagent exposure step can be configured, in certain embodiments, such that side-chain protection groups are preserved, while N-termini protection groups are removed. For instance, in certain embodiments, the protection group used to protect the peptides comprises fluorenylmethyloxycarbonyl (Fmoc). In some such embodiments, a deprotection reagent comprising piperidine (e.g., a piperidine solution) may be exposed to the immobilized peptides such that the Fmoc protection groupsMOFO-360864280 42341812000140 are removed from at least a portion of the immobilized peptides. In some embodiments, the protection group used to protect the peptides comprises tert-butyloxycarbonyl (Boc). In some such embodiments, a deprotection reagent comprising trifluoroacetic acid may be exposed to the immobilized peptides such that the Boc protection groups are removed from at least a portion of the immobilized peptides. In some instances, the protection groups (e.g., tertbutoxycarbonyl, i.e., Boc) may be bound to the N-termini of the peptide.

[0163] In some embodiments, the process of adding amino acid residues to immobilized peptides comprises removing at least a portion of the deprotection reagent. In some embodiments, at least a portion of any reaction byproducts (e.g., protection groups) that may have formed during the deprotection step can be removed. In some instances, the deprotection reagent (and, in certain embodiments byproducts) may be removed by washing the peptides, solid support, and / or surrounding areas with a fluid (e.g., a liquid such as an aqueous or non-aqueous solvent, a supercritical fluid, and the like). In some instances, removing the deprotection reagent and reaction byproducts may improve the performance of subsequent steps (e.g., by preventing side reactions).

[0164] The process of adding amino acid residues to immobilized peptides comprises, in certain embodiments, exposing activated amino acids to the immobilized peptides such that at least a portion of the activated amino acids are bonded to the immobilized peptides to form newly bonded amino acid residues. For example, the peptides may be exposed to activated amino acids that react with the deprotected N-termini of the peptides. In certain embodiments, amino acids can be activated for reaction with the deprotected peptides by mixing an amino acid-containing stream with an activation agent stream, as discussed in more detail below. In some instances, the amine group of the activated amino acid may be protected, such that the addition of the amino acid results in an immobilized peptide with a protected N-terminus.

[0165] In some embodiments, the process of adding amino acid residues to immobilized peptides comprises removing at least a portion of the activated amino acids that do not bond to the immobilized peptides. In some embodiments, at least a portion of the reaction byproducts that may form during the activated amino acid exposure step may be removed. In some instances, the activated amino acids and byproducts may be removed by washing the peptides, solid support, and surrounding areas with a solvent.

[0166] Another method to generate large libraries includes the “split-and-mix” method, which is a quantitative process with reactions driven to completion by applying reagents in excess at each step. This method involved coupling individual amino acids to resin beads,MOFO-360864280 43341812000140 mixing the beads together, separating them in equal portions, and then reacting each portion with a different amino acid. The mixing, separating beads, and reaction steps are repeated until the desired peptide length and diversity are achieved. An important virtue of this method is that a single bead contains a single peptide sequence, which is why the libraries produced in this way are termed one-bead-one-compound (Bozovicar K, et al., (2019). Int J Mol Sci, 21(1):215).

[0167] In a preferred embodiment, methods of peptide library synthesis and platforms are generated based on Bozovicar et al. (Bozovicar K, et al., (2019). Int J Mol Sci, 21 ( 1 ):215) .

[0168] The majority of non-canonical amino acid synthetic strategies rely on well-established reaction manifolds proceeding through closed-shell intermediates, such as asymmetric hydrogenation, electrophilic amidation, Mannich and Strecker-type alkylations, and Petasis borono-Mannich reactions. Recently, reaction manifolds featuring open-shell intermediates have also gained significant attention, prompted by the advances in photo redox catalysis and electrosynthesis. Non-canonical amino acids are typically accessed through the addition of carbon-centered radicals (C-radicals) to glyoxylate imine or dehydro alanine derivatives using redox-active C-radical precursors, such as N-phthalimidoyl esters, trifluoroborates, amines, and others. The radicals are generated at the amino acid backbone, enabling the appending of redox-inactive molecules onto the amino acid side chain. As disclosed in Alvey et al., the entrance to one-electron reaction manifolds with feedstock carboxylic acids as radical precursors and a chiral glyoxylate-derived N-sulfinyl imine as the radical acceptor. The chiral-at-sulfur N-sulfinyl functionality served as an effective chiral auxiliary, providing P- branched non-canonical amino acids with excellent stereoselectivity at the a-stereogenic center. Direct oxidative activation of unfunctionalized carboxylic acids allowed the realization of the developed transformation as an overall redox-neutral reaction, providing stereoselective access to a range of amino acid derivatives with high atom economy and under mild reaction conditions (Alvey GR, et al., (2024). Chem Sci, 15( 19):7316-7323).

[0169] In preferred embodiments, the peptide library is synthesized and purified chemically. In preferred embodiments, the peptide library is not synthesized or purified in any form of bacteria or other eukaryotic or prokaryotic organisms.

[0170] As known in the art, bacteria such as E. coli or yeast can be used to recombinantly synthesize and purify peptide derivatives to create a library (Loftis AR, et al., (2021). Proc Natl Acad Sci USA, 118(34): e2101596118). Plasmid DNA from the selected clones are isolated, transformed, and amplified for plasmid purification (Longwell CK, et al., (2021). ACS Chem Biol, 16( l):58-66) . Bacterial expression systems for expressing recombinantMOFO-360864280 44341812000140 polypeptides are available in, e.g., E. coli, Bacillus sp., Salmonella, and Caulobacter. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well-known in the art and are also commercially available.

[0171] In certain embodiments, the derived peptide can include amino acid sequences that have at least a minimum binding affinity with respect to the wild-type reference peptide. Examples of desired characteristics include increased binding affinity of the receptor as compared to the known reference wild-type peptide that is specific for the receptor.

[0172] An example of such peptide synthesis can be found in Loftis et al., and Quartararo et al., which is incorporated by reference in its entirety (Loftis AR, et al., (2021). Proc Natl Acad Sci USA, 118(34): e2101596118; Quartararo AJ, et al., (2020). Nat Commun, 11:31883).

[0173] It is known in the art that the synthesis of non-canonical amino acids can result in low purity levels or low-efficiency levels.

[0174] In preferred embodiments, the derived peptides are synthesized with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% synthesis success rate.

[0175] In another embodiment, the derived peptides are synthesized with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity levels.Positional Diversity

[0176] As disclosed in the Loftis literature, unlike peptides composed of canonical L-amino acids, peptides synthesized from D-amino acids are not recognized by natural proteases, and should be resistant to proteolysis (Loftis AR, et al., (2021). Proc Natl Acad Sci USA, 118(34): e2101596118).

[0177] As disclosed in US20200267608, positional modifications of amino acids can result in favorable PK enhancements by reducing the likelihood of degradation and clearance from circulation.

[0178] In preferable embodiments, the binding sequence is conserved during the modification of peptide sequences. In some embodiments, the binding region is modified alongside the variable region.Chemical Diversity

[0179] Chemical modifications of interest include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation, the substitution of non-canonical amino acids, or glycosylation of the peptide. In addition, a derivative peptide may be a fusion of a polypeptide to a chemical compound, such as, but notMOFO-360864280 45341812000140 limited to, another peptide, drug molecule or other therapeutic or pharmaceutical agent or a detectable probe.

[0180] As disclosed in Quartararo et al., chemical modifications, such as non-canonical amino acid incorporation, head-to-tail macrocyclization, and chemical stapling, can render peptides more proteolytic ally stable, more cell-penetrant, and even increase binding affinity relative to their natural, underivatized counterparts, which on their own tend to exhibit poor pharmacological properties (Quartararo AJ, et al., (2020). Nat Commun, 11:31883).

[0181] As disclosed in the Tsuboyama literature, charged and uncharged amino acids have an impact on folding and overall protein stability. In regards to solubility, the most likely useful amino acids are the charged amino acids Glu, Asp, and Lys, suggesting selection for solubility, whereas the least likely amino acids are the nonpolar aromatic amino acids Trp, Phe and Tyr, along with Met. These offsets provide a quantitative ‘favourability’ metric incorporating all non-stability evolutionary influences on amino acid composition, including selection for amino acid synthesis cost, codon usage, avoiding oxidation-prone amino acids, net charge and function. These offsets also highlight that biophysical models and protein design methods trained to reproduce native protein sequences should consistently optimize folding stability (Tsuboyama K, et al., (2023). Nature, 620:434-444). Preferable charges at specific peptide positions depend on the derived peptide and its properties. Tsuboyama et al. found that among all sequences tested (wild-type or mutant pairs), pairs with opposite charges and cysteine pairs tended to have positive (favorable) couplings, whereas pairs with the same charge and acidic-aromatic-aliphatic pairs tended to have negative couplings.Average couplings are lower than wild-type couplings because the side chain orientations and environment surrounding wild-type pairs will typically be optimized for that pair (Tsuboyama K, et al., (2023). Nature, 620:434-444). Applying this data should recapitulate expected patterns of side-chain interactions, provide a wealth of data for training machine learning models, and identify a wide range of noteworthy interactions for further usage in derived peptides.

[0182] As described in Example 1, one or more non-canonical amino acids is incorporated in various selected positions in, e.g., a parathyroid hormone peptide by targeting one or more amino acid positions.

[0183] In some embodiments, the derived peptide has not more than 3, 2 or 1 amino acids replaced, deleted or inserted compared to a reference or wild-type peptide or a portion thereof. In other embodiments, the variant has more than 3, 2 or 1 amino acids replaced, deleted or inserted compared to a reference or wild-type peptide or a portion thereof.MOFO-360864280 46341812000140Protein mapping, Protein Design and protein library

[0184] A library is designed based on one peptide sequence, or many peptide sequences to achieve a favorable peptide PK profile. The biologically active portion of the sequence is conserved, whereas at each varied position, one of the thousands of available non-canonical amino acids are incorporated, encompassing a variety of polar, non-polar, charged, and aromatic side-chain functionalities such as those amino acids described in Quartararo et al. (Quartararo AJ, et al., (2020). Nat Commun, 11:31883). Non-canonical amino acids useful for peptide modifications can be seen in Table 1.

[0185] As described in Tsuboyama et al., folding stability can be analyzed using cDNA display proteolysis, which is a powerful high-throughput stability assay that can be used to produce a large dataset of folding stability measurements (Tsuboyama K, et al., (2023). Nature, 620:434-444). This method combines the strengths of cell-free molecular biology and next-generation sequencing and requires no on-site equipment larger than a quantitative PCR (qPCR) instrument. This method is cost-effective and has a wider dynamic range of stability for a large experimental scale. This method will aid in the analysis of peptide interactions to determine favorable PK parameters. These methods can be seen in Tsuboyama et al., and are hereby incorporated by reference.

[0186] As described in Loftis et al., peptide binding ligand identification can be assessed using nano-liquid chromatography-tandem mass spectrometry (nLC MS / MS), and positional preferences can be reviewed using positional frequency analysis (Loftis AR, et al., (2021). Proc Natl Acad Sci USA, 118(34): e2101596118). As described in Bozovicar, et al., a pooled library can be assayed by affinity selection mass spectrometry (AS-MS), and the selected binders can be identified using PCR amplification and sequencing (Bozovicar K, et al., (2019). Int J Mol Sci, 21 ( 1 ) :215). Synthesized derived peptides can be purified by preparative reverse phase HPLC, and analyzed by analytical HPLC and LC-MS. Further confirmational studies of the derived peptide variants can be analyzed with size-exclusion chromatographymass spectrometry (SEC-MS). SEC analysis enables the separation of the folded and denatured proteins. For all folded-derived peptide constructs, the charge distribution can be analyzed. The peptides can be analyzed under native and denaturing conditions through ion mobility mass spectrometry, to ensure the derived peptides still contain a nativelike conformation in solution, compared to the wild-type protein (Charalampidou A, et al., (2024). ACS Cent Sci, 10(3):649-657).

[0187] As described in Zhang et al., derived peptide sequencing can occur through nLC- MS / MS to sequence, validate, and retrieve ion m / z ratios of the peptide hits. BLLassistedMOFO-360864280 47341812000140AS-MS approach can be used to perform affinity selection of the derived peptides, and nLC- MS / MS analysis allows for the analysis of peptides that match the library design (Zhang G, et al., (2021). Chem Sci, 12(32): 10817- 10824).

[0188] Additionally, as described in Quartararo et al., affinity selection-mass spectrometry (AS-MS) can be used as an alternative strategy for target-based discovery of chemically accessed peptide binders. LC-MS / MS can be leveraged to sequence individual synthetic peptides, and to increase the diversity of synthetic -derived peptide libraries amenable to ASMS from approximately ~10 to ~106derived peptides (Quartararo AJ, et al., (2020). Nat Commun, 11:3183).

[0189] As disclosed in Vinogradov et al., the development of new MS / MS-friendly library designs and associated data analysis procedures take advantage of the designs for the interpretation of de novo sequencing outputs. The use of nLC-MS / MS for high-throughput analysis of synthetic peptide mixtures is achieved through the decoding at 600 peptides / hour with a peptide identification rate above 85%. This approach is straightforward, and requires no chemical manipulations or specialty reagents at any stage during the library synthesis or analysis, suggesting that this approach is a feasible high-throughput method for decoding mixtures of synthetic peptides (Vinogradov AA, et al., (2017). ACS Comb Sci, 19(11):694- 701).

[0190] In one embodiment, the direct target interaction of the derived peptide can be analyzed using mass spectrometry.

[0191] In another embodiment, the conformational flexibility or rigidity of the derived peptide can be analyzed using mass spectrometry.

[0192] In another embodiment, the protease resistance of the derived peptide can be analyzed using mass spectrometry.

[0193] In another embodiment, the renal clearance levels of the derived peptide can be analyzed using mass spectrometry.

[0194] In another embodiment, the bioavailability and / or biodistribution of the derived peptide can be analyzed using mass spectrometry.

[0195] In one embodiment, in a reference peptide sequence, a non-canonical amino acid is substituted for a naturally occurring amino acid to improve direct target interaction.

[0196] In one embodiment, in a reference peptide sequence, a non-canonical amino acid is substituted for a naturally occurring amino acid to improve conformational flexibility or rigidity.MOFO-360864280 48341812000140

[0197] In one embodiment, in a reference peptide sequence, a non-canonical amino acid is substituted for a naturally occurring amino acid to improve protease resistance.

[0198] In one embodiment, in a reference peptide sequence, a non-canonical amino acid is substituted for a naturally occurring amino acid to improve the inhibition of renal clearance.

[0199] In one embodiment, in a reference peptide sequence, a non-canonical amino acid is substituted for a naturally occurring amino acid to improve bioavailability, and / or improve biodistribution. As seen in Figure 2, in vitro and in vivo effects are described herein.

[0200] In one embodiment, excluding radiopharmaceutical applications, an unacceptable PK parameter is defined as a half-life value of less than one hour.

[0201] In one embodiment, an unacceptable PK parameter is defined as an oral bioavailability of less than 0.1%, or a blood-brain barrier bioavailability of less than 1%.

[0202] In some embodiments, the derived peptide has an amino acid sequence composition of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% that do not comprise D-amino acids.

[0203] In other embodiments, the derived peptide amino acid sequence has a non-canonical amino acid composition of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% that do not comprise D-amino acids.

[0204] In another non-limiting embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in the derived peptide sequence do not comprise D-amino acids.In vivo Screening of Diverse (Poly)peptide Libraries

[0205] Also disclosed herein are methods of screening for derived peptides of the present disclosure. In some embodiments, an initial screen can be utilized to select for portions of polypeptides that exhibit a property of interest. An example of such screening can be found in Quartararo et al., and in WO2021041953, which is incorporated by reference in its entirety (Quartararo AJ, et al., (2020). Nat Commun, 11:31883). In some embodiments, an initial screen can be used to tile portions of peptides that have reduced pharmacokinetic properties. Peptides from the initial screen can be further engineered as described herein to produce improved derived peptides with enhanced pharmacokinetic properties.

[0206] Additional screening methods include deconvolution which has been developed for screening and identification of hits from chemical peptide libraries. Iterative deconvolution is based on dividing the library into non-overlapping subsets containing peptides with defined residues at the specified positions, while the rest of the structure is randomized. Each subset is then screened separately. The most active subset of compounds is further divided into newMOFO-360864280 49341812000140 subsets to retain the identified optimal residues from the previous screening round and retested for activity. This process is continued until the fittest molecule is identified (Houghten RA, et al., (1985). Prot Natl Acad Sci, 82:5131-5135). Positional scanning addresses sub-libraries individually at each position, which is defined by one single amino acid residue, while the remaining positions are randomized. The positional sub-libraries are assayed in parallel to gather information on the optimal residue for each diversity position, and the fittest member is identified (Pinilla C, et al., (1993). Biotechniques, 13:901-905).

[0207] Advanced candidates selected by screening with improved properties relative to the peptide candidates can be further optimized to improve efficacy and pharmacokinetic parameters. For example, each advanced candidate can be modeled to predict the key interactions between the candidate peptide and the target. Based on the modeling, the length of the candidate can be modified to include key residues, such as non-canonical amino acids. In some embodiments, based on the modeling, the length of the candidate can be modified to minimize the length of the peptide while still including key residues. Further, modifications such as stapling can be employed as described herein to stabilize the secondary structure (e.g. alpha-helical structure) of the derived peptide by reducing the entropic penalty for adopting the structure.

[0208] In some embodiments, in vivo peptide screening comprises analyzing a FPKP of one or more peptide variants in a non-human mammalian subject. In some embodiments, a peptide library designed and manufactured according to embodiment described herein are introduced into a non-human mammalian subject. In some embodiments, the non-human mammalian subject is a mouse, rat, dog, pig or non-human primate. In some embodiments, at least a subset of the unique peptide variants of the peptide library are detected in a sample collected from the non-human mammalian subject. The detection may comprise collecting a blood and / or plasma sample from the non-human mammalian subject at one or more predetermined timepoints, recovering the unique peptide variants and characterizing the unique peptide variants. In some embodiments, the unique peptide variants are characterized using mass spectrometry. The FPKP of unique peptide variants can be calculated based on the timepoints wherein the unique peptide can be detected in samples collected from the non- human mammalian subject.

[0209] In some embodiments, the methods comprise collecting blood and / or plasma from the non-human mammalian subject following administration of a peptide library of the current disclosure at one or more predetermined time intervals. In some embodiments, the one or more predetermined intervals are selected from a group consisting of any time intervalMOFO-360864280 50341812000140 between about 0 minutes and 60 days. In some embodiments, the one or more predetermined interval comprise about 0 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, or about 60 days. In some embodiments, the one or more predetermined intervals comprise about 0 minutes, about 5 minutes, about 20 minutes, and about 60 minutes.

[0210] In some embodiments, the methods comprise detecting unique peptide variants in the blood and / or plasma from the non-human mammalian subject. In some embodiments, detecting the unique peptide variations comprises performing mass spectrometry on a sample from the blood and or plasma.

[0211] In some embodiments, the methods analyzing a FPKP of one or more peptide variants in a non-human mammalian subject by screening the peptide library in a blood, plasma, or serum sample collected from a non-human mammalian subject. In some embodiments, the non-human mammalian subject is a mouse, rat, dog, pig or non-human primate. In some embodiments, at least a subset of the unique peptide variants of the peptide library are detected in the blood, plasma, or serum sample over time such as at the one or more predetermined time points as described herein. In some embodiments, the unique peptide variants are characterized using mass spectrometry. The FPKP of unique peptide variants can be calculated based on the timepoints wherein the unique peptide can be detected in blood, plasma, or serum sample.Machine Learning

[0212] Deep learning offers one route to better capture the complex relationships between sequence and protein behavior and has been the focus of many recent publications. Within the context of discovery and libraries, the generative models such as Generative Adversarial Networks (GANs) and autoencoder networks (AEs) are of particular interest as they have been shown to be viable for generating unique sequences of proteins, nanobodies, and antibody CDRs.MOFO-360864280 51341812000140

[0213] In further examples, one or more implementations described herein may include an autoencoder architecture that can generate protein sequences. In one or more examples, a system can be used to generate protein sequences. In various examples, a system can be implemented to generate amino acid sequences of peptides. After initial training of the model, the model can be further modified by training the model using data that corresponds to amino acid sequences of proteins that have a specified set of characteristics, such as one or more specified biophysical properties.

[0214] In some embodiments, a generative adversarial network (GAN) may generate a set of candidate-derived peptide sequences without using causal inference. In some embodiments, as disclosed in US 11512345, the GAN may generate a set of candidate-derived peptide sequences using causal inference. A GAN refers to a class of deep learning algorithms including two neural networks, a generator and a discriminator, that both compete with one another to achieve a goal. For example, regarding derived peptide sequence generation, the generator goal may include generating derived peptide sequences, including compatible / incompatible sequences of ingredients, and effective / ineffective sequences of ingredients, etc. that the discriminator classifies as feasible candidate derived peptide combination, including compatible and effective sequences of ingredients that may produce desired activity levels for design space. In one embodiment, the generator may use causal inference, including counterfactuals, to calculate numerous alternative scenarios that indicate whether a certain result (e.g., activity level) still follows when any element or aspect of a sequence changes. For example, the generator may be a neural network based on Markov models (e.g., Deep Markov Models), which may perform causal inference. In some embodiments, one or more of the counterfactuals used during the causal inference may be determined and provided by the scientist module. The discriminator goal may include distinguishing candidate drug compounds which include undesirable sequences of ingredients from candidate peptide sequences which include desirable sequences of ingredients.

[0215] Recurrent neural networks include the functionality, in the context of a hidden layer, to process information sequences and store information about previous computations. As such, recurrent neural networks may have or exhibit a “memory.” Recurrent neural networks may include connections between nodes that form a directed graph along a temporal sequence. Keeping and analyzing information about previous states enables recurrent neural networks to process sequences of inputs to recognize patterns (e.g., such as sequences of ingredients and correlations with certain types of activity level). Recurrent neural networks may be similar to Markov chains. For example, Markov chains may refer to stochasticMOFO-360864280 52341812000140 models describing sequences of possible events in which the probability of any given event depends only on the state information contained in the previous event. Thus, Markov chains also use an internal memory to store at least the state of the previous event. These models may be useful in determining causal inference, such as whether an event at a current node changes as a result of the state of a previous node changing.

[0216] The set of candidate drug compounds generated may be input into another machine learning model trained to classify the set of candidate drug compounds as a selected candidate drug compound. The classifier may be trained to rank the set of candidate drug compounds using any suitable ranking (i.e., for example, non-parametric) technique. For example, in some embodiments, one or more clustering techniques may be used to cluster the set of candidate drug compounds. To classify the selected candidate drug compound, the machine learning model may also perform objective optimization techniques while clustering. To classify the selected candidate drug compound having desired levels of certain types of activity, the objective optimization may include using a minimization or maximization function for each candidate drug compound in the clusters.

[0217] In an example embodiment, the system computing entity may be configured to communicate with a peptide database that stores one or more peptide datasets. For example, the system computing entity may communicate with the peptide database to retrieve, receive, access, and / or the like data related to pre-existing peptides to configure (e.g., train) GAN machine learning models to intelligently design aptameric peptides (e.g., to output designed peptides). In some example embodiments, the system computing entity may generate and / or update a peptide database with designed peptides, as described in US20230086091. That is, derived synthetic peptide libraries generated by the system computing entity in accordance with various embodiments of the present disclosure may be stored (e.g., described by data stored) in a peptide database.

[0218] US20230178186A1 demonstrates the GAN library biasing on such properties as a reduction of negative surface area patches, identified as a potential source of aggregation, thermal instability, and possible half-life reductions, and away from MHC class II binding, which may reduce the immunogenicity of the generated antibodies. They show, library biasing to a higher isoelectric point (pl) reduces aggregation and prevents precipitation in therapeutic formulations, and towards longer CDR3 lengths which can increase diversity and has been known to create more effective therapeutics for a class of targets.

[0219] As discussed in Longwell et al., computational structural modeling can be executed using Rosetta Remodel. This system can be used to implement a rotamer library creation,MOFO-360864280 53341812000140 introduce point mutations, and to view and score the model energy properties (Longwell CK, et al., (2021). ACS Chem Biol, 16(l):58-66).PK improvements

[0220] In some embodiments, the derived peptide described herein has an in vivo circulation half-life of at least that of the wild- type counterpart. In some embodiments, the derived peptide has a half-life that is increased over that of a wild-type counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater from the wild-type peptide. In some embodiments, the derived peptide has a half-life or persistence in a cell for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween.

[0221] In other embodiments, the derived peptide has an increased ability to persist in circulation as opposed to the wild-type reference peptide.

[0222] In another embodiment, the derived peptide has an improved blood circulation persistence by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater compared to the wild-type reference peptide.

[0223] In another embodiment, the derived peptide has an improved systemic circulatory half-life by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater compared to the wild-type reference peptide.

[0224] In another embodiment, the derived peptide has an improved target tissue uptake by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater compared to the wild-type reference peptide.

[0225] In another embodiment, the derived peptide has a reduced off-target tissue uptake by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater compared to the wild-type reference peptide.

[0226] In another embodiment, the derived peptide has an improved bioavailability by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater compared to the wild-type reference peptide.

[0227] In a preferred embodiment, derived peptides are localized to the target with a relative biodistribution improvement than the reference peptide. Derived peptides are localized to theMOFO-360864280 54341812000140 target by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater compared to the wild-type reference peptide.

[0228] Preferably, improved PK parameters of the reference peptide improve biodistribution by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater compared to the wild-type reference peptide.

[0229] Peptide preparations suffer from various limitations in their use in medicine (Nestor, J.J., JR. (2007) Comprehensive Medicinal Chemistry II 2: 573-601), resulting in short duration of action, insufficient bioavailability and lack of extracellular or intracellular receptor or target subtype selectivity. Additionally, peptides are often limited to aggregation and are unstable in formulation. Described herein are derived peptides that upon administration of said derived peptides, allow a longer duration of action and / or improved bioavailability.

[0230] In some embodiments, the improved interaction of derived peptides with their receptors is modified in a beneficial manner by sequence truncation, introduction of constraints, and / or incorporation of steric hindrance. In some embodiments, steric hindrance confers receptor selectivity to the modified peptides and / or proteins described herein. In some embodiments, steric hindrance provides protection from proteolysis.

[0231] In some embodiments, the peptide products described herein include covalently attached saccharides and additional hydrophobic groups or non-canonical amino acids that impart surfactant characteristics to the derived peptide, and thereby allow harmonization of the bioavailability, immunogenicity and / or pharmacokinetic behavior of surfactant-modified peptides.

[0232] The derived peptides of the present disclosure can have varying biodistribution patterns in vivo. In some cases, mammalian biodistribution and pharmacokinetic studies are performed using, for example, radiolabeled peptides (e.g.,14C-labeled peptides), in order to determine organ distribution, the uptake and residence times of the peptides in target organs (e.g., brain, liver), and their mode of clearance (e.g., renal or hepatic).

[0233] The stability of derived peptides of this disclosure can be determined by resistance to degradation by proteases. Proteases, also referred to as peptidases or proteinases, are enzymes that can degrade peptides and proteins by breaking bonds between adjacent amino acids. Families of proteases with specificity for targeting specific amino acids can include serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and asparagine proteases. Additionally, metalloproteases, matrix metalloproteases, elastase, carboxypeptidases, Cytochrome P450 enzymes, and cathepsins can also digest peptides andMOFO-360864280 55341812000140 proteins. Proteases can be present at high concentrations in blood, in mucous membranes, lungs, skin, the GI tract, the mouth, nose, eye, and in compartments of the cell. Misregulation of proteases can also be present in various diseases such as rheumatoid arthritis and other immune disorders. Degradation by proteases can reduce the bioavailability, biodistribution, half-life, and bioactivity of therapeutic molecules such that they are unable to perform their therapeutic function. In some embodiments, peptides that are resistant to proteases can better provide therapeutic activity at reasonably tolerated concentrations in vivo.

[0234] Derived peptides of this disclosure can be administered in biological environments that are acidic. For example, after oral administration, derived peptides can experience acidic environmental conditions in the gastric fluids of the stomach and gastrointestinal (GI) tract. The pH of the stomach can range from about 1-4 and the pH of the GI tract ranges from acidic to normal physiological pH descending from the upper GI tract to the colon. In addition, the vagina, late endosomes, and lysosomes can also have acidic pH values, such as less than pH 7. These acidic conditions can lead to the denaturation of peptides and proteins into unfolded states. Unfolding of peptides can lead to increased susceptibility to subsequent digestion by other enzymes as well as loss of biological activity of the peptide. In certain embodiments, the derived peptides of this disclosure can resist denaturation and degradation in acidic conditions as compared to the reference peptide and in buffers, which simulate acidic conditions. In certain embodiments, derived peptides of this disclosure can resist denaturation or degradation in buffer with a pH less than 1, a pH less than 2, a pH less than 3, a pH less than 4, a pH less than 5, a pH less than 6, a pH less than 7, or a pH less than 8. In some embodiments, derived peptides of this disclosure remain intact at a pH of 1-3. In certain embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the derived peptide remains intact after exposure to a buffer with a pH less than 1, a pH less than 2, a pH less than 3, a pH less than 4, a pH less than 5, a pH less than 6, a pH less than 7, or a pH less than 8. In other embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the derived peptide remains intact after exposure to a buffer with a pH of 1-3. In other embodiments, the derived peptides of this disclosure can be resistant to denaturation or degradation in simulated gastric fluid (pH 1-2). In some embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the derived peptide remains intact after exposureMOFO-360864280 56341812000140 to simulated gastric fluid. In some embodiments, low pH solutions such as simulated gastric fluid can be used to determine the derived peptide stability.

[0235] In some embodiments, derived peptides of this disclosure can resist degradation by any class of protease. In certain embodiments, derived peptides of this disclosure resist degradation by pepsin (which can be found in the stomach), trypsin (which can be found in the duodenum), serum proteases, or any combination thereof. In some embodiments, the proteases used to determine peptide stability can be pepsin, trypsin, chymotrypsin, or any combination thereof. In certain embodiments, derived peptides of this disclosure can resist degradation by lung proteases (e.g., serine, cysteinyl, and aspartyl proteases, metalloproteases, neutrophil elastase, alpha- 1 antitrypsin, secretory leucoprotease inhibitor, and elafin), or any combination thereof. In some embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the derived peptide remains intact after exposure to a protease.

[0236] In some embodiments, the derived peptides described herein are resistant to degradation in vivo, in the serum of a subject, or inside a cell. In some embodiments, the derived peptides are stable at physiological pH ranges, such as about pH 7, about pH 7.5, between about pH 5 to 7.5, between about 6.5 to 7.5, between about pH 5 to 8, or between about pH 5 to 7. In some embodiments, the derived peptides described herein are stable in acidic conditions, such as less than or equal to about pH 5, less than or equal to about pH 3, or within a range from about 3 to about 5. In some embodiments, the derived peptides are stable in conditions of an endosome or lysosome, or inside a nucleus.

[0237] Derived peptides of this disclosure can be administered in biological environments with high temperatures. For example, after oral administration, peptides can experience high temperatures in the body. Body temperature can range from 36° C to 40° C. High temperatures can lead to the denaturation of peptides and proteins into unfolded states. Unfolding of peptides and proteins can lead to increased susceptibility to subsequent digestion by other enzymes as well as loss of biological activity of the peptide. In some embodiments, a derived peptide of this disclosure can remain intact at temperatures from 25° C to 100° C High temperatures can lead to faster degradation of peptides. Stability at a higher temperature can allow for storage of the peptide in tropical environments or areas where access to refrigeration is limited. In certain embodiments, 5%-100% of the derived peptide can remain intact after exposure to 25° C for 6 months to 5 years. 5%-100% of a derived peptide can remain intact after exposure to 70° C for 15 minutes to 1 hour. 5%-100% of aMOFO-360864280 57341812000140 derived peptide can remain intact after exposure to 100° C for 15 minutes to 1 hour. In other embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the derived peptide remains intact after exposure to 25° C for at least 6 months to 5 years. In other embodiments, at least 5%-10%, at least 10%-20%, at least 20%- 30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the derived peptide remains intact after exposure to 70° C for 15 minutes to 1 hour. In other embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the derived peptide remains intact after exposure to 100° C for 15 minutes to 1 hour.

[0238] In some embodiments, the derived peptide has a half-life or persistence in circulation for no more than about 3 minutes to about 21 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days.

[0239] In certain embodiments, the derived peptide described herein has a half-life or persistence in circulation for greater than about 3 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween.

[0240] As described in Example 3, a murine assay can be used to assess and select for an extended peptide half-life.

[0241] In some embodiments, the derived peptide described herein modulates a cellular function, e.g., transiently or long-term. In certain embodiments, the cellular function is stably altered, such as a modulation that persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26MOFO-360864280 58341812000140 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer. In certain embodiments, the cellular function is transiently altered, e.g., such as a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours(3 days), 4 days, 5 days, 6 days, or 7 days.

[0242] In some embodiments, the derived peptide described herein is at least about 12 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 75 amino acids, or at least about 100 amino acids.

[0243] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0244] All publications, references cited, patents and sequence database entries mentioned throughout the specification, are hereby expressly incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0245] Also provided herein are systems for designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject. The system comprising one or more processors, and a non-transitory memory coupled to the one or more processors comprising instructions that, when executed by the one or more processors, cause the one or more processors to: receive a therapeutic peptide; select a region of at least 10 amino acids of the therapeutic peptide; select a plurality of sites within the region; and generate at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein one or more of the unique peptide variants comprise a C-terminal and / or N-terminal modification.

[0246] Any aspects of the disclosed techniques herein can be on one or more client devices, one or more server devices, or distributed among one or more client devices and one or more server devices in any order and combination.

[0247] This disclosure contemplates any suitable number of systems such as 600, illustrated in FIG. 6. This disclosure contemplates computing system 600 taking any suitable physicalMOFO-360864280 59341812000140 form. As example and not by way of limitation, computing system 600 may be an embedded computer system, a system-on-chip (SOC), a single-board computer system (SBC) (e.g., a computer-on-module (COM) or system-on-module (SOM)), a desktop computer system, a laptop or notebook computer system, an interactive kiosk, a mainframe, a mesh of computer systems, a mobile telephone, a personal digital assistant (PDA), a server, a tablet computer system, an augmented / virtual reality device, or a combination of two or more of these. Where appropriate, computing system 600 may include one or more computing systems 6000; be unitary or distributed; span multiple locations; span multiple machines; span multiple data centers; or reside in a cloud, which may include one or more cloud components in one or more networks.

[0248] Where appropriate, one or more computing systems 600 may perform without substantial spatial or temporal limitation one or more steps of one or more methods described or illustrated herein. As an example, and not by way of limitation, one or more editing computing systems 600 may perform in real time or in batch mode one or more steps of one or more methods described or illustrated herein. One or more computing system 600 may perform at different times or at different locations one or more steps of one or more methods described or illustrated herein, where appropriate.

[0249] In certain embodiments, the computing system 600 includes a processor 602, memory 604, database 606, an input / output (I / O) interface 608, a communication interface 600, and a bus 602. Although this disclosure describes and illustrates a particular computer system having a particular number of particular components in a particular arrangement, this disclosure contemplates any suitable computer system having any suitable number of any suitable components in any suitable arrangement. In certain embodiments, processor 602 includes hardware for executing instructions, such as those making up a computer program. As an example, and not by way of limitation, the instruction for designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide, processor 602 may retrieve (or fetch) the instructions from an internal register, an internal cache, memory 604, or database 606; decode and execute them; and then write one or more results to an internal register, an internal cache, memory 604, or database 606. In certain embodiments, processor 602 may include one or more internal caches for data, instructions, or addresses. This disclosure contemplates processor 602 including any suitable number of any suitable internal caches, where appropriate. As an example, and not by way of limitation, processor 602 may include one or more instruction caches, one or more data caches, and one or more translation lookaside buffers (TLBs). Instructions in the instruction caches may beMOFO-360864280 60341812000140 copies of the instructions for designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in memory 604 or database 606, and the instruction caches may speed up retrieval of those instructions by processor v02.

[0250] Data in the data caches may be copies of data in memory 604 or database 606 for instructions executing at processor 602 to operate on; the results of previous instructions executed at processor 602 for access by subsequent instructions executing at processor 602 or for writing to memory 604 or database 606; or other suitable data. The data caches may speed up read or write operations by processor 602. The TLBs may speed up virtual-address translation for processor 602. In certain embodiments, processor 602 may include one or more internal registers for data, instructions, or addresses. This disclosure contemplates processor 602 including any suitable number of any suitable internal registers, where appropriate. Where appropriate, processor 602 may include one or more arithmetic logic units (ALUs); be a multi-core processor; or include one or more processors 602. Although this disclosure describes and illustrates a particular processor, this disclosure contemplates any suitable processor.

[0251] In certain embodiments, memory 604 includes main memory for storing instructions for processor 602 to execute or data for processor 602 to operate on. As an example, and not by way of limitation, computing system 600 may load instructions, such as the instruction for designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide, from database 606 or another source (such as, for example, another computing system 600) to memory 604. Processor 602 may then load the instructions from memory 604 to an internal register or internal cache. To execute the instructions, processor 602 may retrieve the instructions from the internal register or internal cache and decode them. During or after execution of the instructions, processor 602 may write one or more results (which may be intermediate or final results) to the internal register or internal cache. Processor 602 may then write one or more of those results to memory 604. In certain embodiments, processor 602 executes only instructions in one or more internal registers or internal caches or in memory 604 (as opposed to database 606 or elsewhere) and operates only on data in one or more internal registers or internal caches or in memory 604 (as opposed to database 606 or elsewhere).

[0252] One or more memory buses (which may each include an address bus and a data bus) may couple processor 602 to memory 604. Bus 602 may include one or more memory buses, as described below. In certain embodiments, one or more memory management units (MMUs) reside between processor 602 and memory 604 and facilitate accesses to memoryMOFO-360864280 61341812000140604 requested by processor 602. In certain embodiments, memory 604 includes random access memory (RAM). This RAM may be volatile memory, where appropriate. Where appropriate, this RAM may be dynamic RAM (DRAM) or static RAM (SRAM). Moreover, where appropriate, this RAM may be single-ported or multi-ported RAM. This disclosure contemplates any suitable RAM. Memory 604 may include one or more memory devices 604, where appropriate. Although this disclosure describes and illustrates particular memory, this disclosure contemplates any suitable memory.

[0253] In certain embodiments, database 606 includes mass storage for data or instructions. In some embodiments, the database 606 stores instructions designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide as well as data that can be used to complete the design. For example, database 606 comprise the amino acid sequence of various peptides, canonical and non-canonical amino acids as described herein and binding patterns for various peptides. As an example, and not by way of limitation, database 606 may include a hard disk drive (HDD), a floppy disk drive, flash memory, an optical disc, a magneto-optical disc, magnetic tape, or a Universal Serial Bus (USB) drive or a combination of two or more of these. Database 606 may include removable or non-removable (or fixed) media, where appropriate. Database 606 may be internal or external to the computing system 600, where appropriate. In certain embodiments, database 606 is non-volatile, solid-state memory. In certain embodiments, database 606 includes readonly memory (ROM). Where appropriate, this ROM may be mask-programmed ROM, programmable ROM (PROM), erasable PROM (EPROM), electrically erasable PROM (EEPROM), electrically alterable ROM (EAROM), or flash memory or a combination of two or more of these. This disclosure contemplates mass database 606 taking any suitable physical form. Database 606 may include one or more storage control units facilitating communication between processor 602 and database 606, where appropriate. Where appropriate, database 606 may include one or more storages 606. Although this disclosure describes and illustrates particular storage, this disclosure contemplates any suitable storage.

[0254] In certain embodiments, RO interface 608 includes hardware, software, or both, providing one or more interfaces for communication between the computing system 600 and one or more RO devices. Computing system 600 may include one or more of these RO devices, where appropriate. One or more of these RO devices may enable communication between a person and the computing system 600. In some embodiments, the RO interface may comprise a user input device. In some embodiments, the user input device may be used for a use to input a reference therapeutic peptide for the library design. As an example, andMOFO-360864280 62341812000140 not by way of limitation, an I / O device may include a keyboard, keypad, microphone, monitor, mouse, printer, scanner, speaker, still camera, stylus, tablet, touch screen, trackball, video camera, another suitable I / O device or a combination of two or more of these. An I / O device may include one or more sensors. This disclosure contemplates any suitable I / O devices and any suitable I / O interfaces 606 for them. Where appropriate, I / O interface 608 may include one or more device or software drivers enabling processor 602 to drive one or more of these I / O devices. I / O interface 608 may include one or more I / O interfaces 606, where appropriate. Although this disclosure describes and illustrates a particular I / O interface, this disclosure contemplates any suitable I / O interface.

[0255] In certain embodiments, communication interface 610 includes hardware, software, or both providing one or more interfaces for communication (such as, for example, packet-based communication) between the computing system 600 and one or more other computer systems 600 or one or more networks. As an example, and not by way of limitation, communication interface 610 may include a network interface controller (NIC) or network adapter for communicating with an Ethernet or other wire-based network or a wireless NIC (WNIC) or wireless adapter for communicating with a wireless network, such as a WI-FI network. This disclosure contemplates any suitable network and any suitable communication interface 610 for it.

[0256] As an example, and not by way of limitation, computing system 600 may communicate with an ad hoc network, a personal area network (PAN), a local area network (LAN), a wide area network (WAN), a metropolitan area network (MAN), or one or more portions of the Internet or a combination of two or more of these. One or more portions of one or more of these networks may be wired or wireless. As an example, computing system 6000 may communicate with a wireless PAN (WPAN) (such as, for example, a BLUETOOTH WPAN), a WI-FI network, a WLMAX network, a cellular telephone network (such as, for example, a Global System for Mobile Communications (GSM) network), or other suitable wireless network or a combination of two or more of these. The computing system 600 may include any suitable communication interface 6100 for any of these networks, where appropriate. Communication interface 610 may include one or more communication interfaces 610, where appropriate. Although this disclosure describes and illustrates a particular communication interface, this disclosure contemplates any suitable communication interface.

[0257] In certain embodiments, bus 612 includes hardware, software, or both coupling components of computing system 6000 to each other. As an example, and not by way ofMOFO-360864280 63341812000140 limitation, bus 612 may include an Accelerated Graphics Port (AGP) or other graphics bus, an Enhanced Industry Standard Architecture (EISA) bus, a front-side bus (FSB), a HYPERTRANSPORT (HT) interconnect, an Industry Standard Architecture (ISA) bus, an INFINIBAND interconnect, a low-pin-count (LPC) bus, a memory bus, a Micro Channel Architecture (MCA) bus, a Peripheral Component Interconnect (PCI) bus, a PCI- Express (PCIe) bus, a serial advanced technology attachment (SATA) bus, a Video Electronics Standards Association local (VLB) bus, or another suitable bus or a combination of two or more of these. Bus 612 may include one or more buses 612, where appropriate. Although this disclosure describes and illustrates a particular bus, this disclosure contemplates any suitable bus or interconnect.

[0258] Also included herein are computer-readable non-transitory storage mediums. Herein, a computer-readable non-transitory storage medium or media may include one or more semiconductor-based or other integrated circuits (ICs) (such, as for example, field- programmable gate arrays (FPGAs) or application-specific ICs (ASICs)), hard disk drives (HDDs), hybrid hard drives (HHDs), optical discs, optical disc drives (ODDs), magnetooptical discs, magneto-optical drives, floppy diskettes, floppy disk drives (FDDs), magnetic tapes, solid-state drives (SSDs), RAM-drives, SECURE DIGITAL cards or drives, any other suitable computer-readable non-transitory storage media, or any suitable combination of two or more of these, where appropriate. A computer-readable non-transitory storage medium may be volatile, non-volatile, or a combination of volatile and non-volatile, where appropriate.Exemplary Embodiments:

[0259] Embodiment 1. A peptide library comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences derived from a single reference sequence, wherein a subset of the derived peptide sequences comprises peptide sequences comprising a functional pharmacokinetic parameter (FPKP) detectably improved relative to the single reference sequence.

[0260] Embodiment 2. The peptide library of embodiment 1, wherein the FPKP is selected from improved systemic circulatory half-life, improved biodistribution, improved target tissue uptake, and improved bioavailability.

[0261] Embodiment 3. The peptide library of embodiment 1, wherein the FPKP is selected from improved protease resistance and / or decreased renal clearance.MOFO-360864280 64341812000140

[0262] Embodiment 4. The peptide library of embodiment 1, wherein the FPKP is selected from improved blood-brain barrier penetration.

[0263] Embodiment 5. The peptide library of embodiment 1, wherein the one or more derived peptide sequence independently contain a non-canonical amino acid, and wherein the non-canonical amino acid contributes to the improved FPKP.

[0264] Embodiment 6. The peptide library of embodiment 1, wherein the one or more derived peptide sequences independently contain a non-canonical amino acid and at least one natural amino acid, wherein the non-canonical amino acid contributes to the improved FPKP.

[0265] Embodiment 7. The peptide library of embodiment 5, wherein the non- canonical amino acid is selected from one or more non-canonical amino acid in Table 1.

[0266] Embodiment 8. The peptide library of embodiment 1, wherein the derived peptide sequence is chemically synthesized.

[0267] Embodiment 9. The peptide library of embodiment 1, wherein the one or more peptide sequences are synthesized from a nucleic acid template.

[0268] Embodiment 10. The peptide library of embodiment 1, wherein the one or more peptide sequences are not capable of being synthesized from a nucleic acid template.

[0269] Embodiment 11. The peptide library of embodiment 1, wherein the one or more peptide sequences are between about 6 amino acids and about 100 amino acids.

[0270] Embodiment 12. The peptide library of embodiment 1, wherein the FPKP is detected in vivo.

[0271] Embodiment 13. The peptide library of embodiment 1, wherein the derived peptide sequence comprises a covalent handle.

[0272] Embodiment 14. The peptide library of embodiment 1, wherein the single reference sequence is a biologically active peptide sequence.

[0273] Embodiment 15: An array comprising the peptide library of embodiment 1 and a solid surface, wherein the derived peptide sequence is individually arrayed upon the solid surface.

[0274] Embodiment 16. A mammalian subject comprising the peptide library of embodiment 1.

[0275] Embodiment 17. A peptide library comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences derived from a single reference sequence or a set of reference sequences, wherein a subset of the derived peptide sequences comprises peptide sequences having a pharmacokinetic parameter detectably improved relative to the singleMOFO-360864280 65341812000140 reference sequence or set of reference sequences, and having a dissociation constant value lower than the reference sequence or set of reference sequences.

[0276] Embodiment 18. The peptide library of embodiment 17, wherein the IC50 value is measured in vivo.

[0277] Embodiment 19. The peptide library of embodiment 17, wherein each derived peptide sequence selectively engages a selected target.

[0278] Embodiment 20. The peptide library of embodiment 19, wherein each derived peptide sequence selectively engages a selected target, and the selected target is also engaged by the single reference sequence or the set of reference sequences.

[0279] Embodiment 21. The peptide library of embodiment 19, wherein the subset of each derived peptide sequence selectively engages the selected target at a higher specificity than the selected target is engaged by the single reference sequence or the set of reference sequences.

[0280] Embodiment 22. A synthetic peptide library comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences independently having at least one amino acid difference from a naturally occurring human peptide sequence.

[0281] Embodiment 23. The synthetic peptide library of embodiment 22, wherein the peptide sequence independently contains a non-canonical amino acid, and wherein the non- canonical amino acid contributes to an improved pharmacokinetic parameter.

[0282] Embodiment 24. The synthetic peptide library of embodiment 23, wherein each non-canonical amino acid is selected from Table 1.

[0283] Embodiment 25. The synthetic peptide library of embodiment 23, wherein at least one of the non-canonical amino acids is not a D-amino acid.

[0284] Embodiment 26. The synthetic peptide library of embodiment 22, wherein the peptide sequence is a linear peptide sequence.

[0285] Embodiment 27. A synthetic peptide library comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptide sequences independently having at least one non- canonical amino acid.

[0286] Embodiment 28. A synthetic peptide comprising at least one non-naturally occurring amino acid, wherein the peptide is capable of binding to a cellular target present in a human subject, and wherein the peptide is isolated from a derived peptide library comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptides present in a mammalian subject.MOFO-360864280 66341812000140

[0287] Embodiment 29. A synthetic peptide capable of binding to a cellular target present in a human subject, isolated from a derived peptide library comprising at least about lxlOA4 peptides present in a mammalian subject.

[0288] Embodiment 30: A mammalian subject comprising a peptide library comprising at least about lxlOA4, lxl0A5, lxlOA6, or lxlOA7 peptides independently comprising at least one non-naturally occurring amino acid.

[0289] Embodiment 31. A method of determining a pharmacokinetic (PK) parameter of a plurality of synthetic peptides, comprising the steps of: introducing into a mammalian subject the plurality of synthetic peptides; detecting thereafter a subset of synthetic peptides present in the plurality of synthetic peptides, thereby determining the PK parameter of the detected synthetic peptides.

[0290] Embodiment 32. A method of identifying a synthetic peptide having an increased pharmacokinetic (PK) parameter relevant to a reference synthetic peptide, comprising the steps of: determining a pharmacokinetic (PK) parameter of a plurality of synthetic peptides, comprising the steps of: introducing into a mammalian subject the plurality of synthetic peptides; detecting thereafter a subset of synthetic peptides present in the plurality of synthetic peptides, thereby determining the PK parameter of the detected synthetic peptides; and selecting a reference synthetic peptide from the detected synthetic peptides; synthesizing chemical variants of the selected reference synthetic peptide; introducing the synthesized chemical variants into a mammalian subject and determining the PK parameter of a plurality of the introduced synthesized chemical variants, comprising the step of: introducing into a mammalian subject the plurality of synthetic peptides; and identifying thereafter one or more synthetic peptides having the increased PK parameter.

[0291] Embodiment 33. A method of identifying a synthetic peptide comprising an acceptable pharmacokinetic (PK) parameter, comprising the steps of: introducing into a mammalian subject a peptide library comprising at least about lxlOA4 synthetic peptide sequences derived from a single reference sequence comprising an unacceptable PK parameter; andMOFO-360864280 67341812000140 detecting thereafter a subset of the synthetic peptide sequences present in the mammalian subject, thereby identifying the synthetic peptide comprising an acceptable PK parameter.

[0292] Embodiment 34. A method of identifying a synthetic peptide comprising an acceptable pharmacokinetic (PK) parameter, comprising the steps of: introducing into a mammalian subject a peptide library comprising at least about lxlOA4 synthetic peptide sequences; and detecting at a desired time point a subset of the synthetic peptide sequences present in the mammalian subject, thereby identifying the synthetic peptide comprising an acceptable PK parameter.

[0293] Embodiment 35. The method of embodiment 33, wherein the peptide library comprises one or more non-canonical amino acids selected from Table 1.

[0294] Embodiment 36. The method of embodiment 31, wherein the pharmacokinetic (PK) parameter of a plurality of synthetic peptides comprises at least 10A4 synthetic peptides, wherein the synthetic peptides comprise non-canonical amino acids in a library, wherein the synthetic peptides are screened in vivo to determine improved pharmacokinetic (PK) parameters.

[0295] Embodiment 37. A method of identifying a target-binding compound having an acceptable pharmacokinetic (PK) parameter, comprising the steps of: providing a first peptide library comprising a plurality of first peptide library members, the first peptide library members derived from a target-binding compound having an unacceptable PK parameter; administering the first peptide library to a mammalian subject; identifying one or more first peptide library members present in the mammalian subject having an acceptable PK parameter; and optionally the steps of providing a second peptide library comprising a plurality of second peptide library members, the second peptide library members derived from the identified target-binding compound having an acceptable PK parameter; administering the second peptide library to a mammalian subject; and identifying one or more second peptide library members present in the mammalian subject having an acceptable PK parameter and having acceptable binding to the target compound.

[0296] Embodiment 38. A method of identifying a target-binding compound having an acceptable pharmacokinetic (PK) parameter, comprising the steps ofMOFO-360864280 68341812000140 providing a first peptide library comprising a plurality of first peptide library members, the first peptide library members derived from a target-binding compound having an unacceptable PK parameter; administering the first peptide library to a mammalian subject; identifying one or more first peptide library members present in the mammalian subject having an acceptable PK parameter; building a machine learning model using one or more machine learning algorithms, and training the model using training data, wherein the training data comprise the chemical composition and acceptable PK parameter of the one or more first peptide library members; and using the trained model to predict the sequences of a plurality of second peptide library members.

[0297] Embodiment 39. The method of embodiment 37 or 38, wherein the acceptable PK parameter is selected from systemic circulatory half-life, biodistribution, target tissue uptake, and bioavailability, wherein the derived peptide is less susceptible to biotransformation in the patient, is excreted from the body of the patient at a slower rate, has increased stability of its tertiary structure, has a longer plasma half-life, has increased oral bioavailability, has higher penetrance across the blood-brain barrier, and has higher accumulation in a target tissue has increased oral bioavailability, has higher penetrance across the blood-brain barrier, and has higher accumulation in a target tissue.

[0298] Embodiment 40. A non-transitory computer-readable medium having instructions stored thereon for causing a suitably programmed information processor to execute any one of the methods in embodiments 37 or 38.

[0299] Embodiment 41. A system comprising: a processor, memory and a non- transitory computer-readable medium having instructions stored thereon for causing a processor to execute any one of the methods in embodiments 37 or 38.

[0300] Embodiment 42. A method of manufacturing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject, comprising:(a) selecting a region of at least 10 amino acids of the therapeutic peptide;(b) selecting a plurality of sites within the region;(c) generating at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non-MOFO-360864280 69341812000140 canonical amino acid, wherein each of the unique peptide variants comprise a C-terminal and / or N-terminal modification; and(d) manufacturing a peptide library comprising the unique peptide variants.

[0301] Embodiment 43. A method of designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject, comprising:(a) selecting a region of at least 10 amino acids of the therapeutic peptide;(b) selecting a plurality of sites within the region;(c) generating at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non- canonical amino acid, wherein one or more of the unique peptide variants comprise a C- terminal and / or N-terminal modification.

[0302] Embodiment 44. A method of determining a functional pharmacokinetic parameter (FPKP) of a plurality of unique peptide variants of a therapeutic peptide, comprising the steps of:(a) introducing into a non-human mammalian subject a peptide library comprising the unique peptide variants, the peptide library comprising at least IxlO3unique peptide variants, each peptide variant comprising at least one modification at one or more of a plurality of sites in a region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein each of the one or more of the unique peptide variants comprise a C-terminal and / or N-terminal modification, wherein less than 75 pg / kg of each unique peptide variant is introduced into the non-human mammalian subject;(b) detecting thereafter a subset of the unique peptide variants present in the plurality of unique peptide variants, thereby determining the FPPK of the detected unique peptide variants.

[0303] Embodiment 45. The method of any of embodiments 42-44, wherein the one or more unique peptide variants comprise an amidated C-terminus and / or acetylated N-terminus.

[0304] Embodiment 46. The method of any of embodiments 42-45, wherein the non- canonical amino acid is selected from the non-canonical amino acids in Table 1 and / or Table 2.

[0305] Embodiment 47. The method of any of embodiments 42 - 46, wherein the non- human mammalian subject is a mouse, rat, dog, pig, or non-human primate.MOFO-360864280 70341812000140

[0306] Embodiment 48. The method of any of embodiments 42- 47 wherein an average mass of a peptide variant is about 1000 to about 10000 DA.

[0307] Embodiment 49. The method of any of embodiments 42-48, wherein the therapeutic peptide is about 10 amino acids to about 100 amino acids.

[0308] Embodiment 50. The method of any of embodiments 42-49, wherein the therapeutic peptide is about 100 to about 5000 amino acids.

[0309] Embodiment 51. The method of any of embodiments 42 - 50, wherein the region is about 10 amino acids to about 100 amino acids.

[0310] Embodiment 52. The method of any of embodiments 42 - 51, wherein the therapeutic peptide comprises a linear peptide sequence or a cyclic peptide sequence.

[0311] Embodiment 53. The method of any of embodiments 42 - 52, wherein selecting a region comprises identifying amino acids of the therapeutic peptide predicted to contribute to binding to a target and / or identifying amino acids of the therapeutic peptides that do not contribute to binding to the target.

[0312] Embodiment 54. The method of any of embodiments 42 - 53, wherein the region comprises a N-terminus of the therapeutic peptide.

[0313] Embodiment 55. The method of any of embodiments 42 - 54, wherein the region comprises a C-terminus of the therapeutic peptide.

[0314] Embodiment 56. The method of any of embodiments 42 - 55, wherein a site in the plurality of sites is within about 1 to about 100 amino acids of another site in the plurality of sites.

[0315] Embodiment 57. The method of any of embodiments 42 - 56, wherein the plurality of sites comprises about 2 to about 25 sites.

[0316] Embodiment 58. The method of any of embodiments 42 - 57, wherein the plurality of sites comprises between about 5% and about 100% of the amino acids in the region.

[0317] Embodiment 59. The method of any of embodiments 42 - 58, wherein each unique peptide variant comprises at least two or more modifications at two or more of the plurality of sites in the region.

[0318] Embodiment 60. The method of any of embodiments 42 - 59, wherein each unique peptide variant comprises at least three or more modifications at three or more of the plurality of sites in the region.MOFO-360864280 71341812000140

[0319] Embodiment 61. The method of any of embodiments 42 - 60, wherein each unique peptide variant comprises at least four or more modifications at four or more of the plurality of sites in the region.

[0320] Embodiment 62. The method of any of embodiments 42 - 61, wherein the at least one modification at one or more of the plurality of sites comprises an amino acid substitution, amino acid insertion, or amino acid deletion.

[0321] Embodiment 63 The method of embodiment 62, wherein the amino acid substitution comprises identifying an amino acid residue at a site and substituting the amino acid residue with a second amino acid residue with; a. a similar charge as the amino acid residue; b. a similar size as the amino acid residue; c. a similar shape as the amino acid residue; d. a similar solubility as the amino acid residue; e. a similar linked hydrophobic moiety; and / or f. a similar chirality as the amino acid residue.

[0322] Embodiment 64. The method of embodiment 62, wherein the amino acid substitution comprises, identifying an amino acid residue at a site and substituting the amino acid residue with a second amino acid residue with; a. a different charge as the amino acid residue; b. a different size as the amino acid residue; c. a different shape as the amino acid residue; d. a different solubility as the amino acid residue; e. a different linked hydrophobic moiety; and / or f. a different chirality as the amino acid residue.

[0323] Embodiment 65. The method of any of embodiment 62, wherein the amino acid substitution comprises: a. identifying an amino acid residue susceptible to dehydration at a site and substituting the amino acid residue with a second amino acid residue less susceptible to dehydration; b. identifying an amino acid residue susceptible to deamidation at a site and substituting the amino acid residue with a second amino acid residue less susceptible to deamidation; c. identifying an amino acid residue susceptible to oxidation at a site and substituting the amino acid residue with a second amino acid residue less susceptible to oxidation; and / orMOFO-360864280 72341812000140 d. identifying an amino acid residue contributing to secondary structure at a site and substituting the amino acid residue with a second amino acid residue to strengthen the secondary structure.

[0324] Embodiment 66. The method of any of embodiments 63-65, wherein the second amino acid comprises a non-canonical amino acid.

[0325] Embodiment 67. The method of any of embodiments 63-66, wherein the amino acid insertion or the amino acid substitution comprises a non-canonical amino acid.

[0326] Embodiment 68. The method of embodiment 66 or 67, wherein the non- canonical amino acid is selected based on a chemical property, a charge, a solubility, a shape of the non-canonical amino acid.

[0327] Embodiment 69. The method of embodiment 68, wherein the non-canonical amino acid is predicted to increase the FPKP of the peptide.

[0328] Embodiment 70. The method of any of embodiments 42 and 45-68, wherein manufacturing the peptide library comprises chemically synthesizing the unique peptide variants.

[0329] Embodiment 71. The method of any of embodiments 42 and 45-70, wherein manufacturing the peptide library comprises synthesizing the unique peptide variants from a plurality of nucleic acid templates.

[0330] Embodiment 72. The method of any of embodiments 42 and 45-71, wherein the peptide library comprises one or more unique peptide variants covalently linked to a covalent handle.

[0331] Embodiment 73. The method of any of embodiments 42 and 45-72, wherein manufacturing the peptide library comprises synthesizing the unique peptide variants on a solid surface array.

[0332] Embodiment 74. The method of any of embodiments 42-73, wherein one or more of the unique peptide variants has an improved FPKP compared to the therapeutic peptide.

[0333] Embodiment 75. The method of any of embodiments 42-74, wherein the FPKP is selected from the group consisting of systemic circulatory half-life, biodistribution, target tissue uptake, bioavailability, protease resistance, metabolism, clearance, and blood-brain barrier penetration.

[0334] Embodiment 76. The method of any one of embodiments 42 and 45-75, further comprising introducing into to the non-human mammalian subject the peptide library.MOFO-360864280 73341812000140

[0335] Embodiment 77. The method of any of embodiments 42-75, wherein one or more of the unique peptide variants has an improved FPKP compared to the therapeutic peptide.

[0336] Embodiment 78. The method of embodiment 77, wherein the improved FPKP comprises reduced renal clearance, increased liver clearance, reduced drug metabolism, increased circulatory half-life, increased brain uptake, and / or increased tumor uptake.

[0337] Embodiment 79. The method of any of embodiments 42-78, further comprising analyzing the FPKP of the unique peptide variants in the non-human mammalian subject.

[0338] Embodiment 80. The method of embodiment 79, wherein analyzing the FPKP of the unique peptide variants comprises measuring an IC50 value in vivo.

[0339] Embodiment 81. The method of embodiment 79 or 80, wherein analyzing the FPKP comprises obtaining blood and / or plasma from the non-human mammalian subject after one or more predetermined time intervals, and detecting a subset of the unique peptide variants from the peptide library in the blood and / or plasma.

[0340] Embodiment 82. The method of embodiment 80, wherein detecting a subset of the unique peptide variants from the peptide library comprises performing mass spectrometry on a sample from the blood and / or plasma.

[0341] Embodiment 83. The method of embodiment 44, wherein detecting the subset of unique peptide variants occurs at after one or more predetermined time intervals.

[0342] Embodiment 84. The method of embodiment 83, wherein detecting a subset of unique peptide variants comprises performing mass spectrometry on a sample obtained from blood and / or plasma collected from the non-human mammalian subject.

[0343] Embodiment 85. The method of any of embodiments 81-84, wherein the one or more predetermined time intervals are selected from a group consisting of any time interval between about 0 minutes and about 60 days.

[0344] Embodiment 86. The method of any of embodiments 81-85, wherein the predetermined time intervals comprise about 0 minutes, about 5 minutes, about 20 minutes, and about 60 minutes.

[0345] Embodiment 87. The method of any of embodiments 81-86, wherein at least about 0.01% of the unique peptide variants are detected.

[0346] Embodiment 88. A kit comprising a peptide library manufactured according to the method of any of embodiments 42 and 45-84.

[0347] Embodiment 89. A peptide library manufactured according to the methods of any of embodiments 42 and 45-88.MOFO-360864280 74341812000140

[0348] Embodiment 90. A peptide library designed according to the methods of any of embodiments 43 and 45-89.

[0349] Embodiment 91. A peptide library comprising at least about IxlO3, about IxlO4, about IxlO5, about IxlO6, or about IxlO7unique peptide variants derived from a therapeutic peptide, wherein a subset of the derived peptide sequences comprises peptide sequences comprising a functional pharmacokinetic parameter (FPKP) detectably improved relative to the therapeutic peptide, wherein the unique peptide variants comprise at least one non- canonical amino acid, wherein one or more of the unique peptide variants comprise a C- terminal and / or N-terminal modification.

[0350] Embodiment 92. The peptide library of embodiment 91, wherein the one or more peptide sequences comprise an amidated C-terminus and / or acetylated N-terminus.

[0351] Embodiment 93. The peptide library of embodiment 92 or 92, wherein the non- canonical amino acid is selected from the non-conical amino acids in Table 1 and / or Table 2.

[0352] Embodiment 94. The peptide library of any of embodiments 90-93, wherein the FPKP displays improved protease resistance and / or decreased renal clearance.

[0353] Embodiment 95. The peptide library of any of embodiments 90-93, wherein the FPKP displays improved blood-brain barrier penetration.

[0354] Embodiment 96. The peptide library of any of embodiments 90-95, wherein each derived peptide sequence independently contains a non-canonical amino acid, and wherein the non-canonical amino acid contributes to the improved FPKP.

[0355] Embodiment 97. The peptide library of any of embodiments 90-96, wherein each derived peptide sequence comprises at least one modification, at least two modification, at least three modifications, or at least four or more modifications compared to the therapeutic peptide.

[0356] Embodiment 98. The peptide library of embodiment 97, wherein the at least one modification, at least two modifications, at least three modifications, or at least four or more modifications compared to the single reference sequence occur at a plurality of sites in a region of the therapeutic peptide.

[0357] Embodiment 99. The peptide library of embodiment 98, wherein the region comprises amino acids of the therapeutic sequence predicted to contribute to binding to a target and / or amino acids of the therapeutic peptide that do not contribute to binding to the target.

[0358] Embodiment 100. The peptide library of embodiment 98 or 99, wherein the region is about 10 amino acids to about 100 amino acids.MOFO-360864280 75341812000140

[0359] Embodiment 101. The peptide library of any of embodiments 98-100, wherein the region comprises a N-terminus of the therapeutic peptide.

[0360] Embodiment 102. The peptide library of any of embodiments 98-101, the region comprises a C-terminus of the therapeutic peptide.

[0361] Embodiment 103. The peptide library of any of embodiments 98-102, wherein a site in the plurality of sites is within about 1 to about 100 amino acids of another site in the plurality of sites.

[0362] Embodiment 104. The peptide library of any of embodiments 98-103, wherein the plurality of sites comprises about 2 to about 25 sites.

[0363] Embodiment 105. The peptide library of any of embodiments 98-104, wherein the plurality of sites comprises between about 5% and about 100% of the amino acids in the region.

[0364] Embodiment 106. The peptide library of any of embodiments 97-105, wherein the at least one modification, at least two modifications, at least three modifications, or the at least four or more modifications comprise an amino acid substitution, an amino acid insertion, or an amino acid deletion.

[0365] Embodiment 107. The peptide library of embodiment 106, wherein the amino acid substitution comprises a substitution of an amino acid residue with a second amino acid residue with: a. a similar charge as the amino acid residue; b. a similar size as the amino acid residue; c. a similar shape as the amino acid residue; d. a similar solubility as the amino acid residue; e. a similar linked hydrophobic moiety; or f. a similar chirality as the amino acid residue.

[0366] Embodiment 108. The peptide library of embodiment 106, wherein the amino acid substitution comprises a substitution of an amino acid residue with a second amino acid residue with: a. a different charge as the amino acid residue; b. a different size as the amino acid residue; c. a different shape as the amino acid residue; d. a different solubility as the amino acid residue; e. a different linked hydrophobic moiety; and / or f. a different chirality ad the amino acid residue.MOFO-360864280 76341812000140

[0367] Embodiment 109. The peptide library of embodiment 106, wherein the amino acid substitution comprises a substitution of an amino acid residue with a second amino acid residue: a. less susceptible to hydration; b. less susceptible to deamidation; c. less susceptible to oxidation; and / or d. predicted to strengthen the secondary structure.

[0368] Embodiment 110. The peptide library of any of embodiments 107-109, wherein the second amino acid comprises a non-canonical amino acid.

[0369] Embodiment 111. The peptide library of any of embodiments 90- 110, wherein the therapeutic peptide is a therapeutic peptide or a subsection of a therapeutic peptide.

[0370] Embodiment 112. The peptide library of any of embodiments 90-110, wherein the therapeutic peptide is about 1000 to about 5000 amino acids.

[0371] Embodiment 113. The peptide library of any of embodiments 90-110, wherein the therapeutic peptide is about 10 to about 100 amino acids.

[0372] Embodiment 114. The peptide library of any of embodiments 90-113, wherein the derived peptide sequences are chemically synthesized.

[0373] Embodiment 115. The peptide library of any of embodiments 90-114, wherein one or more derived peptide sequences are synthesized from a nucleic acid template.

[0374] Embodiment 116. The peptide library of any of embodiments 90-115, wherein one or more of the derived peptide sequences are not capable of being synthesized from a nucleic acid template.

[0375] Embodiment 117. The peptide library of any of embodiments 90-116, wherein the peptide library comprises about 1 x 10'12Mol synthesized peptide from each of the derived peptide sequences.

[0376] Embodiment 118. The peptide library of any of embodiments 90-117, wherein one or more of the derived peptide sequences comprises a covalent handle.

[0377] Embodiment 119. The peptide library of any of embodiments 90-118, wherein the FPKP is detected in vivo.

[0378] Embodiment 120. The peptide library of any of embodiments 90-119, wherein after one or more predetermined time intervals, between about 0.01% and about 99% of the derived peptides sequences are detectable in vivo.MOFO-360864280 77341812000140

[0379] Embodiment 121. The peptide library of embodiment 120, wherein the one or more predetermined time intervals are selected from a group consisting of any time interval between about 40 minutes and about 60 days.

[0380] Embodiment 122. A peptide library comprising at least about IxlO3, about IxlO4, about IxlO5, about IxlO6, or about IxlO7peptide sequences derived from a therapeutic peptide or a set of therapeutic peptides, wherein a subset of the derived peptide sequences comprises peptide sequences having a pharmacokinetic parameter detectably improved relative to the single reference sequence or set of reference sequences, and having a dissociation constant value (KD), half maximal effective concentration (EC50), or half maximal inhibitory concentration (IC50) lower than the reference sequence or set of reference sequences.

[0381] Embodiment 123. The peptide library of embodiment 122, wherein each derived peptide sequence selectively engages a selected target.

[0382] Embodiment 124. The peptide library of embodiment 122 or 123, wherein each derived peptide sequence selectively engages a selected target, and the selected target is also engaged by the therapeutic peptide or the set of therapeutic peptides.

[0383] Embodiment 125. The peptide library of any of embodiments 122-124, wherein the subset of each derived peptide sequence selectively engages the selected target at a higher specificity than the selected target is engaged by the therapeutic peptide or the set of therapeutic peptides.

[0384] Embodiment 126. An array comprising the peptide library of any of embodiments 90-125 and a solid surface, wherein the derived peptide sequence is individually arrayed upon the solid surface.

[0385] Embodiment 127. A synthetic peptide library comprising at least about IxlO3, about IxlO4, about IxlO5, about IxlO6, or about IxlO7peptide sequences independently having at least one amino acid difference from a naturally occurring human peptide sequence.

[0386] Embodiment 128. The synthetic peptide library of embodiment 127, wherein each peptide sequence independently contains a non-canonical amino acid, and wherein the non- canonical amino acid contributes to an improved pharmacokinetic parameter.

[0387] Embodiment 129. The synthetic peptide library of embodiment 128, wherein each non-canonical amino acid is selected from the non-canonical amino acids in Table 1 and / or Table 2.

[0388] Embodiment 130. The synthetic peptide library of embodiment 128 or 129, wherein at least one of the non-canonical amino acids is not a D-amino acid.MOFO-360864280 78341812000140

[0389] Embodiment 131. The synthetic peptide library of any of embodiments 128-130, wherein the peptide sequence is a linear peptide sequence.

[0390] Embodiment 132. A synthetic peptide library comprising at least about IxlO3, about IxlO4, about IxlO5, about IxlO6, or about IxlO7peptide sequences independently having at least one non-canonical amino acid.

[0391] Embodiment 133. The synthetic peptide library of any of embodiments 127-132, wherein the peptide library comprises one or more peptide variants with C-terminal and / or N- terminal modifications.

[0392] Embodiment 134. The synthetic peptide library of any of embodiments 127-133, wherein the peptide library comprises one or more peptide variants with an amidated C- terminus and / or acetylated N-terminus.

[0393] Embodiment 135. A synthetic peptide comprising at least one non-canonical amino acid, wherein the peptide is capable of binding to a cellular target present in a human subject, and wherein the peptide is isolated from a derived peptide library comprising at least about IxlO3, about IxlO4, about IxlO5, about IxlO6, or about IxlO7peptides present in a non-human subject.

[0394] Embodiment 136. A synthetic peptide capable of binding to a cellular target present in a human subject, isolated from a derived peptide library comprising at least about IxlO3peptides present in a non-human subject.

[0395] Embodiment 137. The synthetic peptide of embodiment 135 or 136, wherein the synthetic peptide comprises a C-terminal and / or a N-terminal modifications.

[0396] Embodiment 138. The synthetic peptide of any of embodiments 135-137, wherein the synthetic peptide comprises an amidated C-terminus and / or an acetylated N-terminus.

[0397] Embodiment 139. A mammalian subject comprising the peptide library or synthetic peptide library of any of embodiments 89-134.

[0398] Embodiment 140. A mammalian subject comprising a peptide library comprising at least about IxlO3, about IxlO4, about IxlO5, about IxlO6, or about IxlO7peptides independently comprising at least one non-canonical amino acid.

[0399] Embodiment 141. The mammalian subject of embodiment 139 or 140, wherein the mammalian subject is a non-human mammal.

[0400] Embodiment 142. The mammalian subject of embodiment 140 or 141, wherein the peptide library comprises the peptide library or synthetic peptide library of any of claims 89-134.MOFO-360864280 79341812000140

[0401] Embodiment 143. A method of identifying a synthetic peptide having an increased pharmacokinetic (PK) parameter relevant to a reference synthetic peptide, comprising the steps of: determining a pharmacokinetic (PK) parameter of a plurality of synthetic peptides, comprising the steps of: introducing into a mammalian subject the plurality of synthetic peptides; detecting thereafter a subset of synthetic peptides present in the plurality of synthetic peptides, thereby determining the PK parameter of the detected synthetic peptides; and selecting a reference synthetic peptide from the detected synthetic peptides; synthesizing chemical variants of the selected reference synthetic peptide; introducing the synthesized chemical variants into a mammalian subject and determining the PK parameter of a plurality of the introduced synthesized chemical variants, and identifying thereafter one or more synthetic peptides having the increased PK parameter.

[0402] Embodiment 144. The method of embodiment 143, wherein the plurality of synthetic peptides comprises the peptide library or synthetic peptide library of any of claims 89-134.

[0403] Embodiment 145. A method of identifying a synthetic peptide comprising an acceptable pharmacokinetic (PK) parameter, comprising the steps of: introducing into a mammalian subject a peptide library comprising at least about IxlO3synthetic peptide sequences derived from a single reference sequence comprising an unacceptable PK parameter; and detecting thereafter a subset of the synthetic peptide sequences present in the mammalian subject, thereby identifying the synthetic peptide comprising an acceptable PK parameter.

[0404] Embodiment 146. The method of embodiment 145, wherein after a predefined time intervals between about 1% and about 99% of the synthetic peptides are present in vivo.

[0405] Embodiment 147. The method of embodiment 146, wherein the one or more predetermined time intervals are selected from a group consisting of any time interval between about 40 minutes and about 60 days.

[0406] Embodiment 148. A method of identifying a synthetic peptide comprising an acceptable pharmacokinetic (PK) parameter, comprising the steps of:MOFO-360864280 80341812000140 introducing into a mammalian subject a peptide library comprising at least about IxlO3synthetic peptide sequences; and detecting at a desired time point a subset of the synthetic peptide sequences present in the mammalian subject, thereby identifying the synthetic peptide comprising an acceptable PK parameter.

[0407] Embodiment 149. The method of embodiment 148, wherein the peptide library comprises one or more non-canonical amino acid selected from the non-canonical amino acids in Table 1 and / or Table 2.

[0408] Embodiment 150. The method of embodiment 148 or 149, wherein the peptide library comprises the peptide library or synthetic peptide library of any of claims 89-134.

[0409] Embodiment 151. The method of any of embodiments 148-150, wherein the pharmacokinetic (PK) parameter of a plurality of synthetic peptides comprises at least 103synthetic peptides, wherein the synthetic peptides comprise non-canonical amino acids in a library, wherein the synthetic peptides are screened in vivo to determine improved pharmacokinetic (PK) parameters.

[0410] Embodiment 152. A method of identifying a target-binding compound having an acceptable pharmacokinetic (PK) parameter, comprising the steps of: providing a first peptide library comprising a plurality of first peptide library members, the first peptide library members derived from a target binding compound having an unacceptable PK parameter; introducing into the mammalian subject the first peptide library; identifying one or more first peptide library members present in the mammalian subject having an acceptable PK parameter; and optionally the steps of providing a second peptide library comprising a plurality of second peptide library members, the second peptide library members derived from the identified target binding compound having an acceptable PK parameter; introducing into the mammalian subject the second peptide library; and identifying one or more second peptide library members present in the mammalian subject having an acceptable PK parameter and having acceptable binding to the target compound.

[0411] Embodiment 153. A method of identifying a target binding compound having an acceptable pharmacokinetic (PK) parameter, comprising the steps ofMOFO-360864280 81341812000140 providing a first peptide library comprising a plurality of first peptide library members, the first peptide library members derived from a target-binding compound having an unacceptable PK parameter; introducing into a mammalian subject the first peptide library; identifying one or more first peptide library members present in the mammalian subject having an acceptable PK parameter; building a machine learning model using one or more machine learning algorithms, and training the model using training data, wherein the training data comprise the chemical composition and acceptable PK parameter of the one or more first peptide library members; and using the trained model to predict the sequences of a plurality of second peptide library members.

[0412] Embodiment 154. The method of embodiment 152 or 153, wherein the acceptable PK parameter is selected from systemic circulatory half-life, biodistribution, target tissue uptake, and bioavailability.

[0413] Embodiment 155. A non-transitory computer-readable medium having instructions stored thereon for causing a suitably programmed information processor to execute any one of the methods in embodiments 42-87 and 104-154.

[0414] Embodiment 156. A system comprising: a processor, memory and a non- transitory computer-readable medium having instructions stored thereon for causing a processor to execute any one of the methods in embodiments 42-87 and 143-154.

[0415] Embodiment 157. The method of any of embodiments 42-85, wherein the at least one modification at one or more of the plurality of sites comprises a backbone modification.

[0416] Embodiment 158. The peptide library or synthetic peptide library of any of claims 89-134, wherein one or more of the derived peptide sequences comprise a backbone modification.

[0417] Embodiment 159. The method of embodiment 157 or peptide library or synthetic peptide library of embodiment 158, wherein the backbone modification comprises a betaamino acid, a n-methylated amino acid, an alpha-substituted amino acid, a gamma-substituted amino acid, and / or a peptoid.

[0418] Embodiment 160. The method of any of embodiments 42-87, the peptide library of any of embodiments 89-125, the synthetic peptide library of embodiment 133, or the synthetic peptide of embodiment 137, wherein the C-terminal and / or N-terminal modifications comprise trifluoroacetylation of the N-terminus, C-terminal carboxylic acids,MOFO-360864280 82341812000140C-terminal esterification, N-terminal propionylation, N-terminal monomethylation, N- terminal dimethylation, and / or N-terminal trimethylation.

[0419] Embodiment 161. A system for designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject, comprising: one or more processors, and a non-transitory memory coupled to the one or more processors comprising instructions that, when executed by the one or more processors, cause the one or more processors to: receive a therapeutic peptide; select a region of at least 10 amino acids of the therapeutic peptide; select a plurality of sites within the region; and generate at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein one or more of the unique peptide variants comprise a C-terminal and / or N-terminal modification.

[0420] Embodiment 162 The system of embodiment 161, wherein the one or more unique peptide variants comprise an amidated C-terminus and / or acetylated N-terminus.

[0421] Embodiment 163. The system of embodiment 161 or 162, wherein the non- canonical amino acid is selected from the non-canonical amino acids in Table 1 and / or Table 2.

[0422] Embodiment 164. The system of any of embodiments 161-163, wherein the non- human mammalian subject is a mouse, rat, dog, pig, or non-human primate.

[0423] Embodiment 165. The system of any of embodiments 161-164, wherein an average mass of a peptide variant is about 1000 to about 10000 DA.

[0424] Embodiment 166. The system of any of embodiments 161-165, wherein the therapeutic peptide is about 10 amino acids to about 100 amino acids.

[0425] Embodiment 167. The system of any of embodiments 161-166, wherein the therapeutic peptide is about 100 to about 5000 amino acids.

[0426] Embodiment 168. The system of any of embodiments 161 - 167, wherein the region is about 10 amino acids to about 100 amino acids.

[0427] Embodiment 169. The system of any of embodiments 161 - 168, wherein the therapeutic peptide comprises a linear peptide sequence.MOFO-360864280 83341812000140

[0428] Embodiment 170. The system of any of embodiments 161 - 169, wherein selecting a region comprises identifying amino acids of the therapeutic peptide predicted to contribute to binding to a target and / or identifying amino acids of the therapeutic peptides that do not contribute to binding to the target.

[0429] Embodiment 171. The system of any of embodiments 161 - 170, wherein the region comprises a N-terminus of the therapeutic peptide.

[0430] Embodiment 172. The system of any of embodiments 161 - 171, wherein the region comprises a C-terminus of the therapeutic peptide.

[0431] Embodiment 173. The system of any of embodiments 161 - 172, wherein a site in the plurality of sites is within about 1 to about 100 amino acids of another site in the plurality of sites.

[0432] Embodiment 174. The system of any of embodiments 161 - 173, wherein the plurality of sites comprises about 2 to about 25 sites.

[0433] Embodiment 175. The system of any of embodiments 161 - 174, wherein the plurality of sites comprises between about 5% and about 100% of the amino acids in the region.

[0434] Embodiment 176. The system of any of embodiments 161 - 175, wherein each unique peptide variant comprises at least two or more modifications at two or more of the plurality of sites in the region.

[0435] Embodiment 177. The system of any of embodiments 161 - 176, wherein each unique peptide variant comprises at least three or more modifications at three or more of the plurality of sites in the region.

[0436] Embodiment 178. The system of any of embodiments 161 - 177, wherein each unique peptide variant comprises at least four or more modifications at four or more of the plurality of sites in the region.

[0437] Embodiment 179. The system of any of embodiments 161 - 178, wherein the at least one modification at one or more of the plurality of sites comprises an amino acid substitution, amino acid insertion, or amino acid deletion.

[0438] Embodiment 180 The system of embodiment 179, wherein the amino acid substitution comprises identifying an amino acid residue at a site and substituting the amino acid residue with a second amino acid residue with; a. a similar charge as the amino acid residue; b. a similar size as the amino acid residue; c. a similar shape as the amino acid residue;MOFO-360864280 84341812000140 d. a similar solubility as the amino acid residue; e. a similar linked hydrophobic moiety; and / or f. a similar chirality as the amino acid residue.

[0439] Embodiment 181. The system of embodiment 179, wherein the amino acid substitution comprises, identifying an amino acid residue at a site and substituting the amino acid residue with a second amino acid residue with; a. a different charge as the amino acid residue; b. a different size as the amino acid residue; c. a different shape as the amino acid residue; d. a different solubility as the amino acid residue; e. a different linked hydrophobic moiety; and / or f. a different chirality as the amino acid residue.

[0440] Embodiment 182. The system of any of embodiments 179, wherein the amino acid substitution comprises: a. identifying an amino acid residue susceptible to dehydration at a site and substituting the amino acid residue with a second amino acid residue less susceptible to dehydration; b. identifying an amino acid residue susceptible to deamidation at a site and substituting the amino acid residue with a second amino acid residue less susceptible to deamidation; c. identifying an amino acid residue susceptible to oxidation at a site and substituting the amino acid residue with a second amino acid residue less susceptible to oxidation; and / or d. identifying an amino acid residue contributing to secondary structure at a site and substituting the amino acid residue with a second amino acid residue to strengthen the secondary structure.

[0441] Embodiment 183. The system of any of embodiments 180-182, wherein the second amino acid comprises a non-canonical amino acid.

[0442] Embodiment 184. The system of any of embodiments 180-183, wherein the amino acid insertion or the amino acid substitution comprises a non-canonical amino acid.

[0443] Embodiment 185. The system of embodiment 183 or 184, wherein the non- canonical amino acid is selected based on a chemical property, a charge, a solubility, a shape of the non-canonical amino acid.

[0444] Embodiment 186. The system of embodiment 185, wherein the non-canonical amino acid is predicted to increase the FPKP of the peptide.MOFO-360864280 85341812000140

[0445] Embodiment 187. The system of any of embodiments 161-186, wherein one or more of the unique peptide variants has an improved FPKP compared to the therapeutic peptide.

[0446] Embodiment 188. The system of any of embodiments 161-187, wherein the FPKP is selected from the group consisting of systemic circulatory half-life, biodistribution, target tissue uptake, bioavailability, protease resistance, metabolism, clearance, and blood-brain barrier penetration.

[0447] Embodiment 189. The system of any of embodiments 161-188, wherein one or more of the unique peptide variants has an improved FPKP compared to the therapeutic peptide.

[0448] Embodiment 190. The system of embodiment 189, wherein the improved FPKP comprises reduced renal clearance, increased liver clearance, reduced drug metabolism, increased circulatory half-life, increased brain uptake, and / or increased tumor uptake.MOFO-360864280 86341812000140EXAMPLES

[0449] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice.

[0450] However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0451] Techniques for performing the methods of the present invention are well known in the art and described in standard laboratory textbooks, including, for example, Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997); Molecular Cloning: A Laboratory Manual, Third Edition, J. Sambrook and D. W. Russell, eds., Cold Spring Harbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; O’Brian et al., Antibody Phage Display, Methods and Protocols, Humana Press, 2001; Phage Display: A Laboratory Manual, C. E. Barbas III et al. eds., Cold Spring Harbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; and Antibodies, G. Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can, for example, be performed using site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)); DNA Cloning, Vols. 1 and 2, (D. N. Glover, Ed. 1985); Oligonucleotide Synthesis (M. J. Gait, Ed. 1984); PCR Handbook Current Protocols in Nucleic Acid Chemistry, Beaucage, Ed. John Wiley & Sons (1999) (Editor); Oxford Handbook of Nucleic Acid Structure, Neidle, Ed., Oxford Univ Press (1999); PCR Protocols: A Guide to Methods and Applications, Innis et al., Academic Press (1990); PCR Essential Techniques: Essential Techniques, Burke, Ed., John Wiley & Son Ltd (1996); The PCR Technique: RT-PCR, Siebert, Ed., Eaton Pub. Co. (1998); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C. S. H. L. Press, Pub. (1999); Large-Scale Mammalian Cell Culture Technology, Lubiniecki, A., Ed., Marcel Dekker, Pub., (1990). Border et al., Yeast surface display for screening combinatorial polypeptide libraries, Nature Biotechnology, 15(6):553-7 (1997); Border et al., Yeast surface display for directed evolutionMOFO-360864280 87341812000140 of protein expression, affinity, and stability, Methods Enzymol., 328:430-44 (2000); ribosome display as described by Pluckthun et al. in U.S. Pat. No. 6,348,315, and Profusion™ as described by Szostak et al. in U.S. Pat. Nos. 6,258,558; 6,261,804; and 6,214,553; and bacterial periplasmic expression as described in US20040058403A1.

[0452] Although in the foregoing description, the invention is illustrated with reference to certain embodiments, it is not so limited. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Thus, while the invention is illustrated with reference to biologically active peptide libraries, it extends generally to all peptide and polypeptide libraries.Example 1: Designing a library targeting parathyroid hormone receptor (PTHR)

[0453] The N-terminal 34 residues of parathyroid hormone are selected as the reference peptide: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ ID NO: 1). The molecule is synthesized as described in Mijaslis et al., except that, at select positions, a mixture of amino acids are flowed into the reactor rather than a pure single amino acid solution, as seen in FIG. 1 (Mijalis AJ, et al., (2017). Nat Chem Biol, 13(5):464-466). At positions wherein native residues are determined to not directly interact with the target, the coupling reaction is conducted with a mixture of the native (or wild-type) residue and nonnative residues which impart features such as direct target interaction, conformational flexibility or rigidity, protease resistance, inhibition of renal clearance, improved bioavailability, and / or improved biodistribution. This pooling fashion of amino acids in a single reaction reservoir occurs in a single reaction step to build a combinatorial library. More specifically, at positions E19, V21, R25, K27, Q29, a mixture of a) the native amino acid residue, at 10%, and b) the non-canonical amino acids listed in Table 1, at the given percentages of equimolar stock solutions, are flowed into the reactor for that step of synthesis. The remaining positions are reacted with a solution of the pure native amino acid. By coupling the growing peptides to a mixture of amino acids at specific residue positions, a combinatorial library results with a diversity of 215= 4,084,101. In a further instance, the same library is synthesized except with L7 replaced with the amino acid norleucine.MOFO-360864280 88341812000140Example 2: Flow peptide synthesis and preparative HPLC of a PTHR-targeting libraryAutomated flow peptide synthesis

[0454] As disclosed in Charalampidou A et al, approximately 130 mg of H-Rink amide ChemMatrix resin (loading 0.18 mmol / g) is loaded into a fritted syringe (6 mL), swollen in amine-free DMF for 15 min and is washed altematingly with DCM and DMF. The beads are loaded into the automated flow peptide synthesizer. The general flow rate is 40 mL / min, and the temperature in the loop is 90 °C and 85-90 °C in the reactor. The first step is a 60 second wash step at elevated temperatures with 40 mL of amine-free DMF. The couplingdeprotection cycle is repeated for all additional monomers used in the sequences. The reference sequence consists of the N-terminal 34 residues of parathyroid hormone: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ ID NO: 1). As described in Example 1, at positions E19, V21, R25, K27, Q29, a mixture of the wild-type amino acids and the non-canonical amino acids listed in Table 1 are reacted during the coupling reactions. Orthogonal on-bead deprotection of Alloc and OAll groups

[0455] After synthesis, the beads (loading: 0.0221 mmol, 1 eq.) are washed thoroughly with amine-free DMF (3 x 5 mL) and DCM (3 x 5 mL) before drying them in a vacuum chamber. 5.1 mg Pd(PPh3)4 (0.00442 mmol, 0.2 eq.) is dissolved in 4 mL DCM, and 54 pL of PhSiH3 (0.442 mmol, 20 eq.) is added. The mixture is added to the syringe containing the dried beads. The suspension is incubated for 30 min at room temperature. This step is performed twice. The beads are then washed with DCM (3 x 5 mL), and subsequently with DMF (3 x 5 mL).Cleavage

[0456] The dried beads are mixed with a cleavage solution to remove the remaining protecting groups and release the proteins from the beads. For approximately 200 mg of beads, 5 mL of cleavage solution (82.5 % TFA, 5 % water, 5 % phenol, 5 % thioanisole, 2.5 % EDT) is added and kept on a rolling device for 3 h at room temperature. The proteins are precipitated in 40- 45 mL ice-cold diethyl ether and the precipitate is collected by centrifugation while the supernatant is discarded. Traces of diethyl ether and TFA are removed as much as possible by evaporation with a nitrogen gas flow. The proteins are dissolved in 50 % acetonitrile (0.1% TFA) in water and dried by lyophilization.Preparative mass-directed RP-HPLC

[0457] 30-40 mg of the lyophilized crude proteins are dissolved in 5 mL buffer containing 6 M guanidine hydrochloride and 100 mM Tris at pH 8. All samples are filtrated with a nylon 0.22 pm syringe filter before loading to the column. For all HPLC purifications, a gradient ofMOFO-360864280 8934181200014028 % to 48 % solvent B in 100 min is used. Solvent A is MQ water with 0.1 % TFA and Solvent B is HPLC-grade acetonitrile with 0.1 % TFA. The semipreparative Agilent Zorbax 300SB- C3 column (9.4 mm x 250 mm, 5-pm particle size) column is used at a temperature of 60 °C with a flow rate of 4 mL / min. The pure fractions are combined, lyophilized, weighted and used for further characterization (Charalampidou A, et al., (2024). ACS Cent Sci, 10(3):649-657).Example 3: Designing a linear library of serum albumin binders

[0458] The 18 amino acids of the serum albumin binding peptide were selected as the reference peptide: WWEQDRDWDFDVFGGGTP (SEQ ID NO:2). The molecule was synthesized as describe in Mijaslis et al, except that, at select positions, a mixture of amino acids were flowed into the reactor alongside coupling reagents for that step of synthesis, rather than a pure single amino acid solution, as seen in FIG. 1 (Mijalis AJ, et al., (2017). Nat Chem Biol, 13(5):464-466). Positions, W2 and E3 were selected as positions involved in binding where non-natural amino acids are likely to modulate binding affectivity. D5 and D7 were selected as positions where a range of natural amino acids can be accommodated, and which may tolerate non-natural amino acids that affect properties other than binding. Sites W8, D9, F10, and F13 were selected as sites that are critical for binding where few other natural amino acids can be accommodated. These positions evaluate whether non-natural amino acids can achieve binding properties that are not achievable with natural amino acids. At such positions, the coupling reaction was conducted with a mixture of canonical (Ala) and non-canonical amino acids including but not limited to those found in Table 1 and / or Table 2, at varying equivalences alongside coupling reagents for that step in synthesis. The mixture of non-native residues imparted features such as direct target interaction, confirmational flexibility or rigidity, protease resistance, inhibition of renal clearance, improved bioavailability, and / or improved biodistribution. This pooling fashion of amino acids in a single reaction reservoir occurred in a single reaction step to build a combinatorial library. More specifically, at positions W2, E3, D5, D7, W8, D9, F10, F13, a mixture of canonical and non-canonical amino acids including but not limited to those found in Table 1 and / or Table 2, at varying molar equivalences were flowed into the reactor alongside coupling reagents for that step of synthesis. For example, 5-fluorotryptophan and 7 -azatryptophan was added in place of tryptophan in positions 2, and 8; 1-naphthyl-L-alanine and p-nitro-D- phenylalanine added in place of phenylalanine in positions 10 and 13; and L-citrulline and beta-L-aspartic acid added in place of aspartic acid in positions 5, 7, and 9; in each case,MOFO-360864280 90341812000140 increasing the stability of the molecule. The molar equivalence of each amino acid within the mixture was determined based on the specific mixture composition at each position and is calculated with reference to the radius of gyration, relative to the radius of gyration of the other amino acids present in said mixture. The remaining positions were reacted with a solution of the pure native amino acid and coupling reagents. Additionally, the c-terminus of the peptide was extended to incorporate a biotinylated lysine amino acid to incorporate a functional handle for in vivo and in vitro assessment. Finally, the N-terminus of the peptide was acetylated and the c-terminus of the peptide is Cleaved to reveal a C-terminal amide.

[0459] The modifications yielded a maximum library size of 61,952 members, demonstrating the high diversity enabled with such designs.Example 4: Flow synthesis and SPE of a linear albumin-binding libraryAutomated flow peptide synthesis

[0460] As disclosed in Charalampidou A et al, approximately 130 mg of H-Rink amide ChemMatrix resin (loading 0.18 mmol / g) is loaded into a fritted syringe (6 mL), swollen in amine-free DMF for 15 min and is washed altematingly with DCM and DMF. The beads are loaded into the automated flow peptide synthesizer. The general flow rate is 40 mL / min, and the temperature in the loop is 90 °C and 85-90 °C in the reactor. The first step is a 60 second wash step at elevated temperatures with 40 mL of amine-free DMF. The couplingdeprotection cycle is repeated for all additional monomers used in the sequences. The reference sequence consists of the published Albumin binder:WWEQDRDWDFDVFGGGTP (SEQ ID NO: 2). At positions Wl, W2, E3, D5, D7, W8, D8, F10, F13, a mixture of the wild-type amino acid and non-canonical amino acids listed in Table 1 are reacted during the coupling reactions.Orthogonal on-bead deprotection of Alloc and OAll groups

[0461] After synthesis, the beads (loading: 0.0221 mmol, 1 eq.) are washed thoroughly with amine-free DMF (3 x 5 mL) and DCM (3 x 5 mL) before drying them in a vacuum chamber.5.1 mg Pd(PPh3)4 (0.00442 mmol, 0.2 eq.) is dissolved in 4 mL DCM, and 54 pL of PhSiH3 (0.442 mmol, 20 eq.) is added. The mixture is added to the syringe containing the dried beads. The suspension is incubated for 30 min at room temperature. This step is performed twice. The beads are then washed with DCM (3 x 5 mL), and subsequently with DME (3 x 5 mL).CleavageMOFO-360864280 91341812000140

[0462] The dried beads are mixed with a cleavage solution to remove the remaining protecting groups and release the proteins from the beads. For approximately 200 mg of beads, 5 mL of cleavage solution (82.5 % TFA, 5 % water, 5 % phenol, 5 % thioanisole, 2.5 % EDT) is added and kept on a rolling device for 3 h at room temperature. The proteins are precipitated in 40- 45 mL -80C diethyl ether and the precipitate is collected by centrifugation while the supernatant is discarded. Traces of diethyl ether and TFA are removed as much as possible by evaporation with a nitrogen gas flow. The peptides are dissolved in 50 % acetonitrile (0.1% TFA) in water and dried by lyophilization.Solid Phase Extraction of libraries

[0463] 20-100mg of crude peptide is dissolved in 5mL of lOOmM ammonium acetate in water at pH 9. The sample is then loaded onto a Discovery® DSC- 18 SPE column for purification and isolation as the acetate salt. Following loading, to the column is flowed 20mL of lOOmM ammonium acetate in water at pH 9 twice. These washes are followed by flowing through 20 mL of a 5% Acetonitrile in water with 1% Acetic acid mixture three times, to remove excess acetate salt. The peptides are then eluted using a gradient of solvents comprising of 50% acetonitrile in water with 1% acetic acid, 70% acetonitrile in water with 1% acetic acid, and 95% acetonitrile in water with 1% acetic acid. Fractions are collected and confirmed for desired peptide via LC-MS. Following confirmation of composition, the pure fractions are combined, lyophilized, weighted and used for further characterization (Charalampidou A, et al., (2024). ACS Cent Sci, 10(3):649-657).Example 5: Designing a cyclic library of serum albumin binders

[0464] The 18 amino acids of the SA21 peptide are selected as the reference peptide: RLIEDICLPRWGCLWEDD (C7-C13 disulfide cyclization) (SEQ ID NO: 3). The molecule is synthesized as describe in Mijaslis et al, except that, at select positions, a mixture of amino acids are flowed into the reactor alongside coupling reagents for that step of synthesis, rather than a pure single amino acid solution, as seen in FIG. 1 (Mijalis AJ, et al., (2017). Nat Chem Biol, 13(5):464-466). Positions hypothesized to interact with the protein were identified based on having low diversity in selection for peptide binders (Dennis MS, et al., (2002). J Biol Chem, 277(38):35035-35043). At positions wherein native residues are hypothesized to interact with the target, the coupling reaction is conducted with a mixture of canonical (Ala) and non-canonical amino acids including but not limited to those found in Table 1 and / or Table 2, at varying equivalences are flowed into the reactor alongside coupling reagents forMOFO-360864280 92341812000140 that step in synthesis. At positions wherein native residues are hypothesized to interact with the target, the coupling reaction is conducted with a mixture of non-native residues which impart features such as direct target interaction, confirmational flexibility or rigidity, protease resistance, inhibition of renal clearance, improved bioavailability, and / or improved biodistribution. This pooling fashion of amino acids in a single reaction reservoir occurs in a single reaction step to build a combinatorial library. More specifically, at positions E4, D5, L8, P9, Wi l, L14, W15, E16, D17, a mixture of canonical and non-canonical amino acids including but not limited to those found in Table 1, at varying molar equivalences are flowed into the reactor alongside coupling reagents for that step of synthesis. For example, 5- fluorotryptophan and 7 -azatryptophan are added in place of tryptophan in positions 11 and 15; and L-citrulline and beta-L-aspartic acid added in place of aspartic acid in positions 5 and 17; in each case, increasing the stability of the molecule. The molar equivalence of each amino acid within the mixture is determined based on the specific mixture composition at each position and is calculated with reference to the degree of rotation, relative to the degree of rotation of the other amino acids present in said mixture. The remaining positions are reacted with a solution of the pure native amino acid and coupling reagents. By coupling the growing peptides to a mixture of amino acids at specific reside positions, a combinatorial library results with a diversity up to 25,344 members. Additionally, the c-terminus of the peptide is extended to incorporate a biotinylated lysine amino acid to incorporate a functional handle for in vivo and in vitro assessment. Finally, the N-terminus of the peptide is acetylated and the C-terminus of the peptide is cleaved to reveal a C-terminal amide.Example 6: Flow synthesis and SPE of a cyclic albumin-binding libraryAutomated flow peptide synthesis

[0465] As disclosed in Charalampidou A et al, approximately 130 mg of H-Rink amide ChemMatrix resin (loading 0.18 mmol / g) is loaded into a fritted syringe (6 mL), swollen in amine-free DMF for 15 min and is washed altematingly with DCM and DMF. The beads are loaded into the automated flow peptide synthesizer. The general flow rate is 40 mL / min, and the temperature in the loop is 90 °C and 85-90 °C in the reactor. The first step is a 60 second wash step at elevated temperatures with 40 mL of amine-free DMF. The couplingdeprotection cycle is repeated for all additional monomers used in the sequences. The reference sequence consists of the published Albumin binder: RLIEDICLPRWGCLWEDD (C7-C13 disulfide cyclization) (SEQ ID NO: 3) At positions E4, D5, L8, P9, Wi l, L14,MOFO-360864280 93341812000140W15, E16, D17, a mixture of wild-type amino acid and non-canonical amino acids listed in Table 1 are reacted during the coupling reactions.Orthogonal on-bead deprotection of Alloc and OAll groups

[0466] After synthesis, the beads (loading: 0.0221 mmol, 1 eq.) are washed thoroughly with amine-free DMF (3 x 5 mL) and DCM (3 x 5 mL) before drying them in a vacuum chamber. 5.1 mg Pd(PPh3)4 (0.00442 mmol, 0.2 eq.) is dissolved in 4 mL DCM, and 54 pL of PhSiH3 (0.442 mmol, 20 eq.) is added. The mixture is added to the syringe containing the dried beads. The suspension is incubated for 30 min at room temperature. This step is performed twice. The beads are then washed with DCM (3 x 5 mL), and subsequently with DML (3 x 5 mL).Cleavage

[0467] The dried beads are mixed with a cleavage solution to remove the remaining protecting groups and release the proteins from the beads. Lor approximately 200 mg of beads, 5 mL of cleavage solution (82.5 % TLA, 5 % water, 5 % phenol, 5 % thioanisole, 2.5 % EDT) is added and kept on a rolling device for 3 h at room temperature. The proteins are precipitated in 40- 45 mL -80C diethyl ether and the precipitate is collected by centrifugation while the supernatant is discarded. Traces of diethyl ether and TEA are removed as much as possible by evaporation with a nitrogen gas flow. The peptides are dissolved in 50 % acetonitrile (0.1% TEA) in water and dried by lyophilization.Cyclization

[0468] hollowing cleavage, the peptides were dissolved in 5% acetonitrile in water (with 0.1% trifluoroacetic acid) at ~2 mg / mL (~1 mM) by dropwise addition of ~1 eq. iodine in methanol until a yellow-brown color persisted. After 5-10 minutes at room temperature in the dark, the reaction was quenched with aqueous ascorbic acid to provide a colorless solution again (3.5 eq.) (Lee MA, et al. (2025). Sci Adv, l l(12):eadrl018).Solid Phase Extraction of libraries

[0469] Crude solution following cyclization is then loaded onto an equilibrated Discovery® DSC- 18 SPE column for purification and isolation as the acetate salt. Eollowing loading, to the column is flowed 20mL of lOOmM ammonium acetate in water at pH 9 twice. These washes are followed by flowing through 20 mL of a 5% Acetonitrile in water with 1% Acetic acid mixture three times, to remove excess acetate salt. The peptides are then eluted using a gradient of solvents comprising of 50% acetonitrile in water with 1% acetic acid, 70% acetonitrile in water with 1% acetic acid, and 95% acetonitrile in water with 1% acetic acid. Tractions are collected and confirmed for desired peptide via LC-MS and via Ellmans’sMOFO-360864280 94341812000140 assay. Following confirmation of composition, the pure fractions are combined, lyophilized, weighted and used for further characterization (Charalampidou A, et al., (2024). ACS Cent Sci, 10(3):649-657).Example 7: Designing a library of Natriuretic Peptide Receptor binders

[0470] The 28 residues of Atrial Natriuretic Peptide (or Atrial Natriuretic Factor) were selected as the reference peptide: SLRRSSCFGGRMDRIGAQSGLGCNSFRY (C7-C23 disulfide cyclization) (SEQ ID NO: 4). The molecule was synthesized as described in Mijaslis et al, except that at select positions, a mixture of canonical and noncanonical amino acids were flowed into the reactor alongside coupling reagents for that step of synthesis, rather than a pure single amino acid solution, as seen in FIG. l(Mijalis AJ, et al., (2017). Nat Chem Biol, 13(5):464-466). At positions R3, R4, Rl l, N24, S25, and F26, noncanonical amino acids such as Homoarginine, Citrulline, and 3-(3-pyridyl)-L-alanine were substituted to increase stability. At positions S6, F8, M12, D13, R14, A17, Q18, and L21, noncanonical amino acids such as O-Ethylserine, Selenomethionine, and Nitroarginine were substituted to modify affinity for the NPR1 receptor. At positions D3, T9, Si l, L12, S14, and R16, amino acids such as Norarginine, N,N-dimethyl-L- Glutamine and, 2-Amino-4,4,4-trifluorobutyric acid were substituted to modify the selectivity towards NPR1 vs. NPR3. Alternative amino acids at varying molar equivalences were flowed into the reactor alongside coupling reagents for a single step of synthesis to build a combinatorial library. The remaining positions were reacted with a solution of the pure native amino acid and coupling reagents. By coupling the growing peptides to a mixture of amino acids at specific residue positions, a combinatorial library was generated with a controlled diversity. Additionally, the n-terminus of the peptide was extended to incorporate a biotinylated lysine amino acid to incorporate a functional handle for in vivo and in vitro assessment. Finally, the N-terminus of the peptide was acetylated and the c-terminus of the peptide was cleaved to reveal a C-terminal amide.-Example 8: Flow synthesis of cyclic Natriuretic Peptide Receptor binderAutomated flow peptide synthesis

[0471] As disclosed in Charalampidou A et al, approximately 130 mg of H-Rink amide ChemMatrix resin (loading 0.18 mmol / g) was loaded into a fritted syringe (6 mL), swollen inMOFO-360864280 95341812000140 amine-free DMF for 15 min and was washed altematingly with DCM and DMF. The beads were loaded into the automated flow peptide synthesizer. The general flow rate was 40 mL / min, and the temperature in the loop was 90 °C and 85-90 °C in the reactor. The first step was a 60 second wash step at elevated temperatures with 40 mL of amine-free DMF. The coupling-deprotection cycle was repeated for all additional monomers used in the sequences. The reference sequence consisted of the published natural sequence of human Atrial Natriuretic Peptide:: SLRRSSCFGGRMDRIGAQSGLGCNSFRY (C7-C23 disulfide cyclization) (SEQ ID NO: 4) At positions R3, R4, S6, F8, G9, Rl l, M12, D13, R14, G16, A17, Q18, N19, L21, N24, S25, F26, a mixture of wild-type amino acid and non-canonical amino acids listed in Table 1 were reacted during the coupling reactions.Cleavage

[0472] The dried beads were mixed with a cleavage solution to remove the remaining protecting groups and the proteins were released from the beads. For approximately 200 mg of beads, 5 mL of cleavage solution (82.5 % TFA, 5 % water, 5 % phenol, 5 % thioanisole, 2.5 % EDT) was added and kept on a rolling device for 3 h at room temperature. The proteins were precipitated in 40- 45 mL -80C diethyl ether and the precipitate was collected by centrifugation while the supernatant was discarded. Traces of diethyl ether and TFA were removed as much as possible by evaporation with a nitrogen gas flow. The peptides were dissolved in 50 % acetonitrile (0.1% TFA) in water and dried by lyophilization.Cyclization

[0473] Following cleavage, the peptides were dissolved in 5% acetonitrile in water (with 0.1% trifluoroacetic acid) at ~2 mg / mL (~1 mM) by dropwise addition of ~1 eq. iodine in methanol until a yellow-brown color persisted. After 5-10 minutes at room temperature in the dark, the reaction was quenched with aqueous ascorbic acid to provide a colorless solution again (3.5 eq.) (Lee MA, et al. (2025). Sci Adv, l l(12):eadrl018).Solid Phase Extraction of libraries

[0474] Crude solution following cyclization was then loaded onto an equilibrated Discovery® DSC- 18 SPE column for purification and isolation as the acetate salt. Following loading, 20mL of lOOmM ammonium acetate in water at pH 9 was flowed to the column twice. These washes were followed by flowing through 20 mL of a 5% Acetonitrile in water with 1% Acetic acid mixture three times, to remove excess acetate salt. The peptides were then eluted using a gradient of solvents comprising of 50% acetonitrile in water with 1% acetic acid, 70% acetonitrile in water with 1% acetic acid, and 95% acetonitrile in water with 1% acetic acid. Fractions were collected and confirmed for desired peptide via LC-MS.MOFO-360864280 96341812000140Following confirmation of composition, the pure fractions were combined, lyophilized, weighted and used for further characterization (Charalampidou A, et al., (2024). ACS Cent Sci, 10(3):649-657).Example 9: Peptide library characterizationLC-MS

[0475] To check the mass of the synthesized peptides or proteins, a 1 mg / mL solution was prepared and diluted with 50 % HPLC acetonitrile in water with 0.1% TFA as an additive to a 0.1 mg / mL solution. The solutions were filtered and measured on an Agilent 6545 Accurate- Mass Q-TOF LCMS system. The solvent composition for solvent A was MQ water with 0.1% formic acid as an additive and for solvent B, HPLC grade acetonitrile with 0.1% formic acid is used. Method used: C3-1-95 in 15 min using the Agilent Zorbax 300SB-C3 column (2.1 mm x 150 mm, 5-pm particle size) (Charalampidou A, et al., (2024). ACS Cent Sci, 10(3):649-657).Analytical HPLC

[0476] As disclosed in Charalampidou A, et al., the purity of the samples were analyzed by analytical HPLC. The measurements was performed on Agilent Technologies 1200 Series instrument. The sample was partitioned over Zorbax 300SB- C3, 5 uM, 2.1x150 mm analytical column with 5-95 % acetonitrile gradient in water with 0.1 % formic acid from 2 - 14 minutes. The analytical column was maintained at 40°C.LC-MS / MS

[0477] The sequence or distribution of sequences of a peptide library (the sample) was evaluated by nanoflow HPLC coupled to tandem mass spectrometry. The sample was diluted to 1 nmol in 0.05 mL of 50nM ammonium bicarbonate. Any disulfide bonds were broken by treatment with tris(2-carboxyethyl) phosphine, then free sulfhydryl groups were blocked by treatment with iodoacetamide. Salts were removed from the sample by clean-up on Cl 8 Stage Tips. After being reduced to dryness in a vacuum centrifuge, the sample was suspended in deionized water with 0.1% TFA. 100 pmol of material was loaded onto a Vanquish Neo HPLC system coupled to a ThermoFisher Scientific Eclipse mass spectrometer running in positive ion mode. Peptides were analyzed over a 1-hour gradient using 0.1% TFA in water as solvent A and 80% acetonitrile 0.1% TFA as solvent B. Selected ions were fragmented for tandem mass spectrometry (MS2) using higher-energy collisional dissociation (HCD), and fragment ions were detected in the Orbitrap mass analyzer.

[0478] Hundreds or thousands of individual m / z ratios corresponding to synthesized peptides or proteins were measured depending on the input library size. MS2 spectra were matched toMOFO-360864280 97341812000140 expected peptide sequences using search tools including but not limited to MSFragger (Kong et al., (2017). “MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics.” Nat Methods, 15:513-520), MaxQuant (Cox et al., (2008). “MaxQuant enables high peptide identification rates, individualized p.p.b. -range mass accuracies and proteome-wide protein quantification.” Nat Biotech, 26:1367-1372), SpectroMine (Biognosys AG, Switzerland) or PEAKS (Bioinformatics Solutions, Inc) with target false-discovery rate controlled at 1%. The presence of identified peptides containing each expected amino acid confirmed successful synthesis.Example 10: Characterizing amino acid and library diversity

[0479] To understand the diversity of libraries designed using methods described herein, properties of the canonical amino acids were compared to the properties of the non-canonical amino acids used to prepare libraries. Properties of each animo acid were obtained. The properties in the analysis included, number of heavy atoms, molecular weight, hydrophobicity, degree of branching, rotatable bods, ring count, H- bond donors, H-bond acceptors, and polar surface area. A principal component analysis (PCA) was performed to project the diversity of the amino acids in a two-dimensional space. FIG. 3 shows the canonical amino acids and the amino acids used in the library (including non-canonical amino acids). PCI represents the highest contribution of variation in the dataset and PC2 represents the second highest contribution of variation in the dataset. PCI correlates most strongly with structural features like branching and molecular weight whereas PC2 correlates most strongly with chemical interaction features like bond donors / acceptors and polar surface area. As shown in FIG. 3, the amino acids in the libraries comprise a much larger space along PCI and PC2 compared to the canonical amino acids. These results suggest that the non- canonical amino acid chemical properties contributed a combinatorial expansion in diversity for the libraries. These results suggest that the large peptide libraries synthesized incorporate significantly expanded chemical diversity by virtue of the unrestrained utilization of chemically diverse non-canonical amino acids.

[0480] A similar PCA analysis was completed using the molecular properties for the amino acids that made up various peptides within a library. FIG.4A and 4B show results for the Atrial Natriuretic Peptide (ANP) library described above. According to both FIG. 4A and FIG. 4B, the native peptide with native amino acids is represented as a circle and the peptides in the library are indicated by a plus sign. FIG 4A shows the ANP peptide in PC space andMOFO-360864280 98341812000140FIG 4B shows both the ANP peptide and a related peptide, B-type Natriuretic Peptide, (BNP).Example 11: Assessing library stability and recovery from plasma

[0481] To test the stability and plasma recovery of albumin binding libraries, pure rat plasma was spiked with 1000 pmol of total peptides. To test stability, samples were incubated at 37C for different times includingl hour, 4 hours, 8 hours and 24 hours. TCEP (0.5M) was added at a 1:100 volume ratio and the sample was incubated for 20 minutes at 37C. The sample was mixed with 4-fold volume of acetonitrile, vortex, and incubated for 90 minutes at room temperature with slight agitation. Then, the sample was centrifuged at 1660xg at 4C for 20 minutes and the supernatant was collected and dried completely using the SpeedVac. The dried sample was reconstituted in PBS and the concentration was evaluated using a direct Elisa assay. For the Elisa assay, a 96-well plate was coated with 5 ug / ml human serum albumin overnight at 4C. The next day, the plate was washed 3 times with TBST buffet (Tris buffer with 0.2% tween20 pH 7.4). 200 pl of protein-free blocking buffer was added and the plate was incubated 1 hour at room temperature. The plate was washed once, and 100 pl of diluted peptide sample was added along with a calibration curve generated using known concentrations of the tested library. Following a 1 hour incubation at room temperature, the plate was washed 3 times and a 2 pg / ml HRP-conjugated streptavidin was added for an additional 1 hour incubation at room temperature. Next, the plate was washed 4 times and a TMB substrate was added. After 10 minutes, equal volumes of stop solution was added and the plate was read at 450 nm. The concentration of the plasma extracted sample was extrapolated from the calibration curve.Example 12: Assessing library stability and recovery from plasma

[0482] To test the stability and plasma recovery of natriuretic peptide receptor-binding libraries, pure rat plasma was spiked with 1000 pmol of total peptides. To test stability, samples may be incubated at 37C for different times such as 5 minutes, 20 minutes, and Ihour. The sample was mixed with 1.5-fold volume of acidified acetone, vortexed, and incubated for 90 minutes at room temperature with slight agitation. Then, the sample was centrifuged at 3000xg for 20 minutes and the supernatant was collected and dried. The dried sample was reconstituted in 50 mM ammonium bicarbonate.MOFO-360864280 99341812000140

[0483] TCEP (0.5M) was added at a 1:50 volume ratio and the sample was incubated for 45 minutes at 55C. 22 mM iodoacetamide was added and incubated for 60 minutes. The solution was added to C18 Stage Tips for desalting and cleanup. Resulting samples were dried and reconstituted in 0.1% trifluoroacetic acid (TFA) for analysis on a mass spectrometer. The samples were analyzed using a Vanquish Neo HPLC coupled to an Eclipse mass spectrometer (ThermoFisher Scientific) running in positive ion mode. A 40-minute gradient was used to separate peptides, with selected ions analyzed by HCD fragmentation. Peptides were identified and quantified by bioinformatic analysis using MSFragger (Kong et al., (2017). “MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics.” Nat Methods, 15:513-520).

[0484] The peptide diversity seen at time points 0, 5, 20, and 60 min was determined by mass spectrometry. It was determined if each peptide was still recoverable in the plasma and at what timepoint (data not shown). The results demonstrated that over 1000 unique peptides from the library could be recovered from rat plasma and detected at multiple timepoint. The results also demonstrated that peptides with differing stability in the plasma could be characterized using mass spectrometry of recovered peptides.

[0485] To further demonstrate the methods could be used to recover characteristics of peptides in the library with differing pharmacokinetic properties, the recovery of three exemplary peptides from the library with both n-acetylated and non-acetylated versions were characterized at the timepoints. FIG.5 shows the intensity as measured by mass spectrometry for each peptide normalized by the intensity of the peptide at time point 0. As shown in FIG. 5, the nonacetylated versions of the peptides degraded more rapidly for the three peptides. These results demonstrated both that the methods could be used to compare pharmacokineticsof similar peptides (e.g., capped and uncapped versions) and that n- acetylation increased the stability of exemplary peptides.Example 13: Murine assay identifies, quantifies long-half- life peptides from a large library

[0486] A library is designed and synthesized as described in Examples 3-8, and assayed similar to Eoftis AR, et al., (2021). Proc Natl Acad Sci USA, 118(34): e2101596118. The library is prepared at 7.4 mM. 0.2 mF of the library mixture is administered via the tail vein to female C57BE / 6J mice. At each time point of 30 minutes, 1 hr, 2 hr, and 3 hr, three mice are sacrificed and blood is collected for analysis. For processing, blood samples are washed once with PBS and then centrifuged. The supernatant is transferred to a new microcentrifugeMOFO-360864280 100341812000140 tube and albumin antibody-coated magnetic beads are added. The solution is incubated for 30 min, washed, and is then treated with a sodium dodecyl sulfate and dithiothreitol elution buffer and heated to 70C. The resulting elutions are transferred to a fresh microcentrifuge tube and lyophilized overnight. The lyophilized material is resuspended in 95 / 5 water / acetonitrile (0.1% TFA) containing 6 M guanidine hydrochloride. The suspension is centrifuged and the supernatant is transferred to a new microcentrifuge tube and lyophilized in a 200-pL strip tube overnight. The lyophilized material is resuspended in water (0.1% TFA) and centrifuged at 16,000 g for 5 min. The resulting supernatant is analyzed by LCMS / MS as described in Example 8. Peptides which are present at the 1, 2, and 3 hr time points are considered to have extended half-life.Example 14: Murine assay identifies, quantifies long-half-life peptides from a large library

[0487] A library is designed and synthesized as described in Examples 3 and 4. The resulting library is prepared at 2 mg / mL. The library mixture is administered via the tail vein to C57B / 6 mice to a dose of 2 mg / kg. At each time point of 5 minutes, 15 min, and 1 hr, 0.07 ml of blood is drawn via the saphenous vein and collected into a tube with an anti-coagulant. At 2 hour, terminal blood collection is performed via cardiac puncture and collected into a tube with an anti-coagulant and processed to plasma. Peptides are extracted from blood plasma and analyzed by mass spectrometry as described in Example 12. Peptides which are present at the 2, 4, and 6 hr time points are considered to have extended half-life.Example 15: Rat assay identifies, quantifies long-half-life peptides from a large library

[0488] A library is designed and synthesized as described in Examples 3 and 4. The resulting library is prepared at 2 mg / mL. The library mixture is administered via the tail vein to male Sprague Dawley rats to a dose of 2 mg / kg. At each time point of 5 minutes, 2 hr, and 4 hr, 0.5 ml of blood is drawn via the saphenous vein and collected into a tube with an anti-coagulant. At 6 hour, terminal blood collection is performed via cardiac puncture and collected into a tube with an anti-coagulant and processed to plasma. Peptides are extracted from blood plasma and analzyed by mass spectrometry as described in Example 12. . Peptides which are present at the 2, 4, and 6 hr time points are considered to have extended half-life.MOFO-360864280 101341812000140REFERENCES

[0489] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.MOFO-360864280 102

Claims

341812000140CLAIMS1. A method of manufacturing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject, comprising:(a) selecting a region of at least 10 amino acids of the therapeutic peptide;(b) selecting a plurality of sites within the region;(c) generating at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non- canonical amino acid, wherein each of the unique peptide variants comprise a C-terminal and / or N-terminal modification; and(d) manufacturing a peptide library comprising the unique peptide variants.

2. A method of designing a peptide library for analyzing a functional pharmacokinetic parameter (FPKP) of a therapeutic peptide in a non-human mammalian subject, comprising:(a) selecting a region of at least 10 amino acids of the therapeutic peptide;(b) selecting a plurality of sites within the region;(c) generating at least IxlO3unique peptide variants of the therapeutic peptide, each peptide variant comprising at least one modification at one or more of the plurality of sites in the region of the therapeutic peptide, wherein at least one modification comprises a non- canonical amino acid, wherein one or more of the unique peptide variants comprise a C- terminal and / or N-terminal modification.

3. A method of determining a functional pharmacokinetic parameter (FPKP) of a plurality of unique peptide variants of a therapeutic peptide, comprising the steps of:(a) introducing into a non-human mammalian subject a peptide library comprising the unique peptide variants, the peptide library comprising at least IxlO3unique peptide variants, each peptide variant comprising at least one modification at one or more of a plurality of sites in a region of the therapeutic peptide, wherein at least one modification comprises a non-canonical amino acid, wherein each of the one or more of the unique peptideMOFO-360864280 103341812000140 variants comprise a C-terminal and / or N-terminal modification, wherein less than 75 pg / kg of each unique peptide variant is introduced into the non-human mammalian subject;(b) detecting thereafter a subset of the unique peptide variants present in the plurality of unique peptide variants, thereby determining the FPPK of the detected unique peptide variants.4 The method of any of claims 1-3, wherein the one or more unique peptide variants comprise an amidated C-terminus and / or acetylated N-terminus.

5. The method of any of claims 1-4, wherein the non-canonical amino acid is selected from the non-canonical amino acids in Table 1 and / or Table 2.

6. The method of any of claims 1 - 5, wherein the non-human mammalian subject is a mouse, rat, dog, pig, or non-human primate.

7. The method of any of claims 1- 6 wherein an average mass of a peptide variant is about 1000 to about 10000 DA.

8. The method of any of claims 1-7, wherein the therapeutic peptide is about 10 amino acids to about 100 amino acids.

9. The method of any of claim 1-8, wherein the therapeutic peptide is about 100 to about 5000 amino acids.

10. The method of any of claims 1 - 9, wherein the region is about 10 amino acids to about 100 amino acids.

11. The method of any of claims 1 - 10, wherein the therapeutic peptide comprises a linear peptide sequence or a cyclic peptide sequence.

12. The method of any of claims 1 - 11, wherein selecting a region comprises identifying amino acids of the therapeutic peptide predicted to contribute to binding to a target and / or identifying amino acids of the therapeutic peptides that do not contribute to binding to the target.MOFO-360864280 10434181200014013. The method of any of claims 1 - 12, wherein the region comprises a N-terminus of the therapeutic peptide.

14. The method of any of claims 1 - 13, wherein the region comprises a C-terminus of the therapeutic peptide.

15. The method of any of claims 1 - 14, wherein a site in the plurality of sites is within about 1 to about 100 amino acids of another site in the plurality of sites.

16. The method of any of claims 1 - 15, wherein the plurality of sites comprises about 2 to about 25 sites.

17. The method of any of claims 1 - 16, wherein the plurality of sites comprises between about 5% and about 100% of the amino acids in the region.

18. The method of any of claims 1 - 17, wherein each unique peptide variant comprises at least two or more modifications at two or more of the plurality of sites in the region.

19. The method of any of claims 1 - 18, wherein each unique peptide variant comprises at least three or more modifications at three or more of the plurality of sites in the region.

20. The method of any of claims 1 - 19, wherein each unique peptide variant comprises at least four or more modifications at four or more of the plurality of sites in the region.

21. The method of any of claims 1 - 20, wherein the at least one modification at one or more of the plurality of sites comprises an amino acid substitution, amino acid insertion, or amino acid deletion.22 The method of claim 21, wherein the amino acid substitution comprises identifying an amino acid residue at a site and substituting the amino acid residue with a second amino acid residue with; a. a similar charge as the amino acid residue; b. a similar size as the amino acid residue;MOFO-360864280 105341812000140 c. a similar shape as the amino acid residue; d. a similar solubility as the amino acid residue; e. a similar linked hydrophobic moiety; and / or f. a similar chirality as the amino acid residue.

23. The method of claim 21, wherein the amino acid substitution comprises, identifying an amino acid residue at a site and substituting the amino acid residue with a second amino acid residue with; a. a different charge as the amino acid residue; b. a different size as the amino acid residue; c. a different shape as the amino acid residue; d. a different solubility as the amino acid residue; e. a different linked hydrophobic moiety; and / or f. a different chirality as the amino acid residue.

24. The method of any of claims 21, wherein the amino acid substitution comprises: a. identifying an amino acid residue susceptible to dehydration at a site and substituting the amino acid residue with a second amino acid residue less susceptible to dehydration; b. identifying an amino acid residue susceptible to deamidation at a site and substituting the amino acid residue with a second amino acid residue less susceptible to deamidation; c. identifying an amino acid residue susceptible to oxidation at a site and substituting the amino acid residue with a second amino acid residue less susceptible to oxidation; and / or d. identifying an amino acid residue contributing to secondary structure at a site and substituting the amino acid residue with a second amino acid residue to strengthen the secondary structure.

25. The method of any of claims 22-24, wherein the second amino acid comprises a non- canonical amino acid.

26. The method of any of claims 22-25, wherein the amino acid insertion or the amino acid substitution comprises a non-canonical amino acid.MOFO-360864280 10634181200014027. The method of claim 25 or 26, wherein the non-canonical amino acid is selected based on a chemical property, a charge, a solubility, a shape of the non-canonical amino acid.

28. The method of claim 27, wherein the non-canonical amino acid is predicted to increase the FPKP of the peptide.

29. The method of any of claims 1 and 4-28, wherein manufacturing the peptide library comprises chemically synthesizing the unique peptide variants.

30. The method of any of claims 1 and 4-29, wherein manufacturing the peptide library comprises synthesizing the unique peptide variants from a plurality of nucleic acid templates.

31. The method of any of claims 1 and 4-30, wherein the peptide library comprises one or more unique peptide variants covalently linked to a covalent handle.

32. The method of any of claims 1 and 4-31, wherein manufacturing the peptide library comprises synthesizing the unique peptide variants on a solid surface array.

33. The method of any of claims 1-32, wherein one or more of the unique peptide variants has an improved FPKP compared to the therapeutic peptide.

34. The method of any of claims 1-33, wherein the FPKP is selected from the group consisting of systemic circulatory half-life, biodistribution, target tissue uptake, bioavailability, protease resistance, metabolism, clearance, and blood-brain barrier penetration.

35. The method of any one of claims 1 and 4-34, further comprising introducing into to the non-human mammalian subject the peptide library.

36. The method of any of claims 1-34, wherein one or more of the unique peptide variants has an improved FPKP compared to the therapeutic peptide.MOFO-360864280 10734181200014037. The method of claim 36, wherein the improved FPKP comprises reduced renal clearance, increased liver clearance, reduced drug metabolism, increased circulatory half-life, increased brain uptake, and / or increased tumor uptake.

38. The method of any of claims 1-37, further comprising analyzing the FPKP of the unique peptide variants in the non-human mammalian subject.

39. The method of claim 38, wherein analyzing the FPKP of the unique peptide variants comprises measuring an IC50 value in vivo.

40. The method of claim 38 or 39, wherein analyzing the FPKP comprises obtaining blood and / or plasma from the non-human mammalian subject after one or more predetermined time intervals, and detecting a subset of the unique peptide variants from the peptide library in the blood and / or plasma.

41. The method of claim 3, wherein detecting the subset of unique peptide variant occurs at after one or more predetermined time intervals.

42. The method of claim 40 or 41, wherein the one or more predetermined time intervals are selected from a group consisting of any time interval between about 0 minutes and about 60 days.

43. The method of any of claims 40-42, wherein the predetermined time intervals comprise about 0 minutes, about 5 minutes, about 20 minutes, and about 60 minutes.

44. The method of any of claims 40-43, wherein at least about 0.01% of the unique peptide variants are detected.

45. The method of any of claims 1-44, wherein the at least one modification at one or more of the plurality of sites comprises a backbone modification.MOFO-360864280 10834181200014046. The method of claim 45 or peptide library, wherein the backbone modification comprises a beta-amino acid, a n-methylated amino acid, an alpha-substituted amino acid, a gamma-substituted amino acid, and / or a peptoid.

47. The method of any of claims 1-46, wherein the C-terminal and / or N-terminal modifications comprise trifluoroacetylation of the N-terminus, C-terminal carboxylic acids, C-terminal esterification, N-terminal propionylation, N-terminal monomethylation, N- terminal dimethylation, and / or N-terminal trimethylation.

48. A kit comprising a peptide library manufactured according to the method of any of claims 1 and 4-46.

49. A peptide library manufactured according to the methods of any of claims 1 and 4-46.

50. A peptide library designed according to the methods of any of claims 2 and 4-49.

51. A mammalian subject comprising the peptide library of claim 49 or 50.MOFO-360864280 109

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