Cell-free synthesis platform for synthetically dimierized, clickable fragment antigen-binding (FAB) fragments

Abstract

Disclosed are components, systems, methods, and kits for synthesis of diamerized, clickable antigen binding fragments, such as Fab fragments, and uses thereof. The components, systems, methods, and kits disclosed herein utilize cell-free protein synthesis (CFPS) systems and methods to produce clickable antigen binding fragments.

Description

CELL-FREE SYNTHESIS PLATFORM FOR SYNTHETICALLY DIMIERIZED, CLICKABLE FRAGMENT ANTIGEN-BINDING (FAB) FRAGMENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 519,430 filed on August 14, 2023. The contents of which are incorporated by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number HDTRA1-20- 1-0004 awarded by the Department of Defense, and grant number 1936789 awarded by the National Science Foundation, and grant numbers AI165279 and AI159586 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (702581.02549.xml; Size: 7,911bytes; and Date of Creation: August 05, 2024) is herein incorporated by reference in its entirety.BACKGROUND

[0004] There is broad use of therapeutic antibodies and antibody-equivalent molecules including bi-specific antibodies, nanobodies, darpins, scfvs, etc. In many cases, the Fc effector function is not needed, is detremental, or adds additional off-target properties beyond Fab binding and only the Fab region of the antibody, which imparts binding specificity, is required. Even so, current methods rely on using full-length antibodies, digesting intact antibodies to generate Fc- independent antibody targeting molecules (including Fab and Fab-like structures), recombinantexpression of scfv’s, recombinant expression of nanobodies, using thiol chemistry for conjugation, and chemical synthesis pathways for conjugation.SUMMARY

[0005] The present disclosure generally relates to components, systems, and methods for cell- free protein synthesis of clickable, synthetically dimerized Fab fragments.

[0006] Disclosed herein are systems, methods, compositions, bacterial cells, lysates, and kits for cell-free protein synthesis of clickable, synthetically dimerized antigen binding fragments, such as antibody Fab fragments.

[0007] An aspect of the disclosure comprises a cell-free protein synthesis system for in vitro production of antigen binding fragments such as, but not limited to fragment antigen-binding (Fab) fragments. In some embodiments, the system includes one or more expression templates, wherein each of the one or more expression templates includes a polynucleotide sequence encoding a polypeptide. In some embodiments, the polypeptide comprises (a) a heavy chain polypeptide that includes a heavy chain constant region 1 (CHI) and a heavy chain variable region (VH); (b) a light chain polypeptide that includes a light chain constant region (CL) and a light chain variable region (VL); or (c) both (a) and (b). In some embodiments, the system includes one or more cell-free protein synthesis reagents for expressing the polypeptides.

[0008] In some embodiments of the system, the encoded polypeptide comprises one or more modifications. By way of example, and not by way of limitation, in some embodiments, the encoded polypeptide comprises a synthetic heterodimerization domain and / or an azido-modified amino acid. In some embodiments, the azido-modified amino acid comprises a phenylalanine (pAzF). In some embodiments, the heavy chain polypeptide and the light chain polypeptide each comprise one member of a heterodimerization domain pair, such as, for example, LZA and LZB. In some embodiments, the encoded polypeptide comprises a purification tag.

[0009] In some embodiments of the system, the one or more cell-free synthesis reagents comprises a lysate prepared from a prokaryote, for example, an Escherichia coli (E. co / i) cell lysate. In some embodiments, the lysate is prepared from genetically modified E. coli, wherein at least one genetic modification facilitates incorporation of an azido-modified amino acid into theencoded polypeptide, optionally wherein the modified E. coli is C321.AA.759 E coli strain, and / or wherein the modification facilitates disulfide bond formation.

[0010] In some embodiments of the system, one or more of the system components is in free- dried format. For example, the lysate, expression templates and / or the one or more cell-free synthesis reagents is in a freeze-dried format. In some embodiments, the one or more cell-free synthesis reagents comprises one or more of ATP, GTP, UTP, CTP, or a tRNA mixture from E. coli.

[0011] Another aspect of the disclosure comprises a genetically modified bacterial cell. In some embodiments, the genetically modified bacterial cell comprises one or more expression templates, wherein each of the one or more expression templates includes a polynucleotide sequence for expression of one or more antigen binding fragments such as, but not limited to Fab fragments. In some embodiments, the antigen binding fragment, e.g., polypeptide Fab fragments include (a) a heavy chain polypeptide that includes a heavy chain constant region 1 (CHI), a heavy chain variable region (VH), and a first member of a heterodimerization domain pair; (b) a light chain polypeptide that includes a light chain constant region (CL), a light chain variable region (VL), and a second member of a heterodimerization domain pair; or (c) both (a) and (b). In some embodiments, the bacterial cell is genetically modified to facilitate incorporation of an azidomodified amino acid into the encoded polypeptide Fab fragment, and optionally, wherein the bacterial cell is genetically modified to facilitate disulfide bond formation. Also disclosed herein is a lysate prepared from a genetically modified bacterial cell as disclosed herein.

[0012] Another aspect of the disclosure comprises compositions. In some embodiments, the composition includes an antigen binding fragment such as, but not limited to a fragment antigenbinding (Fab) fragment comprising at least one modified amino acid, and a heterodimerization domain. In some embodiments, the modified amino acid comprises an azido-modified phenylalanine. In some embodiments, a nanoparticle is linked, via the azido modified phenylalanine to the antigen binding fragment. In some embodiments, the nanoparticle comprises a micell or a liposome, and in some embodiments, the nanoparticle is loaded with a therapeutic compound. Additionally or alternatively, in some embodiments, a therapeutic molecule is linked, via the azido modified phenylalanine, to the antigen binding fragment, e g., Fab fragment. In someembodiments, the therapeutic molecule comprises one of a peptide, small molecule, biologic, a prodrug, or a Fab fragment. In some embodiments, a detectable label is linked, via the azido modified phenylalanine, to the antigen binding fragment, e.g., Fab fragment. In some embodiments, the link via the azido modified phenylalanine is a click chemistry linkage. In some embodiments, the composition is formulated as a therapeutic composition for administration to a subject. In some embodiments, the antigen binding fragment comprises a Fab fragment that targets Siglec-6.

[0013] Another aspect of the disclosure comprises a kit. In some embodiments, the kit includes, in a first container, an antigen binding fragment comprising an azido modified phenylalanine. In some embodiments, the antigen binding fragment comprises a Fab fragment. In some embodiments, the Fab fragment is directed to Siglec-6. In some embodiments, the kit further comprises, in a second container, a partner molecule comprising a therapeutic agent. In some embodiments, the partner molecule is a micell, and in some embodiments, the partner molecule is loaded with a therapeutic agent, such a midostaurin.BRIEF DESCRIPTION OF THE FIGURES

[0014] FIGS. 1A-1D. Design and Assembly of anti-Siglec-6 synthetically dimerized Fab fragments. (A) Fab fragments are designed as separate heavy chain and light chain constructs. In addition to the variable region which recognizes Siglec-6 and the kappa constant light chain region and IgG2a (mouse) / IgGl (human) constant heavy chain region for either mouse or human (chimeric) antibodies, each sequence includes a C-terminal heterodimer fusion protein which facilitates assembly of the chains into Fab fragments. A purification affinity superFlag and strep tag is encoded on the C-terminus of the heavy chain and the recoded stop codon, TAG, that encodes for noncanonical amino acid, pAzF, incorporation is on the C-terminus of the light chain, which allows for click chemistry conjugation onto nanoparticles. The opposing positions of each allows for purification that separates unassembled light chains such that only fully assembled Fab fragments can be conjugated to nanoparticles from the elution. (B) Designed Fab fragments are encoded on two separate plasmids and supplemented into cell-free protein synthesis (CFPS) reactions along with necessary building blocks, substrates, and reagents to create an oxidizing environment for assembly. (C) Incorporation of fluorescently labelled lysine enables visualizationof expressed products on a protein gel. The presence of fully assembled sdFab is observed at ~70 kDa with the unassembled chains visible at lower masses. (D) In vitro binding is detected by an in-solution, bead-based assay, AlphaLISA, where a donor bead and acceptor bead associate with one of the binding partners by affinity tag. If binding occurs, beads are brought in close proximity which leads to an energy transfer that emits a luminescent signal that can be detected by plate reader. Co-titrations of each binding partner (CF-expressed sdFabs on the x-axis) and Siglec-6 on the y-axis, exhibit a typical pattern of binding where optimal concentrations yield highest signal over background (signal when at least one binding partner is absent) with a decrease in signal fanning out as a result of the Hook effect at higher concentrations and insufficient concentration at low concentrations. Both the mouse and chimeric anti-Siglec-6 sdFab demonstrate Siglec-6 binding.

[0015] FIG. 2A-2F. Anti-Siglec-6 sdFab cross-reactivity. (A) CF-expressed mouse anti-Siglec- 6 azido-sdFab demonstrates binding by AlphaLISA assay to Siglec-6, but not to Siglec-8 (B) or Siglec-3 (CD33) (C). CF-expressed chimeric anti-Siglec-6 azido-sdFab demonstrates binding to Siglec-6 (D), but not to Siglec-8 (E) or Siglec-3 (F).

[0016] FIG. 3A-3B. Mast cell-specific depletion in a humanized mast cell malignancy mouse model. (A) In a small pilot study, mice engrafted with a human malignant mast cell line (ROSAKIT D816V) were treated with either free midostaurin or midostaurin-loaded micelles conjugated with anti-Siglec-6 sdFabs. (B) When treated with midostaurin-loaded micelles conjugated with anti-Siglec-6 sdFabs, a higher percentage of human mast cell (CD45+)-specific death (purple) was observed when compared to free drug (gray). Preferential killing was observed for Siglec-6 (+) human mast cells (left) when compared to Siglec-6 (-) mouse mast cells (right) when midostaurin was delivered in anti-Siglec-6 sdFab -conjugated micelles indicating Siglec-6- mediated targeting of mast cells with the therapeutic construct (n = 2, * = p < 0.05, 100K cells counted per sample, Bone Marrow tissue).

[0017] FIG. 4A-4D. Provide nucleic acid sequences of exemplary Fab constructs.DETAILED DESCRIPTION

[0018] Overview

[0019] There is a need for reliably imparting cell specificity to therapeutic constructs. For example, conjugating nanoparticles with a cell-specific targeting domain is a generalizable strategy for many disease applications. This would enable cell-specific delivery of drugs and could enhance drug efficacy and minimize off-target effects. For many nanoparticles, including micelles, full-length antibodies are too large and destabilize the nanoparticle.

[0020] The present disclosure generally relates to components, systems, and methods for cell- free protein synthesis of clickable, synthetically dimerized antibody Fab fragments. These fragments are typically about ~70 kDa as opposed to the -150 kDa weight of full-length antibodies, and they comprise the region of the antibody that imparts antigen specificity. Fab fragments are endowed with several favorable, cell-free protein synthesis-enabled features. By way of example but not by way of limitation, Fab fragments can be designed with synthetic heterodimerization domains to aid in assembly, and with an amber suppression codon that allows for noncanonical amino acid incorporation when produced in a recoded E. coll strain. By incorporating a noncanonical azido-modified amino acid (pAzF) into Fab fragments, Fab fragments can be conjugated to partner compounds or molecules by click chemistry. This approach enables construction of azido-sdFabs that can be conjugated onto nanoparticles, for example, to impart cell specific targeting, among other applications. With this approach Fab fragments can be equipped with favorable properties that are difficult to access without cell-free technology.

[0021] As proof-of-concept the inventors show that antigen-binding molecules (e.g., Fabs) can be conjugated to micelles and mediate cell-specific delivery of a small molecule drug. This technology is widely applicable and can expand capabilities of nanoparticle technology toward the application of many disease treatments.

[0022] Definitions and Terminology

[0023] The disclosed components, systems, and methods for generating dimerized clickable Fab Fragments may be further described using definitions and terminology as follows. Thedefinitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0024] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a polynucleotide” or an “allergen protein” should be interpreted to mean “one or more polynucleotides” and “one or more allergen proteins,” respectively, unless the context clearly dictates otherwise. As used herein, the term “plurality” means “two or more.”

[0025] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0026] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0027] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0028] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B andC” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0029] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0030] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0031] The term “disease” refers to any deviation from the normal health of a mammal and includes a state when disease symptoms are present, as well as conditions in which a deviation (e.g., dysbiosis, infection, gene mutation, genetic defect, etc.) has occurred, but symptoms are not yet manifested. The terms “disease”, “condition”, and “disorder” are used interchangeably unless otherwise indicated.

[0032] As used herein, a “symptom” of a disease includes and clinical or laboratory manifestation associated with the disease and is not limited to what a subject can feel or observe.

[0033] As used herein “Fab” or “Fab fragment” are used interchangeably and refer to the fragment antigen-binding region (Fab region), that portion of an antibody or antibody equivalentwhich binds antigen. At minimum, a Fab fragment comprises some portion of the antibody variable regions, plus or minus flanking amino acids at either end. Fab fragments are monovalent, containing only a single antigen binding site, but may have >1 specificity. The molecular weight of a Fab fragment, comprising both a heavy chain component, and a light chain component, is about 50 kDa. As disclosed herein, Fab fragments and modified variants thereof may be synthesized by cell-free methods.

[0034] As used herein, a “modifed Fab fragment” refers to a Fab comprising one or more of: one or more non-natural / non-canonical amino acids, a synthetic heterodimerization region, e.g., C-terminal heterodimer fusion proteins to promote more efficient assembly of the heavy and light chains, yielding synthetically dimerized Fabs (sdFabs)34, affinity tag(s), one or more labels. In some embodiments, sdFabs comprise a C-terminal azide group on the light chain that allows for click conjugation of azido-sdFabs to other structures, components, or compounds. In some embodiments, the other structure, component, or compound comprises a nanoparticle. In some embodiments, a purification tag is encoded on the heavy chain.

[0035] Synthesis and use of a variety of antibody fragments, or antigen binding proteins is also contemplated herein. Different forms of antibody fragments, and antigen binding proteins are well known in the art, and include without limitation, Fabs, sdFabs, scFvs, diabodies, nanobodies scFv- Fcs, scFv-CHs, scFabs, scFv-zippers, affibodies, DARPTNS, single-chain antibodies (VH domain or nanobody), and minibinders, see e.g., Crook, et al., Trends in Biochem. Sci. 45:4:pp 332-346; (Apr 01, 2020); Cao etal., Science 370:6515:pp 426-431 (Oct 23 2020). Any of the above antibody fragments may be synthesized and modified (e.g., to include synthetic heterdimerization regions, “clickable” moieties, affinity tags, labels, etc.) in cell-free reactions, as disclosed herein.

[0036] Antibody sequences, especially VH and VL domain sequences, are readly available and / or can be obtained by methods well known in the art. Antibody and antibody equivalent sequences can be derived from any animal (human, porcine, canine, feline, equine, bovine, camelid, avian, etc.), may be a combination of animal antibody sequences, or may be wholly synthetic sequences (e.g. , developed by artificial intelligence or other in silico methods). Antibody sequences may be derived from any class or subclass of antibodies, as well as any form of antigenbinding protein.

[0037] As used herein “click chemistry” refers to a set of highly efficient, biorthogonal chemical reactions, well-known in the art, between an azide and alkyne that conjugates two species together.28’33

[0038] Polynucleotides and Synthesis Methods

[0039] The terms “nucleic acid” and “oligonucleotide,” as used herein, refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D- ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single- stranded RNA. For use in the present methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs.

[0040] Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68: 109-151; the di ethylphosphorami dite method of Beaucage et al., 1981 , Tetrahedron Letters 22:1859-1862; and the solid support method of U.S. Pat. No. 4,458,066, each incorporated herein by reference. A review of synthesis methods of conjugates of oligonucleotides and modified nucleotides is provided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187, incorporated herein by reference.

[0041] The terms “target,” “target sequence”, “target region”, and “target nucleic acid,” as used herein, are synonymous and refer to a region or sequence of a nucleic acid which is to be amplified, sequenced, or detected.

[0042] As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others.“RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.

[0043] The term “promoter” refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA template that includes the cis-acting DNA sequence.

[0044] As used herein, the term “sequence defined biopolymer” refers to a biopolymer having a specific primary sequence. A sequence defined biopolymer can be equivalent to a genetically encoded defined biopolymer in cases where a gene encodes the biopolymer having a specific primary sequence.

[0045] The polynucleotide sequences contemplated herein may be present in expression vectors. For example, the vectors may comprise: a polynucleotide encoding an ORF of a protein (e.g., a polynucleotide sequence encoding a Fab heavy chain sequence, a Fab light chain sequence, or both). The polynucleotide present in the vector may be operably linked to a prokaryotic or eukaryotic promoter. “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame. Vectors contemplated herein may comprise a heterologous promoter (e.g., a eukaryotic or prokaryotic promoter) operably linked to a polynucleotide that encodes a protein. A “heterologous promoter” refers to a promoter that is not the native or endogenous promoter for the protein or RNA that is being expressed. Vectors as disclosed herein may include plasmid vectors.

[0046] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0047] As used herein, “expression template” refers to a nucleic acid that serves as substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e g., a polypeptide or protein). Expression templates include nucleic acids composed of DNA or RNA. Suitable sources of DNA for use a nucleic acid for an expression template include genomic DNA, cDNA and RNA that can be converted into cDNA. Genomic DNA, cDNA and RNA can be from any biological source, such as a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecal sample, a urine sample, a scraping, among others. The genomic DNA, cDNA and RNA can be from host cell or virus origins and from any species, including extant and extinct organisms. As used herein, “expression template” and “transcription template” have the same meaning and are used interchangeably.

[0048] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably. However, the disclosed methods and compositions are intended to include such other forms of expression vectors, such as viral vectors which serve equivalent functions.

[0049] In certain exemplary embodiments, the recombinant expression vectors comprise a nucleic acid sequence in a form suitable for expression of the nucleic acid sequence in one or more of the methods described herein, which means that the recombinant expression vectors include one or more regulatory sequences which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that thenucleotide sequence encoding one or more RNAs or polypeptides and / or proteins described herein is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription and / or translation system). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0050] Oligonucleotides and polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl -2 -thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methyl cytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5- oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6- diaminopurine and the like. Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and / or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone.

[0051] The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

[0052] Regarding polynucleotide sequences, the terms “percent identity” and “% identity” refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

[0053] Regarding polynucleotide sequences, percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0054] Regarding polynucleotide sequences, “variant,” “mutant,” or “derivative” may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information’s website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250). Such apair of nucleic acids may show, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.

[0055] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code where multiple codons may encode for a single amino acid. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. For example, polynucleotide sequences as contemplated herein may encode a protein and may be codon-optimized for expression in a particular host. In the art, codon usage frequency tables have been prepared for a number of host organisms including humans, mouse, rat, pig, E. coli, plants, and other host cells.

[0056] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0057] The nucleic acids disclosed herein may be “substantially isolated or purified.” The term “substantially isolated or purified” refers to a nucleic acid that is removed from its natural environment, and is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which it is naturally associated.

[0058] Peptides, Polypeptides, Proteins, and Synthesis Methods

[0059] As used herein, the terms “peptide,” “polypeptide,” and “protein,” refer to molecules comprising a chain a polymer of amino acid residues joined by amide linkages. The term “aminoacid residue,” includes but is not limited to amino acid residues contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include nonstandard or unnatural amino acids. The term “amino acid residue” may include alpha-, beta-, gamma-, and delta-amino acids.

[0060] In some embodiments, the term “amino acid residue” may include nonstandard, non- canonical, or unnatural amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, 0-alanine, 0-Amino- propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3 -Hydroxyproline, 4- Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2- Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3- Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4- Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3 -Diaminopropionic acid, Ornithine, and N-Ethylglycine. The term “amino acid residue” may include L isomers or D isomers of any of the aforementioned amino acids.

[0061] Other examples of nonstandard, non-canonical, or unnatural amino acids include, but are not limited, to a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, an O-methyl-L-tyrosine, a p-propargyloxyphenylalanine, a p-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a 3- methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L -tyrosine, a tri-O-acetyl-GlcNAcp - serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L- phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an unnatural analogue of a methionine amino acid; an unnatural analogue of a leucine amino acid; an unnatural analogue of a isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide,hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, 17ufal7hor, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar- containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid; an a, a disubstituted amino acid; a P-amino acid; a y-amino acid, a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan. In some emboiments, the non-standard amino acids comprises an azido modification, e.g., azido-modified phenylalanine (pAzF), for click chemistry.

[0062] As used herein, a “peptide” is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). In some embodiments, apeptide as contemplated herein may include no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. A polypeptide, also referred to as a protein, is typically of length > 100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A polypeptide, as contemplated herein, may comprise, but is not limited to, 100, 101, 102, 103, 104, 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino acid residues.

[0063] A peptide or polypeptide as contemplated herein may be further modified to include non-amino acid moieties. Modifications may include but are not limited to acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein), glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine), azido modifications (e.g., azido-modified phenylalanine (pAzF), for click chemistry).

[0064] Modified amino acid sequences that are disclosed herein may include a deletion in one or more amino acids. As utilized herein, a “deletion” means the removal of one or more amino acids relative to the native amino acid sequence. The modified amino acid sequences that are disclosed herein may include an insertion of one or more amino acids. As utilized herein, an “insertion” means the addition of one or more amino acids to a native amino acid sequence. The modified amino acid sequences that are disclosed herein may include a substitution of one or more amino acids. As utilized herein, a “substitution” means replacement of an amino acid of a native amino acid sequence with an amino acid that is not native to the amino acid sequence. For example, the modified amino sequences disclosed herein may include one or more deletions, insertions, and / or substitutions in order modified the native amino acid sequence of a target protein to include one or more heterologous amino acid motifs that are glycosylated by an N- glycosyltransferase.

[0065] Regarding proteins, a “deletion” refers to a change in the amino acid sequence that results in the absence of one or more amino acid residues. A deletion may remove at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, or more amino acids residues. A deletion may include an internal deletion and / or a terminal deletion (e.g., an N-terminal truncation, a C-terminal truncation or bothof a reference polypeptide). A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a deletion relative to the reference polypeptide sequence.

[0066] Regarding proteins, “fragment” is a portion of an amino acid sequence which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous amino acid residues of a reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues ofa reference polypeptide. Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full-length polypeptide. A fragment may include an N-terminal truncation, a C-terminal truncation, or both truncations relative to the full-length protein. A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a fragment of the reference polypeptide sequence.

[0067] Regarding proteins, the words “insertion” and “addition” refer to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more amino acid residues. A “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include an insertion or addition relative to the reference polypeptide sequence. A variant of a protein may have N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions, and internal insertions. The variant, mutant, or derivative may include non-canonical / non-natural amino acids (e.g. azido modifications for click chemistry), labels, affinity tags, altered or added sequences (e.g., heterodimerization sequences), and the like.

[0068] Regarding proteins, the phrases “percent identity” and “% identity,” refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.Percent identity for amino acid sequences may be determined as understood in the art. (See, e ., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.

[0069] Regarding proteins, percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0070] Regarding proteins, the amino acid sequences of variants, mutants, or derivatives as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, or derivative protein may include conservative amino acid substitutions relative to a reference molecule. “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. The following table provides a list of exemplary conservative amino acid substitutions which are contemplated herein:OriginalResidue Conservative SubstitutionAla Gly, Ser Arg His, Lys Asn Asp, Gin, His Asp Asn, Glu Cys Ala, Ser Gin Asn, Glu, His Glu Asp, Gin, His Gly Ala His Asn, Arg, Gin, Glu He Leu, Vai Leu He, Vai Lys Arg, Gin, Glu Met Leu, He Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Vai Trp Phe, Tyr Tyr His, Phe, TrpVai He, Leu, Thr

[0071] Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain. Non-conservative amino acids typically disrupt (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain.

[0072] The disclosed proteins, mutants, variants, or described herein may have one or more functional or biological activities exhibited by a reference polypeptide (e.g., one or more functional or biological activities exhibited by wild-type protein).

[0073] The disclosed proteins may be substantially isolated or purified. The term “substantially isolated or purified” refers to proteins that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

[0074] Cell-Free Protein Synthesis (CFPS)

[0075] The components, systems, and methods disclosed herein may be applied to cell-free protein synthesis methods as known in the art. See, for example, U.S. Patent Nos. 5,478,730; 5,556,769; 5,665,563; 6,168,931; 6,548,276; 6,869,774; 6,994,986; 7,118,883; 7,186,525; 7,189,528; 7,235,382; 7,338,789; 7,387,884; 7,399,610; 7,776,535; 7,817,794; 8,703,471; 8,298,759; 8,715,958; 8,734,856; 8,999,668; and 9,005,920. See also U.S. Published Application Nos. 2018 / 0016614, 2018 / 0016612, 2016 / 0060301, 2015-0259757, 2014 / 0349353, 2014- 0295492, 2014-0255987, 2014-0045267, 2012-0171720, 2008-0138857, 2007-0154983, 2005- 0054044, and 2004-0209321. See also U.S Published Application Nos. 2005-0170452; 2006- 0211085; 2006-0234345; 2006-0252672; 2006-0257399; 2006-0286637; 2007-0026485; 2007-0178551. See also Published PCT International Application Nos. 2003 / 056914; 2004 / 013151; 2004 / 035605; 2006 / 102652; 2006 / 119987; and 2007 / 120932. See also Jewett, M.C., Hong, S.H., Kwon, Y.C., Martin, R.W., and Des Soye, B.J. 2014, “Methods for improved in vitro protein synthesis with proteins containing non standard amino acids,” U.S. Patent Application Serial No.: 62 / 044,221; Jewett, M.C., Hodgman, C.E., and Gan, R. 2013, “Methods for yeast cell-free protein synthesis,” U.S. Patent No.: 9528137; Jewett, M.C., J. A. Schoborg, and C.E. Hodgman. 2014, “Substrate Replenishment and Byproduct Removal Improve Yeast Cell-Free Protein Synthesis,” U.S. Patent No.: 9951392; and Jewett, M.C., Anderson, M.J., Stark, J.C., Hodgman, C.E. 2015, “Methods for activating natural energy metaboli sm for improved yeast cell-free protein synthesis,” U.S. Patent Application Serial No.: 62 / 098,578. See also Guarino, C., & DeLisa, M. P. (2012). “A prokaryote-based cell-free translation system that efficiently synthesizes glycoproteins” Glycobiology, 22(5), 596-601. The contents of all of these references are incorporated in the present application by reference in their entireties.

[0076] In some embodiments, a CFPS reaction mixture or cell-free protein synthesis (CFPS) reagents may contain one or more of a crude lysate or partially purified cell extract, an RNA translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the RNA translation template. In some embodiments, the CFPS reaction mixture can include exogenous RNA translation template. In other aspects, the CFPS reaction mixture can include a DNA expression template encoding an open reading frame operably linked to a promoter element for a DNA-dependent RNA polymerase. In some aspects, the CFPS reaction mixture can alsoinclude a DNA-dependent RNA polymerase to direct transcription of an RNA translation template encoding the open reading frame. In some aspects, additional NTP’s and divalent cation cofactor can be included in the CFPS reaction mixture. A reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of ordinary skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of ordinary skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components of the invention.

[0077] The disclosed cell-free protein synthesis systems may utilize components that are crude and / or that are at least partially isolated and / or purified. As used herein, the term “crude” may mean components obtained by disrupting and lysing cells and, at best, minimally purifying the crude components from the disrupted and lysed cells, for example by centrifuging the disrupted and lysed cells and collecting the crude components from the supernatant and / or pellet after centrifugation. The term “isolated or purified” refers to components that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

[0078] As used herein, “translation template” for a polypeptide refers to an RNA product of transcription from an expression template that can be used by ribosomes to synthesize polypeptides or proteins.

[0079] In some embodiments, the lysate comprises a recoded lysate or extract, allowing for the incorporation of non-standard / non-canonical amino acids into a polypetide20.

[0080] The term “reaction mixture,” as used herein, refers to a solution containing reagents necessary to carry out a given reaction. A reaction mixture is referred to as complete if it contains all reagents necessary to perform the reaction. Components for a reaction mixture may be storedseparately in separate container, each containing one or more of the total components. Components may be packaged separately for commercialization and useful commercial kits may contain one or more of the reaction components for a reaction mixture. In some embodiments, one or more of the cell-free protein synthesis reagents can be present in the reaction mixture.

[0081] A reaction mixture may include an expression template, a translation template, or both an expression template and a translation template. The expression template (e.g., a DNA template) serves as a substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein). The translation template is an RNA product that can be used by ribosomes to synthesize the sequence defined biopolymer. In certain embodiments the platform comprises both the expression template and the translation template. In certain specific embodiments, the reaction mixture may comprise a coupled transcription / translation (“Tx / Tl”) system where synthesis of translation template and a sequence defined biopolymer from the same cellular extract.

[0082] The reaction mixture may comprise one or more polymerases capable of generating a translation template from an expression template. The polymerase may be supplied exogenously or may be supplied from the organism used to prepare the extract. In certain specific embodiments, the polymerase is expressed from a plasmid present in the organism used to prepare the extract and / or an integration site in the genome of the organism used to prepare the extract.

[0083] Altering the physicochemical environment of the CFPS reaction to better mimic the cytoplasm can improve protein synthesis activity. The following parameters can be considered alone or in combination with one or more other components to improve robust CFPS reaction platforms based upon crude cellular extracts (for examples, SI 2, S30 and S60 extracts).

[0084] The temperature may be any temperature suitable for CFPS. Temperature may be in the general range from about 10° C. to about 40° C., including intermediate specific ranges within this general range, include from about 15° C. to about 35° C., from about 15° C. to about 30° C., from about 15° C. to about 25° C. In certain aspects, the reaction temperature can be about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C.

[0085] The reaction mixture may include any organic anion suitable for CFPS. In certain aspects, the organic anions can be glutamate, acetate, among others. In certain aspects, the concentration for the organic anions is independently in the general range from about 0 mM to about 200 mM, including intermediate specific values within this general range, such as about 0 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM and about 200 mM, among others.

[0086] The reaction mixture may include any halide anion suitable for CFPS. In certain aspects the halide anion can be chloride, bromide, iodide, among others. A preferred halide anion is chloride. Generally, the concentration of halide anions, if present in the reaction, is within the general range from about 0 mM to about 200 mM, including intermediate specific values within this general range, such as those disclosed for organic anions generally herein.

[0087] The reaction mixture may include any organic cation suitable for CFPS. In certain aspects, the organic cation can be a polyamine, such as spermidine or putrescine, among others. Preferably polyamines are present in the CFPS reaction. In certain aspects, the concentration of organic cations in the reaction can be in the general about 0 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about 2 mM. In certain aspects, more than one organic cation can be present.

[0088] The reaction mixture may include any inorganic cation suitable for CFPS. For example, suitable inorganic cations can include monovalent cations, such as sodium, potassium, lithium, among others; and divalent cations, such as magnesium, calcium, manganese, among others. In certain aspects, the inorganic cation is magnesium. In such aspects, the magnesium concentration can be within the general range from about 1 mM to about 50 mM, including intermediate specific values within this general range, such as about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, among others. In preferred aspects, the concentration of inorganic cations can be within the specific range from about 4 mM to about 9 mM and more preferably, within the range from about 5 mM to about 7 mM.

[0089] The reaction mixture may include endogenous NTPs (i.e., NTPs that are present in the cell extract) and or exogenous NTPs (i.e., NTPs that are added to the reaction mixture). In certain aspects, the reaction uses ATP, GTP, CTP, and UTP. In certain aspects, the concentration of individual NTPs is within the range from about 0.1 mM to about 2 mM.

[0090] The reaction mixture may include any alcohol suitable for CFPS. In certain aspects, the alcohol may be a polyol, and more specifically glycerol. In certain aspects the alcohol is between the general range from about 0% (v / v) to about 25% (v / v), including specific intermediate values of about 5% (v / v), about 10% (v / v) and about 15% (v / v), and about 20% (v / v), among others.

[0091] In certain exemplary embodiments, one or more of the methods described herein are performed in a vessel, e.g., a single, vessel. The term “vessel,” as used herein, refers to any container suitable for holding on or more of the reactants (e.g., for use in one or more transcription, translation, and / or glycosylation steps) described herein. Examples of vessels include, but are not limited to, a microtitre plate, a test tube, a microfuge tube, a beaker, a flask, a multi -well plate, a cuvette, a flow system, a microfiber, a microscope slide and the like.

[0092] Producing Synthetic Dimerized. Clickable Fab Fragments

[0093] The components, systems, and methods disclosed herein may be applied to recombinant cell systems and cell-free protein synthesis methods in order to prepare the antigen binding fragments, such as Fab fragments. In certain embodiments, the CFPS synthesis systems can include one or more expression templates and one or more cell-free protein synthesis reagents and / or a CFPS reaction mixtures.

[0094] In some embodiments, the expression templates can include a protein-encoding sequence for one or more antigen binding fragments, such as Fab fragments. Amino acid sequences of interest (e.g. variable domains, hypervariable domains, the Fab sequence, etc. that bind to a target antigen) and their corresponding nucleic acid sequences can be determined or obtained by methods well known in the art. While the present application exemplifies the mast cell surface protein Siglec-6 as a target, the skilled artisan will appreciate that the present technology is not so limited, and that other antigens, epitopes, or molecules of interest may be targeted by the methods and compositions disclosed herein. As used herein with respect to antigenbinding fragments, the term “target” refers to the molecule bound by the antigen binding fragment. Target molecules can include without limiatation, in addition to Siglec-6, Siglec-4, Siglec-7, Siglec-8, KIT, CD33, tyrosine kinases, CXCL12, and other mast cell, and non-mast cell cell surface structures, including without limitation surface proteins, polypeptides, carbohydrates, receptors, lipids, glycolipids, and nucleic acids.

[0095] The antigen binding fragments can be synthesized in the cell-free systems of the present disclosure comprising heterodimerization sequences, to promote more efficient assembly of the heavy and light chains, to yield synthetically dimerized Fabs, for example. Exemplary heterodimerization sequences include the leucine zipper pairs ACID-pl (LZA) / BASEpl(LZB); c- Jun / c-Fos. Incorporation of such sequences accelerates the associatiation of the heavy chain and light chain to form Fab. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed separately and then assembled. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed in one pot for simultaneous expression and assembly. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed from the same plasmid vector in a single pot.

[0096] In addition to providing an economical, efficient, and cell-free method of generating antigen binding fragments and compositions comprising these fragments, the present technology also provides the means to generate antigen binding fragments that can be specifically linked to other components, such as carrier molecules, to transport a cargo to the target. In some embodiments, the cells employed to generate the cell-free mixtures are recoded to allow for incorporation of non-canonical amino acids into a polypeptide of interest. By way of example, an azido-modified amino acid such as phenylalanine (pAzF) can be incorporated into the polypeptide comprising the antigen binding fragment. Such a modification allows the antigen binding fragment to be linked, via click chemistry, to a partner molecule comprising an alkyne. Other amino acids modification that would function in click chemistry are well known in the art and include, without limitation copper-catalyzed click chemistry, strain-promoted click chemistry, and thiol chemistry.

[0097] In some embodiments, the partner molecule comprises a nanoparticle. By way of example but not by way of limitation, such nanoparitcles may include carbon nanotubes, dendrimers, ceramic nanoparticles, metallic nanoparticles, lipid nanoparticles, polymericnanoparticles, polymers, various forms of biological materials, and combinations thereof. In some embodiments, the nanoparticle comprises or is loaded with one or more therapeutic agents (e.g., a nucleic acid, polypeptide, small molecule, etc.) and by linking to the clickable Fab or antigen binding fragment produced by the methods disclosed herein, can be transported to the target molecule. Such targeted transport can help alleviate off-target and side effects, and even lessen the overall dosage. In some embodiments, the partner molecule comprises a small molecule drug, a protein therapeutic and / or biologic, or a prodrug. In some embodiments, the partner molecule comprises a detectable label. In some embodiments, the partner molecule comprises another antigen-binding fragment directed to the same or different target. In some embodiments, the partner molecue comprises a Fab fragment.

[0098] In various embodiments, the expression templates encoding the antigen binding molecule can also include and / or encode additional elements operably linked to the antigen binding molecule. By way of example, such additional elements include but are not limited to purification tags, immune-modulating elements, sites for post-translational modifications, elements that aid expression, elements that aid folding and assembly. In certain embodiments, the expression templates can encode a purification tag, e.g., for purifying the antigen binding fragment. In some emobdiments, one or more additional elements are separated by one or more linker sequences.

[0099] In various embodiments, the expression templates can be present in a vector. In one embodiment, the expression templates can be present in a plasmid vector, e.g., pJLl, pMAFlO, ptrc99, pET, pCAYC.

[0100] One or more of the components of the disclosed systems may be in a preserved form. In some embodiments, one or more components of the disclosed systems are freeze-dried.

[0101] Also disclosed herein are antigen binding fragment proteins or peptides. In various embodiments, the CFPS system can include one or more CFPS reagents or reaction mixtures for expressing the one more antigen binding fragment proteins or peptides. Suitable CFPS reaction mixtures for the disclosed methods may include prokaryotic CFPS reaction mixtures. In some embodiments, suitable CFPS reaction mixtures may include prokaryotic CFPS reaction mixtures comprising a lysate prepared from Escherichia coli. In some embodiments, the E. coll or other cell can be modified for tailoring and / or optimizing cell-free protein synthesis. For instance, insome embodiments, the E. coli or other cell can include a genetic modification to facilitate disulfide bond formation in expressed proteins.

[0102] Additionally or alternatively, in some embodiments, the cells are modified to be recoded, e.g., to utilize an amber codon to incorporate a non-canonical amino acid. A non-limiting example of such a modified cell includes C321. AA.759 E. coli strain.20, 27

[0103] In some embodiments, the cells may be genetically modified to express a protein of interest. In various embodiments, the modified cells can include an expression template for the one or more antigen binding proteins discussed herein. In some embodiments, the modified bacterial cells can produce one or more of the proteins described herein in vivo.

[0104] In some embodiments, a lysate prepared from the modified bacterial cell can be utilized to produce the one or more antigen binding proteins.

[0105] In various embodiments, the CFPS system may include eukaryotic CFPS reaction mixtures. In some embodiments, suitable CFPS reaction mixtures may include eukaryotic CFPS reaction mixtures comprising a lysate prepared from Saccharomyces cerevisiae or other eukaryote. In certain embodiments, the S. cerevisiae or other cell can be modified for tailoring and / or optimizing cell-free protein synthesis as described above for prokaryotic systems.

[0106] In various embodiments, the proteins are expressed by combining the one or more expression vectors with one or more cell-free protein synthesis reagents and / or a CFPS reaction mixture, as discussed above and described in the Example below (e.g., to include by way of example and without limitation: salts, building blocks (e.g., nucleotides, amino acids), buffers, oxidizing reagents, and chaperone proteins, (e.g., Dsbc and FkpA), to facilitate disulfide bond formation, non-canonical amino acid, (e.g., pAzF, along with its tRNA synthetase and / or the charged tRNA).

[0107] In some embodiments, once the protein is expressed, the protein may be purified and / or isolated from the CFPS system using any convenient purification and / or isolation techniques.

[0108] Methods For Assessment of Antigen Binding Fragments

[0109] In various embodiments, the systems and methods described herein can include methods for assessing the function of the antigen binding fragments produced according to the systems and methods described herein. In certain embodiments, the methods can include assessing whether or not the proteins bind to the target antigen from a sample of a subject. In various embodiments, such binding can be assessed using any convenient technique, include but not limited to the use of an AlphaLISA, an ELISA, point-of-use assay, surface plasmon resonance, or biolayer interferometry.

[0110] Methods of Use

[0111] As discussed above, in various embodiments, the systems and methods described herein can be used to provide targeted delivery of a therapeutic composition. Additionally or alternatively, in one or more embodiments, a sample from the subject can be exposed to the antigen-binding proteins using a lateral flow assay, ELISA, point-of-care assay, or other assay, e.g., to detect target molecules from subject sample binding to the antigen binding proteins.

[0112] In various embodiments, the antigen binding proteins produced according to the methods and systems described herein can be used therapeutically. For example, in certain aspects, the one or more antigen binding proteins can be administered to a subject, e.g., subcutaneously, sublingually, by injection, orally, alone or linked to a partner molecule comprising or containing a therapeutic payload. By way of example, therapeutic uses may include cell-specific targeting, cell-specific or tissue-specific drug delivery, dual action therapeutics, conjugation of proteins for bi-specific therapeutics, conjugation of azido-sdFabs to nanoparticles, and conjugation of azido- sdFabs to small molecule drugs.

[0113] The terms point-of-care (POC) testing, point-of-service (POS) testing, point-of-use (POU) testing, and point-of-incidence (POI) testing are used interchangeably in this disclosure unless otherwise indicated. The term point-of-use testing, also known as on-site or near-subject testing, refers to diagnostic testing performed at or near a subject where a sample is obtained and / or at the site where service, care, or treatment is provided.

[0114] In various embodiments, the antigen binding proteins can be prepared at point-of use, e.g., using one or more of the CFPS methods and / or systems described herein, and then a bindingassay from a patient sample can be performed, e.g., at the point-of-care or elsewhere, using an ELISA, AlphaLISA, surface plasmon resonance (SPR), biolayer interferometry (BLI), or flow cytometry assay.

[0115] In various embodiments, one or more antigen binding proteins, e.g., made using one or more of the CFPS methods and / or systems described herein, can be formulated into an ELISA, or lateral flow assay to detect a target antigen for a clinical, at home, or POC test.

[0116] In one or more embodiments, the antigen binding proteins can be made in a centralized facility using CFPS, in a clinical laboratory using CFPS, or at the point-of-care using CFPS.

[0117] Kits comprising one or more components for expressing the antigen binding fragments, or kits comprising the one or more antigen binding fragments are also disclosed herein. In some embodiments, a kit comprises, in separate containers, clickable antigen binding fragments, and a clickable partner molecule. In some embodiments, the partner molecule comprises a therapeutic agent. In some embodiments, the kit comprises instructions for combining the antigen binding fragments and the partner molecule.

[0118] Applications

[0119] Exemplary, non-limiting applications of the disclosed technology are provided below.

[0120] Cell-specific targeting, cell-specific drug delivery, dual action therapeutics, conjugation of proteins for bi-specific therapeutics, lectin targeting, Fab production, Fab conjugation, synthetically dimerized Fab production, synthetically dimerized Fab conjugation, conjugation of azido-sdFabs to nanoparticles, conjugation of azido-sdFabs to small molecule drugs.

[0121] Advantages

[0122] Exemplary, non-limiting advantages of the disclosed technology are provided below.

[0123] This method provides a widely generalizable approach for generating Fab fragments with favorable features and properties. A modification of Fab fragments enabled by this technology is azido-modification, which makes the Fab fragment amenable to click chemistry conjugation. Existing approaches are limited to making full-length antibodies, digesting full-lengthantibodies to generate Fabs, or making recombinant Fabs with a limited range of conjugation chemistries. However, by generating a clickable Fab fragment, the inventors enable a widely applicable approach for making functionalizable Fab fragments for a variety of applications. Clickable Fab fragments also enable a widely applicable approach for generating nanoparticles with a desired target specificity based on Fab sequence chosen and conjugated to the nanoparticle surface.

[0124] This approach for conjugation is highly efficient and highly orthogonal. A cell free system allows an open reaction environment, allowing for addition of non-canonical amino acids without concern for effects on living cells granting wide access to possible chemistries to endow on the Fab fragment. The open reaction environment also gives a tunable system for Fab assembly condition optimizations, such as to create an oxidizing environment, and control over DNA design enables the ability to endow desired properties onto Fab fragments. This platform also grants easy scale up for manufacturing.

[0125] Clickable Fab fragments can endow nanoparticles with desired specificities for improved homing and delivery of nanoparticle formulated therapeutics.

[0126] This approach allows for the efficient and accurate production of a variety of Fab proteins with features that can only be imparted by cell -free protein synthesis. The present technology allows for the production of therapeutic Fab proteins with features that were previously inaccessible, and construction of clickable Fab fragments grants a novel approach for endowing nanoparticle-based therapeutics with desired specificities. This approach provides nanoparticlebased drug formulations with an efficiency and efficacy difficult to achieve without this technology.

[0127] The disclosed technology enables manufacturing of antibody Fab fragments with direct control over design features that are inaccessible without cell-free technology. For example, inclusion of synthetic dimerization domains aids assembly and incorporation of a clickable noncanonical amino acid provides a handle for conjugation that is highly efficient. This tool can impart specificity to nanoparticle-based drugs that would otherwise not be possible. These tools can also be used to functionalize Fab fragments with a small molecule drug, a label, or protein for localization of the functional group. For proof of concept, the inventors demonstrate that cell-freeexpressed, synthetically dimerized, azido-modified, anti-Siglec-6 Fab fragments can be conjugated to micelles containing midostaurin and mediate cell specific delivery.

[0128] There is broad use of therapeutic antibodies and antibody-equivalent molecules including bi-specific antibodies, nanobodies, darpins, scfvs, etc. In many cases, the Fc effector function is not needed and only the Fab region of the antibody, which imparts binding specificity, is required. Current methods rely on digesting intact antibodies to generate Fc-independent antibody targeting molecules (including Fab and Fab-like structures). The cell-free protein synthesis-derived clickable Fab fragments disclosed herein can be used to bind virtually any target where the sequence of a key antibody region is known. The clickable property of these Fab and Fab-like fragments is especially useful for conjugation onto nanoparticles to impart cell specific targeting, but could also be used to functionalize the Fab and Fab-like fragments in a variety of ways.

[0129] This method can be applied in a plug-and-play manner to any affinity type molecule that can be designed from a known, determined, or humanized antibody sequence for any desired target. As a proof-of-concept, disclosed herein the inventors conjugated nanoparticles with anti- Siglec-6 azido-sdFabs. The generated data shows successful mast cell-specific honing mediated by the anti-Siglec-6 azido-sdFab-conjugated nanoparticles.

[0130] Miscellaneous

[0131] The steps of the methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The steps may be repeated or reiterated any number of times to achieve a desired goal unless otherwise indicated herein or otherwise clearly contradicted by context.

[0132] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0133] Additional Embodiments

[0134] Non-limiting exemplary embodiments are presented below:

[0135] 1 De novo synthesis of synthetically dimerized azido-modified antibody Fab fragments by cell-free protein synthesis; Fab fragments, and / or compositions comprising the Fab fragments. In some embodiments, the methods and compositions comprise:

[0136] 1.1 Fab fragments defined as a minimum structure that includes hypervariable regions of antibodies plus or minus flanking amino acids at either end.

[0137] 1.2 DNA design of Fab fragments as described above with any combination of the following features:

[0138] 1.2.1 Synthetic heterodimerization domains;

[0139] 1.2.2 Clickable azido modifications by noncanonical amino acid incorporation;

[0140] 1.2.3 Affinity tags for purification and analysis;

[0141] 1.3 Expression of encoded heavy and light chains with supplemented pAzF in genetically modified E. coli extracts recoded for noncanonical amino acid incorporation.

[0142] 2 The method of embodiment 1 where any hypervariable region sequence of an antibody or species antibody-equivalent with any specificity is known or determined and used in the genetic construct.

[0143] 3. The method of embodiment 1 where sequences from any species are used or mixed.

[0144] 4 The method of embodiment 1 where human or animal antibody sequences are used in the DNA design.

[0145] 5. The method of embodiment 1 where a kappa or lambda light chain is used.

[0146] 6 The method of embodiment 1 where any antibody subclass or isotype is used.

[0147] 7. The method of embodiment 1 where the Fab fragment is made as two separate chains or linked together by amino acids, intervening protein domains, or chemical bonds / interactions.

[0148] 8. The method of embodiment 1 where a scfv, nanobody, darpin, monobody, or other affinity-based protein modeled off antibody domains from any species is endowed with click chemistry capabilities by incorporation of a noncanonical amino acid instead of a Fab fragment.

[0149] 9. The method of embodiment 1 where human and animal antibody sequences are mixed.

[0150] 10. The method of embodiment 1 where a humanized sequence is use.

[0151] 11. The method of embodiment 1 where azido-sdFabs are conjugated to nanoparticles by click chemistry for cell-specific delivery.

[0152] 12. The method of embodiment 1 where Fabs are azido-modified but without synthetic dimerization.

[0153] 13. The method of embodiment 1 where azido-sdFab expression and assembly is analyzed by incorporation of fluorescent lysine and visualization on fluorescent gel.

[0154] 14. The method of embodiment 1 where azido-sdFab in vitro binding is analyzed by AlphaLISA assay.

[0155] 15. The method of embodiment 1 where the Fab specificity is for Siglec-6.

[0156] 16. The method of embodiment 1 where Fab specificity is for KIT, Siglec-8, CD33, or other mast cell target.

[0157] 17. The method of embodiment 1 where the Fab specificity is a target other than Siglec- 6.

[0158] 18. The method of embodiment 1 where Fab specificity is for a non-mast cell target.

[0159] 19. The method of embodiment 1 where extracts are made from other organisms.

[0160] 20. The method of embodiment 1 where heavy and light chains are expressed separately and assembled.

[0161] 21. The method of embodiment 1 where heavy and light chains are expressed in one pot for simultaneous assembly.

[0162] 22. The method of embodiment 1 where oxidizing conditions are created by supplementation of redox reagents and / or chaperone proteins.

[0163] 23. The method of embodiment 1 where noncanonical amino acid incorporation is done with pAzF or another noncanonical amino acid.

[0164] 24. The use of embodiment 1 where conjugation is done by copper-catalyzed click chemistry, strain-promoted click chemistry, or other click chemistry approach.

[0165] 25. The use of embodiment 1 where azido-sdFab fragments are conjugated to nanoparticles

[0166] 26. The use of embodiment 1 where azido-sdFab fragments are conjugated to a small molecule drug.

[0167] 27. The use of embodiment 1 where azido-sdFab fragments are conjugated to a protein therapeutic and / or biologic.

[0168] 28. The use of embodiment 1 where azido-sdFab fragments are conjugated to a labelling entity.

[0169] 29. The use of embodiment 1 where azido-sdFab fragments are conjugated to a prodrug.EXAMPLES

[0170] The following Examples are illustrative and are not intended to limit the scope of the

[0171] Example 1 - Targeting mast cells through cell-free expressed synthetically dimerized anti-Siglec-6 azido-Fabs

[0172] Abstract

[0173] Mast cell malignancies can be devastating diseases and are often refractory to current treatments. Mast cell depletion through Siglec-6 is a logical yet minimally explored strategy for diseases characterized by mast cell overactivity or overproduction. Mast cells share Siglec-6 expression only with placental trophoblasts which are not present outside of pregnancy. Here we combine nanoparticle technology with cell-free protein synthesis (CFPS) technology to develop a therapeutic strategy for cell-specifically depleting mast cells. Nanoparticles are loaded with cytotoxic midostaurin and conjugated with CFPS-enabled clickable, synthetically dimerized anti- Siglec-6 Fab antibody fragments to specifically target Siglec-6-expressing mast cells. In a humanized mast cell malignancy mouse model, we demonstrate human mast cell-specific cell death with preservation of Siglec-6 (-) mouse mast cells. Ultimately, we develop a novel approach for mast cell depletion by leveraging Siglec-6’s cell specificity and combining nanotechnology and cell-free protein synthesis to generate a therapeutic construct that shows mast cell-specific killing in a humanized mast cell malignancy mouse model.

[0174] Introduction

[0175] Mast cell malignancies, including systemic mastocytosis, represent a class of diseases characterized by mast cell overabundance and / or overactivity. Over 90% of systemic mastocytosis cases are associated with a gain-of-function D816V mutation in exon 17 of the cytoplasmic domain of the mast cell KIT receptor (CD117), resulting in constitutive signaling. KIT is a tyrosine kinase receptor that is activated by stem cell factor (scf), which leads to mast cell proliferation and survival.7 2Several tyrosine kinase inhibitors (TKIs) have been FDA-approved for treatment of systemic mastocytosis. Imatinib is the first selective TKI to be approved but depends on the wild type form of Kit for binding? Avapritinib7and Bezluclastinib5are more recent TKI variations designed to have specific activity against the mutant D816V form of KIT. Midostaurin is a broad inhibitor of kinase activity, so while FDA-approved for advanced systemic mastocytosis, it has a wide range of unwanted off-target effects.6Despite these advances, mast cell malignancies remain difficult to treat. A promising strategy for treating these diseases would be malignant mast cell depletion. To deplete mast cells in a cell-specific way without undesired off-target effects, one would need a very specific mast cell surface target. Anti-KIT antibodies have been shown to haveanti -proliferative effects on mast cells but depend on a normal functioning KIT receptor, so they may or may not alter malignant mast cell survival and proliferation.7 8As an alternative, sialic acid-binding immunoglobulin-like lectins (Siglecs) are inhibitory immunoreceptors with differential immune cell expression that can be leveraged to target mast cell-specific Siglecs. Mast cells have high level expression of Siglecs-3.6’9 -7,70 -8-77’12Anti-Siglec-8 antibodies have shown mast cell depletion / suppression72and antibody-drug conjugates targeting Siglec-3 and Siglec-8 have shown promise in promoting mast cell death.74’15Mast cells share Siglec-3 expression with several myeloid-derived immune cell types and Siglec-8 expression with eosinophils and basophils (weakly).76Siglec-6 is a promising target as mast cells only share its expression with placental trophoblasts, which are obviously only present during pregnancy.9'7Anti-Siglec-6 antibodies have shown promise in promoting mast cell inhibition and depletion through antibody dependent cellular phagocytosis.78Conjugating antibodies to nanoparticles for cell specific delivery of a cytotoxic drug could enhance these depletion effects. Furthermore, by using Siglec- 6-mediated delivery, unwanted cytotoxicity could be overcome for otherwise promising tyrosine kinase inhibitors, such as midostaurin.

[0176] However, to achieve the optimal geometry for nanoparticle conjugation and cell engagement, antibody fragments of the antigen binding region (Fab) are desirable over full-length antibodies. Typical approaches for generating Fabs involve digesting full-length antibody into cleavage products, but this often yields incomplete digestion and requires harsh conditions that can compromise protein structure. As an alternative approach, we designed and constructed Fabs de novo using cell-free protein synthesis.79

[0177] Cell-free protein synthesis (CFPS) has emerged as a powerful biomanufacturing platform that offers several key advantages over existing manufacturing approaches.20-26CFPS involves isolating the transcription and translation machinery from E. coll in the form of a crude extract, which serves as an abiotic, open reaction for expressing desired proteins. Importantly, CFPS permits direct control of the reaction environment and supplementation of noncanonical substrates that can endow protein with orthogonal chemistries.27For example, C321.AA.759 is an MG1655-derived E. coli strain that has been recoded to free up an available stop codon (TAG) by amber suppression to allow for incorporation of a 21st noncanonical amino acid.20Here we use an azido-modified phenylalanine (pAzF), which is amenable to click chemistry, which we use fornanoparticle conjugation. Click chemistry is a set of highly efficient, biorthogonal chemical reactions between an azide and alkyne that conjugates two species together.2S~33

[0178] We leverage CFPS technology to synthetically design anti-Siglec-6 Fabs with several favorable features. First, Fab primary protein sequences (based on known hypervariable region sequences of one Siglec-6-specific mAb) are designed with C-terminal heterodimer fusion proteins to promote more efficient assembly of the heavy and light chains yielding synthetically dimerized Fabs (sdFabs)?4Next, through noncanonical amino acid incorporation, sdFabs are equipped with a C-terminal azide group on the light chain that allows for click conjugation of azido-sdFabs to nanoparticles. A purification affinity tag is encoded on the heavy chain. The opposing arrangement of the purification tag on the heavy chain and clickable azido group on the light chain enables a stepwise approach where only fully assembled azido-sdFabs and individual heavy chains would be purified, so that in the elution, the azide group would only be present on fully assembled azido- sdFabs for conjugation onto the nanoparticle.

[0179] An additional favorable feature of the de novo synthesis of azido-sdFabs in a CFPS system is the ability to create an oxidizing reaction environment by supplementation of chaperones and redox reagents, which facilitates formation of disulfide bonds and assembly of heavy and light chains. Also, DNA is simply added directly to the cell-free reaction without need for crossing the membrane through transformation which allows for direct control of the molar ratio between the plasmids encoding the heavy chain and light chain.

[0180] We show that azido-sdFabs made by CFPS are successfully assembled and able to bind Siglec-6 in vitro without cross-reactivity to Siglec-8 or Siglec-3 (CD33).

[0181] Once synthesized, azido-sdFabs are conjugated to micelle nanoparticles that are loaded with a cytotoxic payload, midostaurin. In a humanized mast cell malignancy mouse model, where mice are treated with anti-Siglec-6 azido-sdFab-conjugated micelles containing midosaturin, preferential killing of Siglec-6 (+) human mast cells is observed over Siglec-6 (-) mouse mast cells when compared to treatment with free midostaurin drug.

[0182] By combining cell-free protein synthesis and nanotechnology, we construct a novel therapeutic construct for promoting mast cell-specific depletion, which could offer new treatment options for patients with mast cell malignancies.

[0183] Results

[0184] Design and Assembly of Anti-Siglec-6 azido-sdFabs

[0185] Anti-Siglec-6 azido-sdFabs are designed as two separate heavy and light chain constructs. Each construct is comprised of a CAT linker to standardize expression separated by a cleavage tag from Tobacco Etch Virus (TEV) from the hypervariable region sequence (VH / VL) for a known anti-Siglec-6 antibody. For the mouse azido-sdFab construct, the VH / VL sequence is followed by the mouse kappa constant region for the light chain (CL) and mouse IgG2a constant region for the heavy chain (CH), and for the chimeric azido-sdFab construct, the VH / VL sequence is followed by the human kappa constant regions for the light chain (CL) and the human IgGl constant region for the heavy chain (CH). Each heavy chain construct bears a LZA heterodimer fusion sequence downstream of the Fab sequence, which is separated by a short linker.55The heavy chain C-terminus also has a combined superflag and strep affinity tag for purification and analysis. Each light chain construct bears a LZB heterodimer fusion sequence downstream of the Fab sequence, which is separated by a short linker.55The light chain C-terminus also has an amber suppression codon for noncanonical pAzF incorporation to allow for click chemistry conjugation of azido-sdFabs (FIG. 1A and FIG 4).

[0186] Plasmids encoding the heavy and light chain constructs for either the mouse azido- sdFab or chimeric azido-sdFab were supplemented at equal molar ratios into CFPS reactions with lysate made from the recoded C321.21A.759 E. coli strain.20’27Necessary salts, building blocks and buffers were added as well as key oxidizing reagents, and chaperone proteins, Dsbc and FkpA, to facilitate disulfide bond formation. Noncanonical amino acid, pAzF, along with its tRNA synthetase, was also added to the CFPS reaction (FIG. IB).

[0187] To visualize assembly of CF-expressed azido-sdFabs fluorescent lysine was supplemented for incorporation into the protein and visualization on a fluorescent gel. At ~70 kDa, a band indicative of fully assembled product is visible along with smaller bands representingunassembled heavy and light chains. This indicates that these dseigns aand CFPS platform are suitable for synthesizing fully assembled azido-sdFabs (FIG. 1C).

[0188] In Vitro Binding of Siglec-6 by azido-sdFabs[00189J To confirm that expression and assembly leads to functional azido-sdFabs capable of binding Siglec-6, 1 used an in vitro, bead-based AlphaLISA assay to detect binding. I performed a co-titration of commercially produced Siglec-6 containing an Fc tag that will associate with Protein A donor beads and CFPS reactions expressing each azido-sdFab which will associate with anti -flag acceptor beads. Successful binding is determined by the emission of light resulting from an energy transfer between donor and acceptor beads when brought in close proximity through interactions of their associated binding partners. Typically, signal is highest at some optimal concentration and gradually decreases as concentrations move away from the central optimum. The pattern observed on the co-titration heat maps is indicative of successful binding of both the mouse and chimeric azido-sdFab to Siglec-6 (FIG. ID)

[0190] Cross-reactivity of Anti-Siglec-6 azido-sdFabs

[0191] To verify that anti-Siglec-6 azido-sdFabs are specific to Siglec-6 and do not have crossreactivity with other Siglecs, a similar AlphaLISA co-titration was performed with Siglec-6, Siglec-8, and Siglec-3 (CD33). Successful binding was observed for azido-SdFabs against Siglec- 6 (FIG. 2A, D), but not for Siglec-8 (FIG. 2B, E), or Siglec-3 (FIG. 2C, F) as indicated by signal levels near background when at least one binding partner was absent. This indicates that the azido- sdFabs have the desired specificity for Siglec-6.

[0192] Activity of midostaurin-loaded micelles conjugated with anti-Siglec-6 azido-sdFabs in a humanized mast cell malignancy model.

[0193] In a small pilot study, BALB / c mice engrafted with a human malignant mast cell line (ROSAKIT D816V) were treated with midostaurin-loaded micelles conjugated with anti-Siglec-6 azido-sdFabs as well as free midostaurin as a negative control (FIG. 3A). Bone marrow was harvested, and cells were labelled with mast cell marker, CD45+, with a fluorescent antibody and analyzed by flow cytometry. Midostaurin-loaded micelles conjugated with anti-Siglec-6 azido- sdFabs not only led to a higher percentage of Siglec-6 (+) human mast cell (CD45+) death whencompared to free drug, but also led to reduced percentage of Siglec-6 (-) mouse mast cell (CD45+) death when compared to free drug (FIG. 3B). This observation indicates enhanced specificity when using the therapeutic construct to deliver midostaurin compared to free midostaurin and suggests that midostaurin-loaded micelles conjugated with anti-Siglec-6 sdFabs are capable of preferentially depleting human mast cells through Siglec-6 targeting.

[0194] Discussion

[0195] The therapeutic construct described here leverages the mast cell specificity of Siglec-6 for targeted delivery that overcomes the broad cytotoxicity of otherwise effective TKI, midostaurin. Making this construct requires the combination of two novel technologies, nanotechnology and cell free protein synthesis, to develop midostaurin-loaded micelles conjugated with anti-Siglec-6 azido-sdFabs, which may prove useful in treating mast cell malignancies.

[0196] Using a de novo CFPS approach, the inventors were able to efficiently generate Fab fragments and endow useful properties including heterodimerization assembly, clickability for nanoparticle conjugation, and purification tags for analysis. The inventors were also able to introduce human sequences and demonstrate retained function in Siglec-6 binding to make the drug more relevant clinically. While the azido-sdFabs could be completely humanized before using to treat patients, but the chimeric construct described and tested herein provides a proof of concept that this would be possible and will likely have the desired binding specificity and effects.

[0197] Using a nanoparticle delivery method, a potentially toxic drug is delivered in a much more specific manner, reducing undesired off-target effects, which would otherwise translate into adverse events in patients. While various optimizations could be done to reduce non-specific cell death even further, this work provides a proof of concept that this would be a promising strategy for treating mast cell malignancies through Siglec-6 mediated mast cell depletion.

[0198] Given the limited number of options available to treat patients with mast cell malignancies, the demonstration of midostaurin-loaded micelles conjugated with anti-Siglec-6 sdFabs as a possible new approach is exciting. This approach could also be applied to enhance delivery of other FDA-approved tyrosine kinase inhibitors or other cytotoxic agents.

[0199] Materials and Methods

[0200] Plasmid design and synthesis

[0201] Variable regions and mouse constant regions of azido-sdFab sequences were retrieved from a known anti-Siglec mAb (R & D Systems, MAB2859). Human constant regions were retrieved from Uniprot (heavy chain: P0DOX5, kappa light chain: P01834). Heterodimer sequences are based on previous published methods?5The sflag and strep combination purification tag were designed separated by Pas 12 linkers.54’36(FIG. 4).

[0202] These constructs were cloned into pJLl backbone at Ndel and Sall restriction sites by Twist Biosciences. Plasmid DNA was isolated from glycerol stocks from Twist Biosciences using the ZymoPURE Midi Kit (Zymo Research D4200).

[0203] Harvest and processing of E. coll lysate for CFPS

[0204] E. coli lysate was prepared using previously published methods27with a recoded strain of MG1655-derived E. coli, C321.AA.759,2d engineered for noncanonical amino acid incorporation. A highly productive engineered strain of MG1655-derived E. coli strainC321.AA.759 147 was grown in a Sartorius Biostat C+ 10L fermentor in 10 liters of 2xYTPG media (yeast extract 10 g / L, tryptone 16 g / L, NaCl 5 g / L, K2HPO4 7 g / L, KH2PO4 3 g / L, and glucose 18 g / L, pH = 7.2) at 34 °C with rotation at 250 rpm. T7 RNA polymerase expression was induced with 1 mM isopropyl 0-D- 1 -thiogalactopyranoside (IPTG) (Sigma-Aldrich) at OD = 0.6. At OD = 3.0, cells were put on ice and centrifuged at 8,000 g for 5 min at 4 °C in a chilled JLA 8.1 rotor in a Beckman Avanti J-25I Refrigerated Centrifuge. The supernatant was discarded and cell pellets were washed three times with S30 buffer (10 mM Tris-acetate pH 8.2, 14 mM magnesium acetate, 60 mM potassium acetate) and pelleted by centrifugation at 10,000 g for 2 minutes at 4 °C in a Thermo Heraeus Multifuge X3R Centrifuge. Cell pellets were flash frozen and stored at -80 °C. After thawing, cells were resuspended in 0.8 mL S30 buffer per gram of wet cell mass, distributed in 1.4 mL aliquots, and sonicated (Qsonica) at 50% amplitude for a cycle of 45 s on, 59 s off until -950 Joules was reached. Lysate was kept on ice and spun at 12,000 g for 10 minutes at 4 °C. The supernatant (820 pL) was collected in fresh tubes and incubated at 37 ° with shaking at 250 rpm for 1 hour. Tubes were then spun at 12,000 g for 10 minutes at 4°C and the supernatant collected from this final spin (500 pL) comprises the cell free extract used to assemble CFPS reactions. Extract was flash frozen and stored at -80 °C until ready for use.

[0205] Cell free protein synthesis of azido-sdFabs

[0206] CFPS reactions were assembled based on previously published methods using oxidizing conditions with the addition of 14 pM purified Dsbc, 50 pM FkpA, 1 mM pAzF, 1 uL per 15 uL purified tRNA synthease, and 8 nM of each plasmid (heavy chain and light chain).27’34In addition to the former, CFPS reactions were assembled with the following building blocks and reagents: 146 6 mM magnesium glutamate; 10 mM ammonium glutamate; 130 mM potassium glutamate; 2.646 mM ATP; 1.874 mM each of GTP, UTP, and CTP; 0.075 mg / mL folinic acid, 0.376 mg / mL E. coli tRNA mixture from strain MRE600 (Roche Applied Science); 0.33 mM nicotinamide adenine dinucleotide (NAD); 0.27 mM coenzyme-A (CoA); 4 mM oxalic acid; 1 mM putrescine; 1.5 mM spermidine; 57 mM HEPES at pH=7.2; 2 mM of each of the 20 standard amino acids; 30 mM phosphoenolpyruvate (PEP) (Roche Applied Science); and 27% v / v of E. coli crude lysate (prepared above). Reagents were purchased from Sigma-Aldrich unless otherwise stated. Oxidizing conditions were assembled as described above with an additional 30 min pre-incubation of the extract in 14.3 pM iodoacetamide (IAM) before supplementation into the CFPS reaction and with the additional following reagents: 4 mM oxidized L-glutathione GSSG, 1 mM reduced L-glutathione. CFPS reactions were incubated for 20 hours at room temperature.

[0207] Fluorotect Gel

[0208] Licor Protein Sample Loading Buffer (LLCOR Biosciences 928-40004) was mixed with CFPS reaction containing expressed azido-sdFab and heat denatured at 70 °C for 3 minutes. Samples were then run on a NuPAGE 4-12% Bis-Tris gel (Invitrogen, NP0321BOX) at 150 V with MOPS buffer (Thermo Fisher Scientific, NP0001). Gel was imaged on a LI-COR-Odyssey Fc imaging system on the 600 and 800 channels and formatted in ImageStudio.

[0209] AlphaLISA Assay

[0210] A 2x serial dilution was prepared for CFPS reactions expressing azido-sdFabs in AlphaLISA buffer (50 mM HEPES pH 7.4 with 150 mM NaCl, 0.015% v / v TritonX-100, and 1 g / L BSA). A 2x serial dilution was also prepared for commercial Siglec-6 (ACRObiosystems SI6- H5256), Siglec-8 (ACRObiosystems SI8-H5254), and Siglec-3 (ACRObiosystems CD3-H5257). AlphaLISA reactions were assembled using an acoustic liquid handler (Echo 525) from a 384-well polypropylene 2.0 plus plate (Labcyte PPL-0200) on AQ_GPSA setting into a 384-well plate (PerkinElmer, 6008280) and allowed to incubate for at least 1 hour. Bead components were then distributed and allowed to incubate for at least 1 additional hour. Signal was read on a plate reader (Tecan Infinite Ml 000).

[0211] References

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[0244] 33. Rillahan, C. D., Schwartz, E., McBride, R., Fokin, V. V., and Paulson, J. C. (2012) Click and pick: identification of sialoside analogues for Siglec-based cell targeting, Angew Chem IntEdEngl 51, 11014-11018.

[0245] 34. Hunt, A. C., Vogeli, B., Kightlinger, W. K., Yoesep, D. J., Kruger, A., and Jewett, M. C. (2021) A high-throughput, automated, cell-free expression and screening platform for antibody discovery, bioRxiv.

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[0247] 36. Grawe, A., Ranglack, J., Weyrich, A., and Stein, V. (2020) iFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains, Nucleic Acids Res 48, e24.

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[0250] 39. Des Soye, B. J., Gerbasi, V. R., Thomas, P. M., Kelleher, N. L., and Jewett, M. C. (2019) A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli, Cell Chem Biol 26, 1743-1754 el749.

[0251] 40. Martin, R. W., Des Soye, B. J., Kwon, Y. C., Kay, J., Davis, R. G., Thomas, P. M., Majewska, N. I., Chen, C. X., Marcum, R. D., Weiss, M. G., Stoddart, A. E., Amiram, M., Ranji Charna, A. K., Patel, J. R., Isaacs, F. J., Kelleher, N. L., Hong, S. H., and Jewett, M. C. (2018) Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids, Nat Commun 9, 1203.

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[0253] 42. Duan, S., C.J. Koziol-White, W.F. Jester, Jr., C M. Nycholat, M.S. Macauley, R.A. Panettieri, Jr., and J.C. Paulson, CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen. J Clin Invest, 2019. 129(3): p. 1387-1401. PMC6391081.

[0254] 43. Rillahan, C D., E. Schwartz, R. McBride, V.V. Fokin, and J.C. Paulson, Click and pick: identification of sialoside analogues for siglec-based cell targeting. Angew Chem Int Ed Engl, 2012. 51(44): p. 11014-8. PMC3480989.

[0255] 44. Kolb, H.C , Finn, M. G., Sharpless, K. Barry, Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition, 2001. 40: p. 2004-2021.

[0256] 45. AU20160002317 Cytoplasmic Expression of Fab Proteins.

[0257] 46. US10118950 Platforms for cell-free protein synthesis comprising extracts from genomically recoded strains having genetic knock-out mutations in release factor 1 (RF-1) and endA.

[0258] 47. US10465221 Genomically recoded organisms lacking release factor 1 (RF1) and engineered to express a heterologous RNA polymerase.

[0259] 48. US20180016614 Highly Productive One-Pot System for the Incorporation of Non- Standard Amino Acids into Cell-Free Synthesized Proteins.

[0260] 49. US20160060301 METHODS FOR IMPROVED IN VITRO PROTEIN SYNTHESIS WITH PROTEINS CONTAINING NON STANDARD AMINO ACIDS.

[0261] 50. US20060269556A1 Mast cell activation using siglec 6 antibodies.

[0262] 51. WO2005124358A2 Diagnosis and treatment of siglec-6 associated diseases.

[0263] 52. Baskin, J. M. and C. R. Bertozzi (2007). "Bioorthogonal Click Chemistry: Covalent Labeling in Living Systems." OSAR & Combinatorial Science 26(11-12): 1211-1219.

[0264] 53. Bull, C., et al. (2017). "Steering Siglec-Sialic Acid Interactions on Living Cells using Bioorthogonal Chemistry." Angew Chem Int Ed Engl 56(12): 3309-3313.

[0265] 54. Kolb, H. C., et al. (2001). "Click Chemistry: Diverse Chemical Function from a Few Good Reactions." Angew Chem Inti Ed 40: 2004-2021.

[0266] 55. Rillahan, C. D., et al. (2012). "Click and pick: identification of sialoside analogues for Siglec-based cell targeting." Angew Chem Int Ed Engl 51(44): 11014-11018.

[0267] 56. Baskin, J. M. and C. R. Bertozzi (2007). "Bioorthogonal Click Chemistry: Covalent Labeling in Living Systems." QSAR & Combinatorial Science 26(11-12): 1211-1219.

[0268] 57. Baskin, J. M., et al. (2007). "Copper-free click chemistry for dynamic in vivo imaging." PNAS 104(43): 16793-16797.

[0269] 58. Dehnert, K. W ., et al. (2012). "Imaging the sialome during zebrafish development with copper-free click chemistry." Chembiochem 13(3): 353-357.

[0270] 59. Hudak, J. E., et al. (2012). "Synthesis of heterobifunctional protein fusions using copper-free click chemistry and the aldehyde tag." Angew Chem Int Ed Engl 51(17): 4161-4165.

[0271] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0272] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0273] Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is aninconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

Claims

CLAIMSWe claim:

1. A cell-free protein synthesis system for in vitro production of fragment antigenbinding (Fab) fragments, comprising: one or more expression templates, wherein each of the one or more expression templates comprises a polynucleotide sequence encoding a polypeptide comprising:(a) a heavy chain polypeptide comprising: a heavy chain constant region 1 (CHI) and heavy chain variable region (VH);(b) a light chain polypeptide comprising: a light chain constant region (CL) and a light chain variable region (VL); or(c) both (a) and (b); one or more cell-free protein synthesis reagents for expressing the polypeptides.

2. The system of claim 1, wherein the encoded polypeptide comprises(a) a synthetic heterodimerization domain; and / or(b) an azido-modified amino acid.

3. The system of claim 2, wherein the light chain polypeptide comprise the azidomodified amino acid.

4. The system of claim 3, wherein the azido-modified amino acid comprises a phenylalanine (pAzF).

5. The system of any one of claims 1-3, wherein the heavy chain polypeptide and the light chain polypeptide each comprise one member of a heterodimerization domain pair.

6. The system of claim 5, wherein the heterodimerization pair comprises LZA and LZB.

7. The system of any of the previous claims wherein the encoded polypeptide comprises a purification tag.

8. The system of any of the previous claims wherein the one or more cell-free synthesis reagents comprises a lysate prepared from a prokaryote.

9. The system of claim 8, wherein the prokaryote is E. coli.

10. The system of claim 8 or 9, wherein the lysate is prepared from genetically modified E. coli, wherein at least one genetic modification facilitates incorporation of an azido-modified amino acid into the encoded polypeptide, optionally wherein the modified E. coli is C321.AA.759 E. coli strain.

11. The system of claim 10, wherein the lysate was prepared from genetically modified E. coli, wherein at least one genetic modification facilitates disulfide bond formation.

12. The system of any one the previous claims, wherein the one or more expression templates and / or the one or more cell-free synthesis reagents is in a freeze-dried format.

13. The system of any one of the previous claims, wherein the one or more cell-free synthesis reagents comprises one or more of ATP, GTP, UTP, CTP, or a tRNA mixture from E. coli.

14. A bacterial cell comprising one or more expression templates, wherein each of the one or more expression templates comprise a polynucleotide sequence for expression of one or more polypeptide Fab fragments comprising:(a) a heavy chain polypeptide comprising: a heavy chain constant region 1 (CHI) and heavy chain variable region (VH), and a first member of a heterodimerization domain pair;(b) a light chain polypeptide comprising: a light chain constant region (CL) and a light chain variable region (VL), and a second member of a heterodimerization domain pair; or(c) both (a) and (b);wherein the bacterial cell is genetically modified to facilitate incorporation of an azido-modified amino acid into the encoded polypeptide Fab fragment, and optionally, wherein the bacterial cell is genetically modified to facilitate disulfide bond formation.

15. A lysate prepared from the bacterial cell of claim 14.

16. The lysate of claim 15, wherein the lysate is freeze-dried.

17. A composition comprising a fragment antigen-binding (Fab) fragment, wherein the Fab fragment comprises:(a) at least one modified amino acid; and(b) a heterodimerization domain.

18. The composition of claim 17, wherein the modified amino acid comprises an azidomodified phenylalanine.

19. The composition of claim 18, wherein the heterodimerization domain comprises LZA and LZB.

20. The composition of any one of claims 18 or 19, further comprising a nanoparticle linked, via the azido modified phenylalanine, to the Fab fragment, optionally wherien the fab fragment targets Siglec-6.

21. The composition of claim 20, wherein the nanoparticle is a micell or a liposome.

22. The composition of claim 21, wherein the nanoparticle is loaded with a therapeutic compound.

23. The composition of any one of claims 18 or 19, further comprising a therapeutic molecule linked, via the azido modified phenylalanine, to the Fab fragment.

24. The composition of claim 23, wherein the therapeutic molecule comprises one of a peptide, small molecule, biologic, a prodrug, or a Fab fragment.

25. The composition of any one of claims 18 or 19, further comprising a detectable label linked, via the azido modified phenylalanine, to the Fab fragment.

26. The composition of any one of claims 20-25, wherein the link via the azido modified phenylalanine is a click chemistry linkage.

27. The composition of any one of claims 17-26 formulated as a therapeutic composition for administration to a subject.

28. The composition of any one of claims 17-27, wherein the Fab fragment targets Siglec-6.

29. A kit comprising: in a first container, an antigen binding fragment comprising an azido modified phenylalanine.

30. The kit of claim 29, wherein the antigen binding fragment comprises a Fab fragment.

31. The kit of claim 30, wherein the Fab fragment is directed to Siglec-6.

32. The kit of any one of claims 29-31, further comprising, in a second container, a partner molecule comprising a therapeutic agent.

33. The kit of claim 31 or 32, wherein the partner molecule comprises a midostaurin- loaded micell.

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