Genetically encoded lysosome targeting chimeras for cell-mediated delivery

Abstract

Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. Fully genetically-encodable lysosome-targeting chimeras (GELYTAC) are provided, which allows for example integration into therapeutic cells for targeted delivery at diseased sites. The glycopeptide portion of LYTACs was replaced with the protein insulin like growth factor 2 (IGF2). After showing initial efficacy with wild type IGF2, the potency of GELYTAC was improved by mutation, resulting in an engineered GELYTAC construct that can secrete from cells, including from human primary T-cells, to drive the uptake of various targets into receiver cells.

Description

PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 GENETICALLY ENCODED LYSOSOME TARGETING CHIMERAS FOR CELL-MEDIATED DELIVERY CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No.63 / 660,049, filed June 14, 2024, which is incorporated by reference for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under contracts CA278686 and GM058867 awarded by the National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] The Sequence Listing written in file “110221-1509199-011110WO”.xml created on June 9, 2025, 15,196 bytes in size, is hereby incorporated by reference in its entirety for all purposes. BACKGROUND OF THE INVENTION

[0004] Lysosome-targeted degradation is an emerging therapeutic modality that facilitates the degradation of membrane and soluble extracellular proteins. Compared to traditional therapeutic modalities, such as small molecule or antibody-based inhibitors, targeted protein degradation offers increased potential potency and broadens the druggable proteome. The first generation of this technology came in the form of lysosome-targeting chimeras (LYTACs), which are bifunctional molecules comprised of an antibody that binds to a cell surface or secreted protein of interest (POI) conjugated to a ligand that binds a lysosome trafficking receptor such as the insulin growth factor 2 receptor (IGF2R, also known as CI-M6PR). Since then, other technologies have been developed, such as antibody-based proteolysis targeting chimeras, proteolysis-targeting antibodies, and cytokine receptor-targeting chimeras, among others. These use similar bifunctional molecules to recruit POIs either to lysosome trafficking receptors or KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 plasma membrane-associated ubiquitin ligases and have opened a new chapter in the field of targeted protein degradation.

[0005] There is increasing interest in new technologies that target therapeutic molecules to the sites of their desired action. This has recently been accomplished by encoding the production of therapeutic proteins in the genomes of targeted cell therapies. For example, therapeutic T cells or natural killer cells have been engineered to secrete ingle-chain variable fragments (scFvs), bispecific T cell engagers, or cytokines within tumor microenvironments, thereby concentrating the activity of these proteins at the desired tissue site. Though LYTACs are a promising therapeutic modality to drive the degradation of extracellular proteins, LYTAC contain synthetic glycopeptides that cannot be genetically encoded, limiting cell-mediated delivery applications. BRIEF SUMMARY OF THE INVENTION

[0006] Provided herein are nucleic acids encoding a fusion polypeptide comprising a target binding domain and an IGF2R-binding portion of a human IGF2 protein, wherein the IGF2R- binding portion comprises a substitution corresponding to S39X in SEQ ID NO:1, wherein X is an amino acid other than serine. In some embodiments, the S39X substitution is an S39P substitution. In some embodiments, IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 4. In some embodiments, IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 2.

[0007] In some embodiments, the IGF2R-binding portion comprises at least one further substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution. In some embodiments, the at least one substitution that reduces binding to IGF1R is R24A, F48X, T58A, Y59X, and / or T62A, wherein X is either serine or alanine. In some embodiments, IGF2R- binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6.

[0008] In some embodiments, the IGF2R-binding portion further comprises one or both of a substitution corresponding to E6R and F19L relative to SEQ ID NO: 1. In some embodiments, IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5. In some embodiments, IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 3. In some embodiments, the IGF2R-binding portion comprises at least one further substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding 2 KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 wildtype amino acid at the at least one further substitution. In some embodiments, the at least one substitution that reduces binding to IGF1R is R24A, F48X, T58A, Y59X, and / or T62A, wherein X is either serine or alanine. In some embodiments, IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7.

[0009] In some embodiments, the IGF2R-binding portion comprises at least one substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution. In some embodiments, the at least one substitution that reduces binding to IGF1R is R24A, F48X, T58A, Y59X, and / or T62A, wherein X is either serine or alanine. In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8.

[0010] In some embodiments, the nucleic acid comprises two or more IGF2R-binding portions. In some embodiments the two or more IGF2R-binding portions have identical or non- identical amino acid sequences.

[0011] In some embodiments, the target binding domain comprises an immunoglobulin variable region. In some embodiments, the target binding domain is a single chain antibody (scFv) or a nanobody. In some embodiments, the target is an immunosuppressive molecule. In some embodiments, the target is an autoantibody. In some embodiments, the target is PD-1. In some embodiments, the target is a cytokine or an interleukin. In some embodiments, the target isselected from the group consisting of TGF- , IL-6R, HER2, and EGFR, optionally EGFRcomprising a T790M mutation. In some embodiments, the nucleic acid comprises RNA.

[0012] Also provided is an expression cassette comprising a promoter operably-linked to any of the nucleic acids described herein. Also provided is a cell comprising any of the nucleic acids provided herein or the expression cassettes provided herein. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a primary cell. In some embodiments, the cell is a primary human immune cell or primary human hematopoietic stem cell. In some embodiments, the primary human immune cell is a primary human T-cell or natural killer cell. In some embodiments, the cell expresses the fusion polypeptide. In some embodiments, the cell secretes the fusion polypeptide. Also provided herein is the fusion polypeptide encoded by any of the nucleic acids provided herein. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0013] Further provided herein are methods of degrading a target protein, the method comprising contacting the target protein with a fusion polypeptide described herein in the presence of a first cell comprising an IGF2R transmembrane protein under conditions in which the fusion polypeptide binds the target protein and is bound by the IGF2R transmembrane protein and internalized into the cell. In some embodiments, the contacting occurs in vitro or in vivo. In some embodiments, the contacting occurs in vivo in a human.

[0014] In some embodiments, the fusion polypeptide is expressed from a second cell. In some embodiments, the second cell is a human cell. In some embodiments, the human cell is a primary cell. In some embodiments, the cell is a human immune cell or human hematopoietic stem cell. In some embodiments, the human immune cell is a primary human T-cell or natural killer cell. In some embodiments, the second cell expresses a chimeric antigen receptor (CAR). In some embodiments, the CAR binds to a tumor antigen or a B-cell antigen.

[0015] In some embodiments, the contacting used in the methods provided herein comprises introducing the fusion polypeptide into a human. In some embodiments, the contacting comprises introducing a nucleic acid encoding the fusion polypeptide into the human, wherein the fusion polypeptide is expressed in the human. In some embodiments, the nucleic acid is a viral vector. In some embodiments, the contacting comprises introducing the second cell described herein into the human. DEFINITIONS

[0016] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0017] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0018] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements.

[0019] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0020] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0021] As used herein, the term “target molecule” refers to any molecule (e.g., a protein or an antibody) for which the targeted binding domain has an affinity. In some embodiments, the target binding domain specifically binds to the target molecule.

[0022] As used herein, the term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or functional fragments thereof that specifically binds and recognizes an antigen. The term "antibody," as used herein, also includes antibody fragments that retain binding specificity, including but not limited to Fab, F(ab’)2, Fv, and scFv. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are 5 KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0023] An exemplary immunoglobulin (antibody) structural unit comprises two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. Thus, the terms "variable heavy chain" or "VH" refer to the variable region of an immunoglobulin heavy chain, including an Fv, scFv, dsFv or Fab; while the terms "variable light chain" or "VL" refer to the variable region of an immunoglobulin light chain, including an Fv, scFv, dsFv or Fab. Nanobodies, including humanized nanobodies or other single domain antibodies are also examples of antibodies. Single domain antibodies are described, for example, in International Application Publication No. WO 94 / 04678. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species (e.g., camel, llama, dromedary, alpaca and guanaco) or other species besides Camelidae.

[0024] The term "variable region" or “immunoglobulin variable region” refers to a domain in an antibody heavy chain or light chain that gives an antibody its specificity for binding to an antigen. Typically, an antibody variable region comprises four conserved "framework" regions interspersed with three hypervariable "complementarity determining regions."

[0025] The term "complementarity determining region" or "CDR" refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0026] As noted, the part of a variable region not contained in the CDRs is called the framework. The "framework regions" of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three- dimensional space. Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBASE2” germline variable gene sequence database for human and mouse sequences.

[0027] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art. The position and length of the CDRs have been precisely defined by Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987, and others. See, e.g., Johnson and Wu, Nucleic Acids Res. 2000 Jan 1; 28(1): 214–218; Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia & Lesk, (1987) J. Mol. Biol. 196, 901-917; Chothia et al. (1989) Nature 342, 877-883; Chothia et al. (1992) J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J.Mol.Biol 1997, 273(4)); and MacCallum et al., J. Mol. Biol., 262:732-745 (1996). Also see international ImMunoGeneTics database (IMGT), AbM, and observed antigen contacts.

[0028] The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and refer to one or more fragments of an antibody that retains the ability to specifically bind to an antigen. Examples of antibody-binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains), F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), a single chain Fv (scFv), a disulfide-linked Fv (dsFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), nanobodies, and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen. Antibodies and antigen- binding portions thereof include domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 Exemplary configurations of variable and constant domains that may be found within an antigen- binding fragment of an antibody of the present invention include: (a) VH-CHI; (b) VH-CH2; (c) VH-CH3; (d) VH - CH1-CH2; (e) VH-Ch1-Ch2-Ch3; (f) VH-Ch2-Ch3; (g) VH-CL; (h) VL- CH1; (i) VL-Ch2; (X) VL-Ch3; (j) VL-CH1-CH2; (k) VL-CH1-CH2-CH3; (l) VL-CH2-CH3; and (m) VL-CL (see, e.g., FUNDAMENTAL IMMUNOLOGY (Paul ed., 4th ed.2001), Gruber et al. (1994) J Immunol.152:5368-5374; McCartney, et al., 1995 Protein Eng.8:301-314; Shukra et al., 2014, “Production of recombinant antibodies using bacteriophages” Eur J Microbiol Immunol (Bp). 4(2): 91–98; Todorovska, 2001, “Design and application of diabodies, triabodies and tetrabodies for cancer targeting” J Immunol Methods; 248(1-2):47-66; Salvador et al., 2019, “Nanobody: outstanding features for diagnostic and therapeutic applications” Anal Bioanal Chem.411(9):1703-1713; Gill et al., 2006, “Biopharmaceutical drug discovery using novel protein scaffolds.” Curr Opin Biotechnol., (6):653-8; and Ubah et al., 2016, “Phage Display Derived IgNAR V Region Binding Domains for Therapeutic Development” Curr Pharm Des. 22(43):6519-6526, each of which is incorporated by reference herein.

[0029] The term "specifically binds" refers to a molecule (e.g., target binding domain) that binds to a target molecule with greater affinity, avidity, more readily, and / or with greater duration to that target in a sample than it binds to a non-target compound. In some embodiments, an target binding domain thereof that specifically binds a target binds to the target molecule with at least 2-fold greater affinity than non-target molecules, e.g., at least 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold or greater affinity.

[0030] The term "isolated," as used with reference to a nucleic acid or protein , denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated nucleic acid or protein (e.g., antibody) is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0031] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0032] The term "amino acid" refers to refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, - carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0033] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0034] As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably. Use of the term "polynucleotide" includes oligonucleotides (i.e., short polynucleotides). This term also refers to deoxyribonucleotides, ribonucleotides, and naturally occurring variants, and can also refer to synthetic and / or non-naturally occurring nucleic acids (i.e., comprising nucleic acid analogues or modified backbone residues or linkages), such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide- nucleic acids (PNAs), and the like. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see, e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)).

[0035] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.

[0036] A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.

[0037] Modified nucleotide base pairing encompasses not only the standard adenosine- thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base / sugar or linker may be incorporated into nucleic acids of the present disclosure. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0038] A “5 untranslated region” (5 UTR) refers to a region of an mRNA that is directlyupstream (i.e., 5 ) from the start codon (i.e., the first codon of an mRNA transcript translated by aribosome) that does not encode a polypeptide.

[0039] A “3 untranslated region” (3 UTR) refers to a region of an mRNA that is directlydownstream (i.e., 3 ) from the stop codon (i.e., the codon of an mRNA transcript that signals atermination of translation) that does not encode a polypeptide.

[0040] A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e.,3 ), from the 3 UTR, that contains multiple, consecutive adenosine monophosphates. A polyAtail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation.

[0041] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0042] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith & Waterman Add. APL. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol.48:443 (1970), by the search for 11 KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 similarity method of Pearson & Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0043] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10 and Altschul et al. (1977) Nucleic Acids Res.25: 3389-402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1977)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation I of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0044] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0045] An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. The promoter can be a heterologous promoter. In the context of promoters operably linked to a polynucleotide, a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).

[0046] A “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0047] A promoter is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence, for example, a polynucleotide encoding a polypeptide provided herein. For example, a promoter is operably linked to a polynucleotide if it affects, either positively or negatively, the transcription of the polynucleotide.

[0048] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a target cell into which it has been optionally introduced. A “vector” as used here refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0049] The terms “first cell” and “receiver cell” are used interchangeably herein to refer to a cell comprising an IGF2R transmembrane protein. In the methods described herein, a first cell ultimately internalizes and degrades the fusion polypeptide bound to a target molecule.

[0050] “Subject,” “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc. For example, a “cancer patient” or “AML patient” can refer to an individual that has been diagnosed with cancer, is currently following a therapeutic regimen, or is at risk of recurrence, e.g., after surgery to remove a tumor. In some embodiments, the cancer patient has been diagnosed with cancer and is a candidate for therapy. Cancer patients can include individuals that have not received treatment, are currently receiving treatment, have had surgery, and those that have discontinued treatment.

[0051] The term “chimeric antigen receptor” or “CAR” refers to a polypeptide comprising (1) a target antigen binding domain (e.g., a binding portion of an antibody, such as scFV) ; (2) a hinge region; (3) a transmembrane domain (TM); and (4) an intracellular domain comprising atleast one signal transduction domain (e.g.,CD3 ). CARs optionally further comprise one or morecostimulatory region (e.g., 4-1BB or CD28). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG.1A depicts a schematic illustration of an embodiments of the present disclosure. A genetically encoded LYTAC (GELYTAC) is shown. GELYTAC is a fusion protein consisting of a binder (i.e., nanobody or scFv) to the target of interest and IGF2, a 7.5 kDa protein that binds to IGF2R.

[0053] FIG.1B depicts a schematic illustration of an embodiment of the present disclosure. FIG. 1B shows an embodiments in which GELYTACs can be utilized as a recombinant protein, where the target is internalized via GELYTAC binding to IGF2R and then degraded in the lysosome. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0054] FIG.1C depicts a schematic illustration of an embodiment of the present disclosure in which GELYTAC is secreted by cells (e.g., therapeutic T cells) to act on local targets.

[0055] FIG.2A shows mCherry internalization as a function of GELYTAC concentration. Median fluorescence intensity (MFI) of K562 cells was measured 24 hrs after treatment with mCherry GELYTAC (circles) and recombinant mCherry (100 nM). Negative controls are mCherry nanobody only (triangles) and a GELYTAC targeting EGFR (squares). Errors bars represent the standard deviation (SD) from 3 biological replicates. *** = p < 0.001 (determined using parametric t-test).

[0056] FIG.2B shows GELYTAC-mediated mCherry internalization by wild type and IGF2R KO K562 cells. The experiment was performed at 275 nM of GELYTACs and 100 nM mCherry for 4 hrs. Errors bars represent the SD from 3 biological replicates. *** = p < 0.001 (determined using parametric t-test).

[0057] FIG.3A shows the effect of point mutations in IGF2 on GELYTAC-mediated internalization of mCherry. Fold change in mCherry mean fluorescent intensity was calculated in relation to GELYTAC concentration. mCherry GELYTACs having a series of point mutants of IGF2 with varying affinities for IGF2R were used.

[0058] FIG.3B shows EC50 calculated from the data in FIG.3A plotted against Kd values.

[0059] FIG.3C shows GELYTAC-mediated internalization (original and G2) of mCherry (100 nM) by K562 cells, performed as in FIG.2A. Errors bars represent the standard deviation (SD) from 3 biological replicates.

[0060] FIG.4A left panel shows GELYTAC-mediated internalization (original and G2) of mCherry when GELYTACs were secreted by HEK293T cells. Control GELYTAC targets IL6R instead of mCherry. K562 receiver cells were separated and analyzed by flow cytometry. Errors bars represent the SD from 3 biological replicates. **** = p < 0.0001 (determined using parametric t-test). FIG. 4A right panel shows GELYTAC-mediated internalization (original and G2) in cocultures comprising either wild type or IGF2R KO K562 cells were treated with mCherry (100 nM) for 24 hrs and analyzed as in FIG. 4A right panel. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0061] FIG.4B left panel shows mCherry (10 nM) clearance in media after incubation of GELYTAC secreting HEK293T cells with K562 receiver cells. Media was analyzed for mCherry fluorescence 72 hrs after mCherry addition using a plate reader. This experiment was repeated 5 times. Errors bars represent the SD from 5 biological replicates. **** = p < 0.0001 (determined using parametric t-test). FIG.4B right panel shows mCherry (10nM) degradation in both cells and media of GELYTAC secreting HEK293T cells coculture with K562 receiver cells. Analysis of media and cells was performed the same as in FIG. 4B left panel.

[0062] FIG.4C is a schematic illustration of an embodiment of the present disclosure in whichGELYTACs target TGF- utilizing a TGF- scFv derived from Fresolimumab.

[0063] FIG.4D left panel shows GELYTAC-mediated internalization of AlexaFluor-467-tagged TGF- . Cocultured cells were treated with AlexaFluor-647 tagged TGF- (100 nM) for24 hrs. Then, K562 receiver cells were separated and analyzed by flow cytometry. HEK293T cells secreting G2 GELYTAC were tested. Control GELYTAC targets mCherry instead of TGF- . Errors bars represent the SD from 3 biological replicates. ** = p < 0.01 (determined usingparametric t-test). FIG. 4D right panel shows biotinylated TGF- degradation in cells andmedia. To quantify TGF- (1 nM supplemented biotinylated TGF- ) degradation, both cells andmedia were collected and analyzed for biotin signal via streptavidin-800 blot. This experiment was repeated 6 times. Errors bars represent the SD from 6 biological replicates. ** = p < 0.01 (determined using parametric t-test).

[0064] FIG.4E is a schematic illustration of an embodiment of the present disclosure in which GELYTACs target IL6R utilizing a IL6R nanobody derived from ALX-0061.

[0065] FIG.4F shows GELYTAC-mediated internalization of AlexaFluor-467-tagged IL6R. Cocultured cells were treated with AlexaFluor-647 tagged IL6R (100 nM) for 24 hrs. Then, K562 receiver cells were separated and analyzed by flow cytometry. HEK293T cells secreting G2 GELYTAC were tested. Control GELYTAC targets mCherry instead of IL6R. Errors bars represent the SD from 3 biological replicates. ** = p < 0.01 (determined using parametric t-test).

[0066] FIG.5A is a schematic illustration of an embodiment of the present disclosure in which adherent human primary T cells secrete GELYTAC that acts on suspended K562 cells and T cells to internalize and degrade targets. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0067] FIG.5B left panel shows GELYTAC-mediated internalization of mCherry in K562 cells, with GELYTAC secreted by T cells. Cocultured cells were treated with mCherry (100 nM) for 62 hrs. Then, K562 cells were analyzed by flow cytometry. T cells secreting G2 GELYTAC were tested. Control GELYTAC targets IL6R instead of mCherry. Errors bars represent the SD from 3 biological replicates. *** = p < 0.001 (determined using parametric t- test). FIG.5B right panel shows GELYTAC-mediated internalization of mCherry in T cells, with GELYTAC secreted by T cells. Cocultured cells were treated with mCherry (100 nM) for 62 hrs. Then, T cells were analyzed by flow cytometry. T cells secreting G2 GELYTAC were tested. Control GELYTAC targets IL6R instead of mCherry. Errors bars represent the SD from 3 biological replicates. *** = p < 0.001 (determined using parametric t-test).

[0068] FIG.5C left panel shows GELYTAC-mediated internalization of AlexaFluor-467-tagged TGF- in K562 cells, with GELYTAC secreted by T cells. Cocultured cells were treatedwith AlexaFluor-647 tagged TGF- (100 nM) for 52 hrs. Then, K562 receiver cells wereanalyzed by flow cytometry. T cells secreting G2 GELYTAC were tested. Control GELYTACtargets mCherry instead of TGF- . Errors bars represent the SD from 3 biological replicates. **= p < 0.01 (determined using parametric t-test). FIG.5C middle panel shows GELYTAC- mediated internalization of AlexaFluor-467-tagged IL6R in K562 cells, with GELYTAC secreted by T cells. Cocultured cells were treated with AlexaFluor-647 tagged IL6R (100 nM) for 9 hrs. Then, K562 receiver cells were analyzed by flow cytometry. T cells secreting G2 GELYTAC were tested. Control GELYTAC targets mCherry instead of IL6R. Errors bars represent the SD from 3 biological replicates. * = p < 0.1 (determined using parametric t-test).

[0069] FIG.5C right panel shows GELYTAC-mediated internalization of AlexaFluor-467- tagged IL6R in T cells, with GELYTAC secreted by T cells. Cocultured cells were treated with AlexaFluor-647 tagged IL6R (100 nM) for 9 hrs. Then, T cells receiver cells were analyzed by flow cytometry. T cells secreting G2 GELYTAC were tested. Control GELYTAC targets mCherry instead of IL6R. Errors bars represent the SD from 3 biological replicates. * = p < 0.1 (determined using parametric t-test). KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 DETAILED DESCRIPTION OF THE INVENTION

[0070] Fusion polypeptides allowing for targeted degradation of proteins are provided. The fusion polypeptides described herein comprise a target binding domain and an IGF2R-binding portion of a human IGF2 protein comprising at least one mutation as described herein that improves binding to IGF2R or reduces binding to IGF1R, or both. As explained herein, it has been discovered that a mutation at the position corresponding to S39 in human IGF2 protein (SEQ ID NO: 1) improves binding of IGF2 to IGF2R. For example, a mutant comprising the S39P, E6R and F19L substitutions in SEQ ID NO: 1 improved binding to IGF2R. In addition, mutations of the IGF2R-binding portion of a human IGF2 protein are provided that reduce binding to non-IGF2R proteins such as, for example, IGF1R. Also provided herein are nucleic acids encoding the fusion polypeptides as well as methods of using the fusion proteins, for example to cause degradation of a target molecule in vivo or in vitro in the presence of a cell expressing the IGF2R receptor. I. Compositions

[0071] This disclosure provides for nucleic acids encoding a fusion polypeptide comprising a target binding domain and an IGF2R-binding portion of a human IGF2 protein, comprising at least one mutation as described herein that improves binding to IGF2R or reduces binding to IGF1R, or both. Also provided are compositions comprising the fusion protein and cells expressing the fusion protein. A. Nucleic Acids

[0072] Provided herein are nucleic acids encoding a fusion polypeptide comprising a target binding domain and an IGF2R-binding portion of a human IGF2 protein, wherein the IGF2R- binding portion comprises a S39X substitution in the wildtype IGF2 sequence (SEQ ID NO: 1). The human IGF2 protein binds to IGF2R, a lysosome trafficking surface receptor expressed by multiple cell types. The IGF2-IGF2R complex is ultimately internalized and trafficked to the lysosome in in the receiver cell. The nucleic acids provided herein encode a fusion polypeptide comprising a portion of human IGF2 which binds IGF2R, and a target binding domain to bind a target protein of interest. The fusion polypeptide binds the target protein, via the target binding domain, and the IGF2R, via the IGF2R-binding portion, leading to internalization and lysosomal degradation of the target protein. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0073] The fusion polypeptides described herein comprise a target binding domain linked to the IGF2R-binding portion of a human IGF2 protein. The target binding domain can be covalently linked to the IGF2R-binding portion of a human IGF2 protein directly or via an intervening linker amino acid sequence. Any amino acid sequence with affinity for a target molecule can be used as the target binding domain. The term “domain” is not intended to reference any particular structure. In some embodiments, the target binding domain is a natural or modified ligand or receptor or other natural or modified binding molecule for the target molecule. In other embodiments, the target binding domain can comprise an immunoglobulin variable region. Exemplary immunoglobulin variable regions will have affinity for the target molecule and will have at least one set of three complementarity determining regions (CDRs). In some embodiments, the immunoglobulin variable region will be a heavy chain variable region, e.g., part of a single domain antibody, e.g., a VHH or nanobody. In some embodiments, the target binding domain will comprise two immunoglobulin variable regions, e.g., a heavy chain and a light chain variable region, e.g., as a single-chain antibody, e.g., an scFv.

[0074] Any linker amino acids sequence can be used to link the target binding domain to the IGF2R-binding portion of a human IGF2 protein. In some embodiments, linkers can include flexible Glycine-Serine (GS) linkers (e.g., a linker in which a majority or all of the amino acids are glycine or serine) and XTEN (e.g., SESATPES) linkers. Amino acid linker sequences can be any desired length, and in some embodiments, are 1-50, 1-20, or 1-10 amino acids in length. In some embodiments, the linker is a GS linker 10 amino acids in length. In some embodiments, the linker is a GS linker 20 amino acids in length.

[0075] The target may be any molecule (e.g., a protein) bound by the target binding domain. The target binding domain may be varied depending on the desired target. A non-limiting list of examples is provided in Table 1 below. In some embodiments, the target is an immunosuppressive molecule. In some embodiments, the target is an autoantibody. In some embodiments, the target is PD-1. In some embodiments, the target is a cytokine or interleukin(e.g., IL6R). In some embodiments, the target is one of the following: TGF- , IL-6R, HER2, orEGFR. In some embodiments, the target is EGFR comprising a T790M mutation. Table 1. Exemplary target and target binding domain pairings KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0076] Provided herein are substitutions in IGF2 which improve binding affinity of the fusion polynucleotide to IGF2R. The IGF2R-binding portion in the fusion polypeptide described comprises a substitution corresponding to S39X the amino acid sequence of human wildtype human IGF2 (SEQ ID NO:1). X in the S39X substitution may be any amino acid other than serine (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine). The S39X substitution improves binding affinity of the fusion polynucleotide to IGF2R.

[0077] In some embodiments, the IGF2R-binding portion is a full-length IGF2 protein comprising a S39X substitution (e.g., SEQ ID NO: 4). In some embodiments, the IGF2R- binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 4 (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 4). In some embodiments, the IGF2R- binding portion is a fragment of the IGF2 protein comprising a S39X substitution (e.g., SEQ ID NO: 2). In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 2 (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2). In some embodiments, X in the S39X substitution is proline and the substitution is S39P.

[0078] In some embodiments, the IGF2R-binding portion further comprises one or both of a substitution corresponding to E6X and F19X relative to SEQ ID NO: 1, where X is any amino acid other glutamic acid at residue 6 of SEQ ID NO: 1 and phenylalanine at residue 19 of SEQ ID NO: 1. In some embodiments, the IGF2R-binding portion further comprises one or both of a substitution corresponding to E6R and F19L relative to SEQ ID NO: 1. In some embodiments, the IGF2R-binding portion is a full-length IGF2 protein comprising an S39X substitution and further comprising one or both of a substitution corresponding to E6R and F19L (e.g., SEQ ID NO: 5). In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 identical to SEQ ID NO: 5 (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 5). In some embodiments, the IGF2R-binding portion is a fragment of the IGF2 protein comprising an S39X substitution and further comprising one or both of a substitution corresponding to E6R and F19L (e.g., SEQ ID NO: 3). In some embodiments, the IGF2R- binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 3 (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 3).

[0079] In some embodiments, the IGF2-binding protein further comprises at least one substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution position. In some embodiments, the at least one substitution that reduces binding to IGF1R is R24A, F48X, T58A, Y59X, and / or T62A. In F48X and Y59X substitutions, X is any amino acid other than the wildtype position in SEQ ID NO: 1. In F49X substitutions, X may be any amino acid other than phenylalanine (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, or valine). In Y59X substitutions, X may be any amino acid other than tyrosine (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine threonine, tryptophan, or valine). One or more of the substitutions that reduce binding to IGF1R may be present, e.g., only R24A, each of R24A, F48X, and T58A, or each of R24A, F48X, T58A, Y59X, and T62A.

[0080] In some embodiments, the IGF2R-binding portion further comprising at least one substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution position is a full-length IGF2 protein comprising a S39X substitution, wherein X is an amino acid other than serine (e.g., SEQ ID NO: 6). In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6 (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 6). In some embodiments, the IGF2R-binding portion further comprising at least one substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution position is a fragment KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 of the IGF2 protein also comprising a S39X substitution (e.g., SEQ ID NO: 2 further comprising a R24A substitution).

[0081] In some embodiments, the IGF2R-binding portion further comprising at least one substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution position is a full-length IGF2 protein comprising a S39X substitution and further comprising one or both of a substitution corresponding to E6X and F19X relative to SEQ ID NO: 1, where X is any amino acid other glutamic acid at residue 6 of SEQ ID NO: 1 and phenylalanine at residue 19 of SEQ ID NO: 1. In some embodiments, the IGF2R-binding portion further comprising at least one substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution position is a full-length IGF2 protein comprising a S39X substitution and further comprising one or both of a substitution corresponding to E6R and F19L. In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7 (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 7). In some embodiments, the IGF2R-binding portion further comprising at least one substitution that reduces binding to IGF1R compared to the IGF2R- binding portion having a corresponding wildtype amino acid at the at least one further substitution position is a fragment of the IGF2 protein comprising a S39X substitution and further comprising one or both of a substitution corresponding to E6R and F19L (e.g., SEQ ID NO: 3 further comprising a R24A substitution).

[0082] In some embodiments, the IGF2-binding protein comprises, without the S39X, E6R, and / or F19L substitutions, at least one substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution position. In some embodiments, the at least one substitution that reduces binding to IGF1R is R24A, F48X, T58A, Y59X, and / or T62A. In F48X and Y59X substitutions, X is any amino acid other than the wildtype position in SEQ ID NO: 1. In F49X substitutions, X may be any amino acid other than phenylalanine (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, or valine). In Y59X substitutions, X may be any amino acid other than tyrosine (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine threonine, tryptophan, or valine). One or more of the substitutions that reduce binding to IGF1R may be present, e.g., only R24A or each of R24A, F48X, T58A, Y59X, and T62A.

[0083] In some embodiments, the IGF2R-binding portion comprising at least one substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution position is a full-length IGF2 protein (e.g., SEQ ID NO: 8). In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8 (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 8).

[0084] Many IGF molecules, including IGF-2, are bound by one of the six serum IGF-binding proteins (see e.g., Allard & Duan, Front Endocrine., 9(117):1-12 (2018)). IGF2 is bound by IGF-binding proteins 1, 5, and 6. In some embodiments, the IGF2R-binding portion comprises at least one substitution to reduce binding to serum IGF-binding proteins as described in WO 2022 / 271981. In some embodiments, the IGF2R-binding portion comprises a F26S substitution relative to SEQ ID NO: 1. In some embodiments, the IGF2R-binding portion comprises a F19S and / or E9L substation relative to SEQ ID NO: 1. In some embodiments, the IGF2R-binding portion comprises one or more substitutions that confer increased resistance to furin cleavage compared to a IGF2R-binding portion not having the one or more substitutions that confer increased resistance to furin cleavage. In some embodiments, the IGF2R-binding portion comprises an R37A substitution relative to SEQ ID NO: 1. IGF2R-binding portions of interest include those described in WO 2022 / 271981, the disclosure of which is incorporated herein by reference in its entirety.

[0085] In certain embodiments, the fusion polynucleotide encoded by the nucleic acids provided herein comprises two or more IGF2R-binding portions. The two or more IGF2R- binding portions may be those described above or elsewhere herein (e.g., any of SEQ ID NOs: 1- 8). In some embodiments, the two or more IGF2R-binding portions have identical amino acid sequences (e.g., SEQ ID NOs: 2 and 2). In some embodiments, the two or more IGF2R-binding portions have non-identical amino acid sequences (e.g., SEQ ID NOs: 4 and 7). KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0086] In some embodiments, the two or more IGF2R-binding portions are SEQ ID NOs: 1 and 1.

[0087] In certain embodiments, a nucleic acid encoding the fusion polypeptide as described herein is used for expression of the polypeptide in vitro (e.g., for production of the polypeptide), ex vivo, or in vivo. Such nucleic acids may be either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or analogs thereof. In some embodiments, the nucleic acid is RNA and is further modified, for example, to increase stability and / or reduce immunogenicity. In some embodiments, the RNA comprises one or more nucleotide modifications as described in U.S. Patent No. 10,272,150. In some embodiments, the RNA comprises pseudouridine and / or 5- methyl-cytidine, e.g., as described in U.S. Patent No. 10,272,150. In some embodiments, the RNA includes one or more stabilizing elements (e.g., an untranslated region at the 5’ or 3’ end, or a modified 5’ cap structure), e.g., as described in U.S. Patent No. 10,272,150. Exemplary nucleic acids encoding the fusion polypeptides described herein can be determined by one skilled in the art based on the fusion polypeptide amino acid sequences provided herein (e.g., SEQ ID NOs: 2-8).

[0088] The nucleic acids may be introduced into cells ex vivo or in vivo, or administered to a subject in vivo. For production of the polypeptide in vitro, one can use prokaryotic or eukaryotic cells for polypeptide production.

[0089] Nucleic acids for administration to a subject can be formulated for pharmaceutical administration. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier can vary depending on the mode of administration, which can be as described herein for administration of the polypeptide.

[0090] Nucleic acid (e.g., DNA or RNA or analogs thereof) compositions can be administered once, or multiple times as needed to induce the desired response. Multiple administrations can be administered, for example, bi-weekly, weekly, bi-monthly, monthly, or more or less often, as needed, for a time period sufficient to achieve the desired response. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0091] Nucleic acids can be administered in solution (e.g., a phosphate-buffered saline solution) by injection, usually by an intravenous, subcutaneous, or intramuscular route. Dosages depend on the route of administration and can be readily determined by one of skill in the art. B. Expression Cassettes and Vectors

[0092] Also provided is an expression cassette comprising a promoter operably linked to any of the polynucleotides described above or elsewhere herein. As described above, a promoter is “operably linked” to a polynucleotide when it is placed into a functional relationship with the polynucleotide sequence. Numerous promoters can be used in the constructs described herein.

[0093] In some embodiments the promoter is tissue-specific (i.e., it directs transcription at high levels only in particular types of cells or tissues). Exemplary tissue-specific promoters include, inter alia, a synapsin, camKIIa, glial fibrillary acidic protein (GFAP), retinal pigment epithelium (RPE), albumin (ALB), thyroxine binding globulin (TBG), myelin basic proteins (MBP), muscle creatine kinase (MCK), cardiac troponin T (TnT), or alpha-myosin heavy chain (aMHC), and the like. In some embodiments, the promoter is inducible (i.e., it directs transcription only under certain circumstances). For example, an inducible promoter used in the expression cassettes herein may be tetracycline inducible. In some embodiments, the promoter is constitutive (i.e., it directs transcription at relatively similar levels across all cell and tissue types). Exemplary constitutive promoters include, inter alia, a CMV promoter, CAG promoter, CBA promoter, EF1a promoter, PGK promoter, and the like.

[0094] The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue- preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a sequence by appropriately selecting and positioning promoters and other regulatory regions relative to that sequence. Exemplary promoters useful in the expression cassettes described herein include EF1a, CMV, and TBG promoters.

[0095] In some embodiments, the nucleic acids or expression cassettes provided herein can be administered as a vector. In some embodiments, the vector is a viral vector, wherein the nucleic acid encoding a fusion polypeptide comprising a target binding domain and an IGF2R-binding portion of a human IGF2 protein comprising a S39X substitution can be contained within a viral KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 vector and administered as a viral particle. Exemplary viral vectors include but are not limited to adenovirus vectors (e.g., Ad2, Ad5, Ad7), adeno-associated viral vectors, herpes simplex viral vectors, retroviral vectors, pox viral vectors (such as vaccinia and avian poxvirus vectors, such as the fowlpox and canarypox vectors), lentiviral vectors, alphavirus vectors, poliovirus vectors, measles vectors, and other positive and negative stranded RNA viruses, viroids, and virusoids, or portions thereof. The viral vector used herein may have (e.g., a replicating retroviral vector) or lack replicative capacity. In some embodiments, the nucleic acid may be codon optimized for delivery as a viral vector.

[0096] In other embodiments, the nucleic acid may be administered as a non-viral vector, including, but not limited to, as a plasmid, in a nanoparticle, (e.g., a lipid nanoparticle), or in a liposome. C. Cells

[0097] Also provided herein are cells comprising the nucleic acid as described herein, or the expression cassette as described herein. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a primary cell, e.g., a primary human immune cell or a primary human hematopoietic cell. In some embodiments, the primary human immune cell is a primary human T-cell or natural killer cell. In some embodiments, the cell is genetically engineered to express another recombinant molecule, e.g., a chimeric antigen receptor (CAR), such as a human CAR T-cell or CAR-expressing natural killer cell. The cell expressing a chimeric antigen receptor may target, for example, one or more antigens associated with a tumor or a B cell (e.g., CD19, CD20, or CD33). The nucleic acid or expression cassette may be introduced to the cell using one of the methods as described herein (e.g., as a vector or as RNA).

[0098] The nucleic acid or expression cassette is introduced into the cell such that the fusion polypeptide is expressed and / or secreted by the cell. In some embodiments, introducing comprises contacting the cell with a vector comprising a nucleic acid described herein such that the fusion polypeptide is expressed and / or secreted by the cell. In some embodiments, delivering comprises contacting the cell with a nucleic acid (e.g., RNA) encoding the fusion polypeptide such that the fusion polypeptide is expressed and / or secreted by the cell. Delivering may occur in vitro, ex vivo, or in vivo. In some embodiments, the fusion polypeptide is secreted by the cell KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 (i.e., is soluble) and binds a target molecule and IGF2R on a receiver cell, and the target molecule-IGF2R complex is internalized and lysosomally degraded by the receiving cell. In some embodiments, the cell expressing or secreting the fusion polypeptide described herein is in a human. Accordingly, the nucleic acids described herein can be administered to a human in need of a condition requiring targeted protein degradation in an appropriate dosage to ameliorate or treat at least one symptom thereof. D. Pharmaceutical Compositions

[0099] Also provided are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and the nucleic acid as described herein. In some embodiments, the pharmaceutical composition comprises a nucleic acid or expression cassette encoding the fusion polypeptide as described herein. In some embodiments, the pharmaceutical composition comprises a cell comprising the nucleic acid or expression cassette encoding the fusion polypeptide as described herein. The pharmaceutical composition can additionally contain other therapeutic agents that are suitable for treating or preventing a given disorder. Pharmaceutically carriers can enhance or stabilize the composition, or to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0100] A pharmaceutical composition as described herein can be administered by a variety of methods known in the art. The route and / or mode of administration vary depending upon the desired results. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., polypeptide described herein, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0101] Typically, a therapeutically effective dose or efficacious dose of the polypeptides, nucleic acids, or vectors described herein is employed in the pharmaceutical compositions. The KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 polypeptides, nucleic acids, or vectors can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the desired response (e.g., a therapeutic response). In determining a therapeutically or prophylactically effective dose, a low dose can be administered and then incrementally increased until a desired response is achieved with minimal or no undesired side effects. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0102] Actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors. E. Fusion Polypeptides

[0103] Also provided is a fusion polypeptide encoded by the nucleic acids described herein, comprising a target binding domain and a IGF2R-binding portion. As described above, the fusion polypeptide comprises a substitution corresponding to S39X in wildtype human IGF2 (SEQ ID NO:1), wherein X is an amino acid other than serine. In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 4. In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 2. In some embodiments, X in the S39X substitution is proline and the substitution is S39P. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 In some embodiments, the IGF2R-binding portion further comprises one or both of a substitution corresponding to E6X and F19X relative to SEQ ID NO: 1, where X is any amino acid other glutamic acid at residue 6 of SEQ ID NO: 1 and phenylalanine at residue 19 of SEQ ID NO: 1. In some embodiments, the IGF2R-binding portion further comprises one or both of a substitution corresponding to E6R and F19L relative to SEQ ID NO: 1. In some embodiments, the IGF2R- binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 3. In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5. In some embodiments, the IGF2-binding protein further comprises at least one substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution position. In some embodiments, the at least one substitution that reduces binding to IGF1R is R24A, F48X, T58A, Y59X, and / or T62A. In F48X and Y59X substitutions, X is any amino acid other than the wildtype position in SEQ ID NO: 1. In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6. In some embodiments, the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7. In some embodiments, the IGF2-binding protein comprises, without the S39X, E6R, and / or F19L substitutions, at least one aforementioned substitution that reduces binding to IGF1R. In some embodiments, the IGF2R- binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8. II. Methods

[0104] Further provided herein are methods of degrading a target molecule comprising contacting the target molecule with a fusion polypeptide described herein in the presence of a first cell comprising an IGF2R transmembrane protein under conditions in which the fusion polypeptide binds the target protein and binds the IGF2R transmembrane protein and is internalized into the cell. The fusion polypeptide-target molecule complex is ultimately degraded by the first cell. The first cell may be any cell which comprises an IGF2R transmembrane protein.

[0105] In some embodiments, the fusion polypeptide used in the methods described herein may be expressed from a second cell. In some embodiments, the second cell is a human cell. In some embodiments, the second cell is a primary cell, e.g., a primary human immune cell or a primary human hematopoietic cell. In some embodiments, the primary human immune cell is a KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 primary human T-cell or natural killer cell. In some embodiments, the second cell is genetically engineered to express another recombinant molecule, e.g., a chimeric antigen receptor (CAR), such as a human CAR T-cell or CAR-expressing natural killer cell. In some embodiments, the CAR binds to a tumor antigen or a B-cell antigen (e.g., CD19, CD20, or CD33). In some embodiments, the CAR binds to receptor tyrosine-protein kinase erbB-2 / HER2, CD22, B-cell maturation antigen (BCMA), and epidermal growth factor receptor (EGFR). Additional exemplary CAR targets that can be used in the CARs described herein can be found in Table 1 of Labanieh & Mackall, Persp., 614: 635-648 (2023).

[0106] In embodiments in which the fusion polypeptide is expressed from a second cell, the fusion polypeptide may be secreted from the second cell. In some embodiments, the second cell is programmed to secrete the fusion polypeptide, and the target molecule is bound of the second cell. Upon binding the target molecule, the fusion polypeptide binds to IGFR2 transmembrane protein on a first cell, which in turn internalizes and degrades the fusion and bound target.

[0107] The methods described herein comprise contacting the target molecule with a fusion polypeptide. The contacting used in the methods described herein may occur in vitro or in vivo. In some embodiments, the contacting occurs in vivo in a human. In some embodiments, the contacting comprises introducing the fusion polypeptide into the human. The fusion polypeptide used in the methods herein may be introduced, e.g., into a human, in a nucleic acid, vector, expression cassette, or cell expressing and / or secreting the fusion polypeptide as described above or elsewhere herein. The fusion polypeptide may be introduced to an isolated cell, isolated tissue, or to a subject (e.g., a human) in vivo.

[0108] In some embodiments, the contacting the target molecule with a fusion polypeptide comprises introducing a nucleic acid encoding the fusion polypeptide into the human, wherein the fusion polypeptide is expressed in a cell in the human. In some embodiments, the nucleic acid encoding the fusion polypeptide is contained in a vector, and the contacting comprises introducing the vector into the human, such that the vector enters a cell wherein the fusion polypeptide is expressed. In embodiments in which the fusion polypeptide is expressed and / or secreted from a second cell, the contacting comprises introducing the second cell into the human. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 EXAMPLES Materials and Methods

[0109] Recombinant GELYTACs production. BL21 DE3 E. coli (Agilent) were transformed with a vector containing sequences encoding for GELYTACs or nanobody only controls with pelB signal sequence for localization to the periplasm for disulfide bond formation. A colony was picked into 10mL of LB (supplemented with 2% glucose and kanamycin) overnight at 37 °C. The next day, the starter culture was added to 1L of 2xYT (supplemented with antibiotics) and grown at 37 °C to an OD600 ~ 1.0-1.3. The culture was then induced at 1mM and grown overnight at 16 C at 225 rpm. The next day, the culture was centrifuged at 7000g for 10 min and the supernatant discarded. The cell pellet was then resuspended thoroughly with 20mL 1x TES (0.2M Tris, pH=8, 0.5 mM EDTA, 0.5 M sucrose) and then the mixture was added to 20mL of ice cold ddH2O (supplemented with protease inhibitor). The mixture was then incubated overnight at 4 C with shaking. The next day the mixture was centrifuged at 16,000xg. The supernatant was then filtered using a 5uM filter and then purified using Ni NTA column (Cytiva / GE Healthcare) on an FPLC (AKTA Pro). Following Ni-NTA purification, the mixture was then purified using size exclusion chromatography (Superdex 75 Increase 10 / 300 GL) to and only the monomer was isolated.

[0110] For FIGs, 2A, 2B, 3A, the GELYTACs were mixed with mCherry containing media before cells (also in mCherry media) were added. The mCherry GELYTACs dose response curve shown in FIG. 2A is data as the mCherry GELYTACs (original) dose response curve in FIG.3C, and was set up alongside the mCherry GELYTACs (G2) does curve in FIG.3C.

[0111] Yeast display. The yeast culture and display protocols and library generation protocols are described in e.g., Branon et al., Nat. Biotechnol., 36(9), 880–898 (2018).

[0112] Cell Culture (excluding primary T cell culture). All cell lines used were less than passage 20. HEK 293T cells (ATCC) and 293GP retroviral packaging line (gift from Surgery Branch (National Cancer Institute, National Institutes of Health)) were cultured in a DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% Glutamax (Gibco), 100 units / mL penicillin, and 100 mg / mL streptomycin at 37 °C under 5% CO2. K562’s (ATCC) was cultured in in RPMI (Sigma Aldrich) supplemented with 10% FBS, 1% Glutamax (Gibco), 100 units / mL penicillin, and 100 mg / mL streptomycin at 37 °C under 5% CO2. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0113] HEK293T coculture with K562. 12 well plates were coated with HFN (1mL of HFN from Sigma added to 50mL PBS), by adding 700uL of PBS to the wells. A 70-90% confluent HEK293T T75 flask were lifted, by aspirating media and adding ~1-2mL trypsin. Cells were diluted to 3.5e5 cells / mL, and 1mL was added to each well and slightly agitated before returning it in the incubator. On the next day, cells were transfected with plasmids encoding for GELYTACs or controls.

[0114] To make the transfection mix, 1000ng of plasmids encoding for GELYTACs or controls were added to 100uL of blank DMEM. The mixture was mixed by flicking the tube, and then 5uL of polyethylene imine (PEI). The tube was flicked gently to homogenize, and allowed to incubate for 20 min. After incubation, the transfection mix was added directly to cells dropwise, cells were returned to incubator for 12-18 hrs. After 12-18 hrs, media + the transfection mix were aspirated and replated with 1 mL of RPMI mCherry with 3.0e5 GFP K562 / mL. After 3 hrs 100nM of soluble antigen (i.e., mCherry, TGF- , or IL6R) was spiked into the coculture.

[0115] At the time of analysis, the GFP K562s were analyzed by flow cytometry for median fluorescence of mCherry or AlexaFluor-647 tagged proteins. GFP was used to distinguish between K562 cells and HEK293T cells.

[0116] Retrovirus production. Retroviral supernatant was packaged using 293GP cells and theRD114 envelope plasmid. In brief, 11 g RD114 and 22 g of the corresponding MSGV1transfer plasmid that contain GELYTACs or controls were delivered to 293GP cells grown on 150mm HFN dishes (Corning) to 80% confluency by transient transfection with Lipofectamine 2000 (Thermo Fisher). Media was replenished every 24 hrs. Virus production was performed side-by-side for comparable GELYTACs and control constructs. Retroviral supernatant was harvested 48-hr post transfection. Supernatant from replicate dishes were pooled, centrifuged to deplete cell debris, and stored at -80C until use. A similar protocol is described in e.g., Labanieh et al., Cell, 185(10), 1745-1763.e22, (2022) and Yamada-Hunter et al., bioRxiv [Preprint], (2023), doi: 10.1101 / 2023.06.20.545790.

[0117] T cell activation. Anonymous healthy donor buffy coats were collected by and purchased from the Stanford Blood Center under an IRB-exempt protocol. Primary human T cells (CD3+) were isolated using the RosetteSep Human T cell Enrichment kit (Stem Cell 32 KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 Technologies) according to the manufacturer’s protocol using Lymphoprep density gradient medium and SepMate-50 tubes. All purified T cells were cryopreserved in CryoStor CS10 medium (Stem Cell Technologies).

[0118] At Day 0, primary human T cells were thawed and activated with anti-CD3 / CD28 Human T-Expander Dynabeads (Thermo Fisher) at a 3:1 bead to cell ratio. On Day 2 virus coated culture plates were prepared on non TC-treated 12-well plates that had been pre-coated with RetroNectin (Takara Bio) according to the manufacturer’s instructions, by incubating with1mL of retroviral supernatant (2x107-5x107 TU / mL) and centrifugation at 3200 RPM, 32 °C fortwo hrs. The supernatant was subsequently aspirated from the wells and 0.5x106 T cells were added in 1mL of T cell media comprised of: AIM V (Thermo Fisher), 5% fetal bovine serum (FBS), 100 U / mL penicillin (Gibco), 100 mg / mL streptomycin (Gibco), 2 mM L-glutamine (Gibco), 10 mM HEPES (Gibco), and 100 U / mL rhIL2 (Peprotech). After addition of the T cells, the plates were gently spun down at 1200 RPM for 2 min then incubated for 24hrs at 37 °C 5% CO2. This transduction process was repeated at Day 3. Dynabeads were removed on Day 4 by magnetic separation. Cells were maintained between 0.4 - 2x106 cells / mL and expanded until Day 10. A similar protocol is described in e.g., Labanieh et al., Cell, 185(10), 1745-1763.e22, (2022) and Yamada-Hunter et al., bioRxiv [Preprint], (2023), doi: 10.1101 / 2023.06.20.545790.

[0119] T cell coculture with K562 cells. Coculture experiments were conducted with primary T cells 7-14 days post retroviral transduction. For mCherry and Il6R GELYTACs, 0.6e6 T cellswere cocultured with 0.3e6 K562’s. For TGF- GELYTACs, 0.9e6 T cells were cocultured with0.1e6 K562 cells. Coculture was set up with complete RPMI supplemented with 100 U / mL rhIL2.

[0120] At the time of analysis, the GFP K562’s and T cells were analyzed by flow cytometry for median fluorescence of mCherry or AlexaFluor-647 tagged proteins. GFP and / or differences in forward and sider scatter were used to distinguish between K562 cells and T cells.

[0121] Flow cytometry. 300uL of K562 and or T cell culture was transferred to 96-well V- bottom plate and spun down (500g for 1min). Cells were then washed 1x PBS w / 0.5% supplemented bovine serum albumen (BSA). Cells were then incubated with SYTOX Blue (1:1000 dilution) for 5 min. Flow cytometry was performed a BioRad ZE5 flow cytometer, and analysis was performed using the FlowJo software package. Gating was performed on single KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 cells and live cells. This instrument is equipped with a 405-nm violet laser, a 488-nm blue laser, a 561-nm green laser and a 639-nm red laser.

[0122] For FIGs.2A, 2B, and 4A, K562 cells were washed with PBS then incubated with 5% trypsin for 1 min at 37 °C. After trypsin treatment, trypsin was quenched with PBS supplemented with 0.5% BSA and then subsequently subjected to the standard flow protocol described above. This was done to cleave GELYTACs and receptors on the cell surface that may contribute to mCherry signal from non-internalized mCherry.

[0123] Western blot protocols. Conditioned media from transfected HEK293T cells or transduced T cells was collected (cells were removed by centrifugation) and LDS Sample Buffer (4X) (for reduced blot 4x loading buffer was supplemented with 10% 1M DTT) and boiled at 95 °C for 10 min. The recommended volume (15uL for 26-well gel or 30uL for 18-well gel) was loaded on a SDS–PAGE (10% Bis-Tris gel), then transferred to a nitrocellulose membrane. After transfer, the blot was blocked with Odyssey Blocking Buffer (PBS or TBS) (LI-COR) for 1 h at room temperature with gentle shaking. Membranes were stained with M2 anti-FLAG(Sigma Aldrich) for 1 hr at room temperature or at 4 °C with gentle shaking, then washed threetimes with PBS-T for five minutes each. The membrane was then incubated with 800CW goat anti-mouse IgG (1:10,000) in Odyssey Blocking Buffer (PBS or TBS) for 1 h at room temperature with gentle shaking. Membranes were washed three times with TBS-T, then imaged using an OdysseyCLxImager (LI-COR). Quantification of band intensities was performed using Image Studio Software (LI-COR).

[0124] Determining GELYTACs and controls concentration in coculture assay. Conditioned media from transfected HEK293T cells or transduced T cells were collected and subjected to the Western blot protocol described above. Alongside the supernatant samples, were a series of standards (samples of recombinant FLAG-tagged GELYTACs at set concentration). A standard curve was determined by plotting signal of the recombinant GELYTAC band versus the mass of recombinant GELYTAC loaded.

[0125] To calculate the mass of the secreted GELYTACs loaded onto the gel, the intensity of the secreted GELYTACs band was divided the slope of the standard curve. From calculated mass, the concentration can be calculated. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004

[0126] Confocal imagining. K562 cells were plated on HFN coated glass cover slip bottom well plates with phenol red free complete RPMI. On the next day cells were treated with 275 nM of recombinant mCherry GELYTAC or controls and 100 nM of mCherry for 1 hr. The well plates were then imaged live using a Zeiss AxioObserver microscope. This microscope is equipped with 603 oil immersion objectives, outfitted with a Yokogawa spinning disk confocal head, Cascade II:512 camera, a Quad-band notch dichroic mirror (405 / 488 / 568 / 647), and 405 (diode), 491 (DPSS), 561 (DPSS) and 640 nm (diode) lasers (all 50 mW). DAPI (405 laser excitation, 445 / 40 emission), Alexa Fluor488 (491 laser excitation, 528 / 38 emission) and AlexaFluor647 (640 laser excitation, 700 / 75 emission) and differential interference contrast (DIC) images were acquired through a 60x oil-immersion lens. Acquisition times ranged from 100 to 2,000 ms. All images were collected and processed using SlideBook 6.0 software (Intelligent Imaging Innovations). A similar protocol is described in e.g., Qin, Cell 186(15), 3307-3324.e30 (2023).

[0127] Knock-out cell line generation. Knock-out K562 cell lines were generated by electroporating the sgRNA (Synthego) Cas9 (ID Technologies) complex using a Lonza 4D Nucleofector. The cell line was generated using Synthego’s protocols.

[0128] Top-Down Protein Mass Spectrometry. After protein expression and purification, the protein samples were further buffer exchanged using a methanol / chloroform / water precipitation and resolubilization method (see, e.g., Rogers et al., Anal. Chem., 95 (35), 13091-13100 (2023)). Here, 300 L of 10 mM TCEP prepared in cold LC / MS-grade water (4 °C) was added to 100 Lof protein solution. Then, 400 L of cold methanol ( 20 °C) was added to the protein solutionand vortexed for 30 s followed by 100 L of cold chloroform ( 20 °C) and an additional 30 s ofvortexing. The sample was centrifuged for 10 min at 18,000g at 4 °C after which a biphasic mixture was created with a protein pellet present at the interface. The top layer of the solutionwas discarded without disturbing the protein pellet. Then, 400 L of cold methanol ( 20 °C)was added to the sample and gently vortexed. The sample was centrifuged for 10 min at 18,000 g at 4 °C after which the supernatant was discarded. The cold methanol and centrifugation washing step was repeated two additional times. Protein pellets were resolubilized with 4 μL of80% formic acid ( 20 °C) and diluted to 1% formic acid with 80:20 water: acetonitrile. Top-down LC-MS was carried out using an Agilent 1260 Infinity II high performance liquid KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 chromatography (HPLC) system coupled to an Agilent 6230 ToF LC / MS (Agilent Technologies). Samples were injected onto an Agilent PLRP-S column (2.1 × 50 mm, 5 μm particle size, 1,000 Å pore size) using a gradient of 10% to 90% mobile phase B (0–5 min, 10% B; 5–15 min, 20%–65% B; 15–18 min, 65%–90% B, 18–22 min, 90% B; 22–25 min, 10% B; mobile phase A set to 0.2% formic acid in water; mobile phase B set to 0.2% formic acid in acetonitrile). Flow rate was set to 200 L / min with a column temperature of 60 °C. Mass spectra were taken at a scan rate of 1 Hz over a 200–3200 m / z scan range with the ToF set to extended dynamic range. The mass spectrometer was operated using a dual Agilent jet stream (AJS) high-sensitivity ion source with the following instrument parameters: gas temperature (275°C), drying gas (12 L / min), nebulizer (40 psi), sheath gas temperature (400 °C), sheath gas flow (12 L / min), VCap(3000 V), nozzle voltage (2000 V), fragmentor (250 V), skimmer (65 V), and Oct 1 RF Vpp (750 V). Mass spectra were output from the MassHunter (Agilent Technologies) software and analyzed using MASH Native. A similar protocol is described in Donnelly et al., bioRxiv [Preprint] (2024), doi: 10.1101 / 2024.01.03.574113. Results Example 1. Design of GELYTACs.

[0129] To design an all-protein, genetically-encodable bifunctional molecule for targeted degradation of extracellular proteins, a small protein binder was fused to IGF2 peptide via a flexible linker (FIG.1A). IGF2 binds to domain 11 of IGF2R with a KD of 4.5 nM (see, e.g., Brown et al., EMBO, 27(1), 265–276 (2008)) and is subsequently shuttled to the lysosome (see, e.g., Oka et al., JBC, 260(16), 9435–9442 (1985) and Zavorka et al., Onctotarget, 7(38), 10–16, (2016)). This biologic can either be administered recombinantly or be delivered by therapeutic cells as a cell therapy (FIGs. 1B-C).

[0130] As a proof of concept, a GELYTAC targeting a model protein, mCherry, was generated. The nanobody LAM4 which binds to mCherry with a KD of 180 pM was selected (see, e.g., Fridy et al., Nat. Methods, 11(12), 1253–1260 (2014)) and fused it to IGF2. mCherry GELYTAC was produced by recombinant expression in E. coli. To test mCherry GELYTAC’s ability to selectively target and mediate internalization of mCherry into cells, K562 leukemia cells were treated with a mixture of mCherry protein (100 nM) and mCherry GELYTAC (from KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 0-1000 nM) for 24 hrs (FIG.2A). Flow cytometry showed a 30-fold increase in mCherry fluorescence in K562 cells at 350 nM of mCherry GELYTAC while controls with mCherry nanobody only or an alternate-targeting GELYTAC (EGFR GELYTAC) showed minimal mCherry uptake in K562 cells across all concentrations. In agreement with the flow cytometry data, fluorescence microscopy showed that mCherry colocalized with stained lysosomes, in cells treated with mCherry GELYTAC. A time course analysis showed that mCherry uptake continues at 48 hrs of mCherry GELYTAC treatment.

[0131] To elucidate the mechanism of action, flow cytometry experiments were repeated using IGF2R knockout K562 cells. In these samples, no uptake of mCherry was observed (FIG. 2B) in the presence of mCherry GELYTAC. These data suggest that GELYTAC effectively targets soluble extracellular proteins for lysosomal targeting of via recruitment of IGF2R. Example 2. Directed Evolution of GELYTACs.

[0132] GELYTAC was designed such that it can be secreted by therapeutic cells. However, unlike recombinant proteins, for which dosing concentrations can be specified, GELYTAC concentrations cannot be controlled. Thus, one aim was to make GELYTACs more effective at lower concentrations by lowering their EC50 through directed evolution, which is facilitated by the genetically-encoded nature of GELYTAC.

[0133] First, to test whether higher affinity of IGF2 translates into a more efficacious GELYTAC, mCherry GELYTACs were created using a series of point mutants of IGF2 with varying affinities for IGF2R (FIG.3A). A negative association between IGF2 variants’ KD’s and mCherry internalization by the corresponding mCherry GELYTAC was observed (FIG.3B). This provided the rationale to engineer an improved IGF2 binder for improving GELYTAC potency through yeast surface display directed evolution.

[0134] Using the mCherry GELYTAC as a scaffold, diversity in the IGF2 domain was introduced by error prone PCR. The library was displayed on the yeast cell surface via fusion to the C-terminus of the yeast mating protein Aga2p (see, e.g., Lam et al., Nat. Methods, 12(1), 51– 54 (2014)). To select for clones capable of binding to IGF2R with high affinity, the yeast library was incubated with tagged recombinant IGF2R (Fc-fused or monomeric). Staining with anti- myc antibody was used to quantify GELYTAC expression level. Two-dimensional KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 fluorescence-activated cell sorting (FACS) sorting was used to enrich library members with high IGF2R / anti-myc intensity ratio. To increase selection stringency in the last rounds, staining protocols were modified to either include competitive washes or eliminate the avidity effects from Fc-fused IGF2R by shifting to monomeric IGF2R, as shown in Table 2 below. Ultimately, after 6 rounds of sorting over 2 generations, a clone with the IGF2 mutations E6R, F19L, and S39P (termed ‘G2’) was isolated. G2 exhibited improved binding to IGF2R’s ectodomain at expression levels matched to that of wild-type IGF2 (FIG.3C). S39P is a crucial mutation for G2’s improved binding, as confirmed by comparing G2’s triple mutations to the E6R and F19L double mutant using flow cytometry. Table 2. Clone selection conditions.

[0135] To verify that the benefits of directed evolution translate into a cellular context, the efficacy of the original GELYTAC was compared to the evolved G2 GELYTAC in mCherry uptake by K562 cells. Dramatic reduction of the EC50 from 97 nM to 22 nM was observed (FIG. 3C). Finally, to determine if both the original and G2 GELYTACs can be secreted by HEK293T cells, an anti-FLAG western blot of the supernatant of HEK293T cells transfected with these constructs was performed. Not only did both constructs secrete well, the evolved G2 GELYTAC also exhibited decreased propensity to oligomerize. This increases the amount of biologically active material, which is likely to contribute to improvements in the in efficacy of downstream cell-based therapy applications where GELYTACs cannot be purified. Example 3. Cell-based secretion of GELYTACs and knockdown of other targets.

[0136] Next, an alternate mode of GELYTAC administration was explored: secretion by HEK293T sender cells to drive uptake of soluble targets into receiver K562 cells. To achieve this, HEK293T cells transfected with mCherry GELYTAC (original or G2) were cocultered KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 with, control GELYTAC (alternate-targeting, IL6R GELYTAC), or mCherry nanobody only with GFP-expressing K562 cells for 24 hrs in media supplemented with 100 nM mCherry. The presence of all constructs in the conditioned media were verified at 24 hrs by Western blot and comparable secretion amongst the constructs was observed. Another band of slightly lower mass was also observed in both GELYTAC secretion samples. To determine the identity of the second band, both GELYTACs were analyzed by top-down mass spectrometry (MS); while both contained a portion of truncated GELYTAC (C-terminal truncation of 28 and 27 amino acids for original and G2, respectively), the predominant species corresponded to the full length.

[0137] Next, the receiver cells were analyzed cells using flow cytometry and observed a ~6- fold and ~9-fold increase in mCherry median fluorescence intensity in K562 cells cocultured with HEK293Ts secreting original and G2 mCherry-GELYTACs, respectively (FIG.4A). The same experiment was performed with the original and G2 mCherry GELYTACs on IGF2R KO K562 cells, and no apparent uptake by the IGF2R KO cells was observed, demonstrating that uptake of proteins in the coculture model is mediated by IGF2R, as observed for treatment with the recombinant GELYTAC.

[0138] While observed robust mCherry uptake into receiver cells had been observed as a proxy for protein degradation, to determine if secreted mCherry GELYTACs could mediate effective clearance and degradation of mCherry, mCherry fluorescence was analyzed in the coculture supernatant for mCherry clearance and the combined supernatant plus cells for mCherry degradation using a plate reader. After incubation of GELYTAC secreting HEK293T cells with K562 receiver cells, it was observed that ~25% of mCherry is cleared from the media (FIG.4B, left panel) and ~20% of mCherry is degraded (FIG.4B, right panel).

[0139] Finally, GELYTAC design optimized for targeting mCherry can be generalized to internalization of more therapeutically relevant targets by simply exchanging the nanobody. Tothis end, GELYTACs targeting soluble proteins TGF- and IL6R were developed. TGF- andIL6R are immunosuppressive factors in the tumor microenvironment, so depleting these soluble factors could help improve the efficacy of cancer immunotherapy treatments, such as immune- checkpoint blockade and CAR-T therapy (see, e.g., Labanieh et al., Nature, 614(7949), 635–648 (2023)). Local degradation of these targets is attractive because systemic targeting of immunosuppressive factors has been shown to be toxic (see, e.g., Ala et al., Curr. Cardiol. Rep. KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-00421(12), 1–9 (2019)). The TGF- GELYTAC was designed by fusing our evolved IGF2 with aTGF- scFv derived from the clinical candidate antibody Fresolimumab (FIG. 4C) (see, e.g.,Moulin et al. Structures of a pan-specific antagonist antibody complexed to different isoforms ofTGF reveal structural plasticity of antibody-antigen interactions. Protein Sci., 23(12), 1698–1707 (2014)). The TGF- GELYTAC was tested in a coculture system with HEK293T secretingthe TGF- GELYTAC, GFP-expressing K562, and media supplemented with 100 nMAlexaFluor647 (AF-647)-tagged TGF- . Like in the mCherry assays, a ~2.6-fold increase inAF-647 fluorescence was observed in K562 cells (FIG. 4D, left panel). To determine if TGF-GELYTAC could mediate the degradation of TGF- , 1 nM of biotinylated TGF- was addedtoK562 cells cocultured with HEK293T cells secreting mCherry GELYTAC plus TGF- scFv, orTGF- GELYTAC, and analyzed via streptavidin western blot a mixture of the cell lysate andsupernatant. The TGF- GELYTAC mediated ~52% reduction in biotinylated TGF- whencompared to the combined mCherry GELYTAC and TGF- scFv control (FIG. 4D, right panel).

[0140] To test clearance and degradation of another target, IL6R, a GELYTAC consisting of evolved IGF2 fused to a clinical candidate IL-6R nanobody (ALX-0061) was developed (FIG. 4E) (see, e.g., Van Roy et al., Arthritis Res. Ther., 17(1) (2015)). In the coculture assay, media was supplemented with 100 nM AF-647-tagged IL-6R and ~10-fold increase in AF-647 signal was observed in the K562 cells when cocultured with IL6R GELYTAC secreting HEK293T cells (FIG.4F).

[0141] In these experiments, the MFI increase mediated by secreted TGF- GELYTAC waslower compared to mCherry and IL6R GELYTACs. This could be due to binding of TGF- tothe TGF- 1 receptor or other receptors resulting in increased background signal. Developmentof tighter binding nanobodies could potentially improve efficacy of this class of GELYTAC that competes with endogenous cell-surface receptors for target binding. Overall, these results demonstrate the modularity and potential of GELYTAC to recognize a wide range of targets by simply exchanging the POI binder.

[0142] To potentially enable spatial specificity for GELYTACs, GELYTAC could be integrated into adoptively transferred T cell therapy, such as CAR-T therapy. In adoptive T cell therapy, engineered primary T cells home to tumors and proliferate in the tumor microenvironment based on recognition of specific tumor antigens. Furthermore, primary T cells KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 can be engineered to secrete factors under the control of logic gates determined by recognition of tumor-specific factors (see, e.g., Roybal et al., Cell, 167(2) 419–432 (2016)). Thus, secretion from T cells could enable spatial specificity at the tumor site. To demonstrate the feasibility of this approach, donor human primary T cells were transduced with retrovirus encoding mCherry G2 GELYTAC (FIG.5A). Robust GELYTAC secretion was observed, and upon co-incubation of GELYTAC-secreting T cells with K562 cells in media supplemented with 100 nM mCherry, 5.25-fold and 2.2-fold increases in mCherry MFI in K562 and T cells, respectively, was observed (FIG. 5B). No significant increase in mCherry MFI was observed in controls with IL6R GELYTAC or mCherry nanobody. Interestingly, while there were differences in secretion of mCherry GELYTACs across primary T cells from 3 different donors, the degree of mCherry uptake in T cells between the 3 donors was similar. Since the primary T cells were engineered to secrete GELYTACs, the IGF2 receptors were likely already saturated by GELYTACs on the membrane, so uptake in engineered T cells was more desensitized to increases or decreases in secretion.

[0143] Lastly, TGF- and IL6R GELYTACs were tested in the primary T cell coculturesystem. For these experiments, human primary T cells secreting GELYTACs were cocultured with K562 tumor cells and incubated with 100 nM of AF-647 tagged target molecules. ForTGF- GELYTACs, a 1.6-fold increase in TGF- uptake into K562 cells was observed (FIG.5C, left panel). For IL6R GELYTACs, a 2.2-fold increase and 5.2-fold increase was observed in IL6R uptake into K562s and primary T cells, respectively (FIG. 5C, middle and right panels,respectively). For both TGF- and IL6R experiments, no major increase in AF-647 fluorescencewas observed in the mCherry GELYTAC or binder-only controls.

[0144] These results from human primary T cells demonstrate the potential for secreted GELYTACs to work in multiple cell types. Additionally, the ability of sender cells to act on themselves creates the possibility of feedback regulation of GELYTAC secretion. Example 4. IGF1R Ablating Mutants.

[0145] To decrease undesirable binding to IGF1R, mutations that ablate IGF1R binding were determined. Amino acid positions of SEQ ID NO: 1 that have contacts with IGF1R, shown in Table 3 below, were mutated to a serine or alanine, though any amino acid could be used. 41 KILPATRICK TOWNSEND 797228011PATENT Attorney Docket No. 110221-1509199-011110WO Client Ref. No. CZB-302S-PC / S24-004 Table 3. Amino acid positions that contact IGF1R

[0146] Point mutants were generated via Gibson assembly cloning. Plasmids that were successfully cloned were transformed into chemically competent yeast (S. cerevisiae strain EBY100). Transformation and generation of chemically competent yeast were generated using the Zymo Frozen- EZ Yeast Transformation II Kit.

[0147] After transformation, yeast were plated on SDCAA agar plates. After 3 days, colonies appeared, and a colony was picked into SDCAA. After the 2-3 days, the SDCAA became saturated and 1mL of SDCAA was added to 3mL of SGCAA to initiate display on the yeast surface overnight. The next day, 300uL of saturated yeast culture was taken spun down at 3000 g for 2 min. The supernatant was removed and washed 1x w / PBS-B (PBS supplemented w / 2% bovine serum albumen). Next, the yeast were incubated with PBS-B for 30 min – 1hr. After blocking, yeast were incubated with 100nM of Alexa647 tagged recombinant IGF1R ectodomain (R&D systems). After 1hr of incubation, yeast cells were washed 3x w / PBS-B and then incubated with 2.5uL of Anti-myc PE (for expression) in 100uL of PBS-B. Yeast were again washed 3x w / PBS-B and then analyzed via flow cytometry.

[0148] Mutants that showed ablation to IGF1R are the following: Arg24Ala, Phe49Ala, Phe48Ser, Thr58Ala, Tyr59Ala, Tyr59Ser, Thr62Ala.

[0149] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. KILPATRICK TOWNSEND 797228011INFORMAL SEQUENCE LISTING SEQ ID NO:1: Wildtype Human IGF2 AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYC ATPAKSE SEQ ID NO:2:Fragment of IGF2 mutant with S39X AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRXR SEQ ID NO:3: Fragment IGF2 mutant with S39X, E6R, and F19L (G2) AYRPSRTLCGGELVDTLQLVCGDRGFYFSRPASRVSRRXR SEQ ID NO:4: Full length IGF2 mutant with S39X AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRXRGIVEECCFRSCDLALLETYC ATPAKSE SEQ ID NO:5: Full length IGF2 mutant with S39X, E6R and F19L (G2) AYRPSRTLCGGELVDTLQLVCGDRGFYFSRPASRVSRRXRGIVEECCFRSCDLALLETY CATPAKSE SEQ ID NO: 6: Full length IGF2 mutant with S39X and IGF1R mutations: R24A, F48X, T58A, Y59X, and / or T62A, wherein X is any amino acid AYRPSETLCGGELVDTLQFVCGDAGFYFSRPASRVSRRXRGIVEECCXRSCDLALLEAX CAAPAKSE SEQ ID NO: 7: Full length IGF2 mutant with S39X, E6R, and F19L, and IGF1R mutations: R24A, F48X, T58A, Y59X, and / or T62A, wherein X any amino acid AYRPSRTLCGGELVDTLQLVCGDAGFYFSRPASRVSRRXRGIVEECCXRSCDLALLEAX CAAPAKSE SEQ ID NO: 8: Full length IGF2 mutant with only IGF1R mutations: R24A, F48X, T58A, Y59X, and / or T62A, wherein X is any amino acid AYRPSETLCGGELVDTLQFVCGDAGFYFSRPASRVSRRSRGIVEECCXRSCDLALLEAXC AAPAKSE KILPATRICK TOWNSEND 797228011

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a target binding domain and an IGF2R-binding portion of a human IGF2 protein, wherein the IGF2R-binding portion comprises a substitution corresponding to S39X in the wildtype human IGF2 protein sequence (SEQ ID NO:1), wherein X is an amino acid other than serine.

2. The nucleic acid of claim 1, wherein the substitution is an S39P substitution.

3. The nucleic acid of claim 1 or 2, wherein the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO:

4.

4. The nucleic acid of claim 1 or 2, wherein the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO:

2.

5. The nucleic acid of any one of claims 1-4, wherein the IGF2R-binding portion comprises at least one further substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution.

6. The nucleic acid of claim 5, wherein the at least one substitution that reduces binding to IGF1R is R24A, F48X, T58A, Y59X, and / or T62A, wherein X is either serine or alanine.

7. The nucleic acid of claim 6, wherein the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO:

6.

8. The nucleic acid of any of claims 1-4, wherein the IGF2R-binding portion further comprises one or both of a substitution corresponding to E6R and F19L as determined from the wildtype human IGF2 protein sequence (SEQ ID NO: 1).

9. The nucleic acid of any one of claims 1-4, wherein the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO:

5. KILPATRICK TOWNSEND 79722801110. The nucleic acid of any one of claims 1-4, wherein the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO:

3.

11. The nucleic acid of any one of claims 8-10, wherein the IGF2R-binding portion comprises at least one further substitution that reduces binding to IGF1R compared to the IGF2R-binding portion having a corresponding wildtype amino acid at the at least one further substitution.

12. The nucleic acid of claim 11, wherein the at least one substitution that reduces binding to IGF1R is R24A, F48X, T58A, Y59X, and / or T62A, wherein X is either serine or alanine.

13. The nucleic acid of claim 12, wherein the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO:

7.

14. A nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a target binding domain and an IGF2R-binding portion of a human IGF2 protein, wherein the IGF1R-binding portion comprises at least one substitution corresponding to SEQ ID NO: 1 that reduces binding to IGF1R relative to the wildtype position of SEQ ID NO: 1, wherein the at least one substitution that reduces binding to IGF1R is R24A, F48X, T58A, Y59X, and / or T62A, wherein X is either serine or alanine.

15. The nucleic acid of claim 14, wherein the IGF2R-binding portion is at least 90, 95, 98, 99, or 100% identical to SEQ ID NO:

8.

16. The nucleic acid of any one of claims 1-15, wherein the fusion polypeptide comprises two or more IGF2R-binding portions.

17. The nucleic acid of claim 16, wherein the two or more IGF2R-binding portions have identical or non-identical amino acid sequences.

18. The nucleic acid of any one of claims 1-16, wherein the target binding domain comprises an immunoglobulin variable region. KILPATRICK TOWNSEND 79722801119. The nucleic acid of claim 18, wherein the target binding domain is a single chain antibody (scFv) or a nanobody.

20. The nucleic acid of any one of claims 1-19, wherein the target is an immunosuppressive molecule.

21. The nucleic acid of any one of claims 1-19, wherein the target is an autoantibody.

22. The nucleic acid of any one of claims 1-19, wherein the target is PD-1.

23. The nucleic acid of any one of claims 1-19, wherein the target is a cytokine or an interleukin.

24. The nucleic acid of any one of claims 1-19, wherein the target is selectedfrom the group consisting of TGF- , IL-6R, HER2, and EGFR, optionally EGFR comprising aT790M mutation.

25. The nucleic acid of any one of claims 1-24, wherein the nucleic acid comprises RNA.

26. An expression cassette comprising a promoter operably-linked to the nucleic acid of any one of claims 1-25.

27. A cell comprising the nucleic acid of any one of claims 1-25 or the expression cassette of claim 26.

28. The cell of claim 27, wherein the cell is a human cell.

29. The cell of claim 28, wherein the human cell is a primary cell.

30. The cell of claim 29, wherein the cell is a primary human immune cell or primary human hematopoietic stem cell.

31. The cell of claim 30, wherein the primary human immune cell is a primary human T-cell or natural killer cell. KILPATRICK TOWNSEND 79722801132. The cell of any one of claims 27-31, wherein the cell expresses the fusion polypeptide.

33. The cell of claim 32, wherein the cell secretes the fusion polypeptide.

34. The fusion polypeptide of any one of claims 1-25.

35. A method of degrading a target protein, the method comprising, contacting the target protein with the fusion polypeptide of claim 34 in the presence of a first cell comprising an IGF2R transmembrane protein under conditions in which the fusion polypeptide binds the target protein and is bound by the IGF2R transmembrane protein and internalized into the cell.

36. The method of claim 35, wherein the contacting occurs in vitro or in vivo.

37. The method of claim 36, wherein the contacting occurs in vivo in a human.

38. The method of any one or claims 35-37, wherein the fusion polypeptide is expressed from a second cell.

39. The method of claim 38, wherein the second cell is a human cell.

40. The method of claim 39, wherein the human cell is a primary cell.

41. The method of claim 39 or 40, wherein the cell is a human immune cell or human hematopoietic stem cell.

42. The method of claim 41, wherein the human immune cell is a primary human T-cell or natural killer cell.

43. The method of any one of claims 38-42, wherein the second cell comprises a chimeric antigen receptor (CAR).

44. The method of claim 43, wherein the CAR binds to a tumor antigen or a B-cell antigen. KILPATRICK TOWNSEND 79722801145. The method of claim 37, wherein the contacting comprises introducing the fusion polypeptide into the human.

46. The method of claim 37, wherein the contacting comprises introducing a nucleic acid encoding the fusion polypeptide into the human, wherein the fusion polypeptide is expressed in the human.

47. The method of claim 46, wherein the nucleic acid is a viral vector.

48. The method of any one of claims 38-43, wherein the contacting comprises introducing the second cell into the human. KILPATRICK TOWNSEND 797228011

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