FAP-ACTIVATED SERUM EXTENDED HALF-LIFE THERAPEUTIC

CONJUGATES

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of US 63/108,020, filed October 30, 2020 and US 63/197,926, filed June 7, 2021, which are incorporated by reference herein in their entireties.

BACKGROUND

Various therapeutic drugs lack practical efficacy due to immune-related toxicities and other dose-limiting factors, for example, drugs that induce innate immune responses; however, these drugs are frequently too toxic for systemic use. This is because systemic innate immune activation greatly limits the dose that may be administered. Therefore, the maximum tolerated doses do not reach a therapeutic level in many subjects.

SUMMARY

Disclosed are serum half-life extended drug conjugates of therapeutic or imaging agents, which drug conjugates have a linker including an FAPα substrate that can be selectively cleaved by fibroblast activating protein alpha (FAPα ) to release the therapeutic moiety at the site of cleavage. Upon cleavage by FAPα of the FAP substrate, the drug conjugate releases the therapeutic moiety in its active form or in a form that is readily metabolized (or otherwise converted) to its active form. Pharmaceutical compositions comprising the drug conjugates, as well as methods of using the drug conjugates to treat a disorder characterized by FAPα upregulation are also disclosed.

In some aspects, are therapeutic conjugates designed to release a therapeutically effective amount of a drug (or other therapeutic moiety) in a tumor microenvironment of a subject without inducing acute toxicity in the subject. Surprisingly, the conjugates of the present disclosure are capable of local delivery of a therapeutically effective amount of a drug, even in the absence of a cell-binding (e.g., targeting) moiety. Instead of a targeting moiety or other cell-specific homing mechanism, the conjugates of the present disclosure include a half-life extension moiety and a cleavable linker that enables local accumulation and release of the drug (or other therapeutic moiety) specifically in a tumor microenvironment. The half-life extension moiety enables local accumulation of the drug, while the specificity is achieved using a cleavable linker that is cleaved specifically by FAPα - an enzyme present at high levels in tumor microenvironments. Thus, specific cleavage of the linker by FAPα results in local release of a therapeutically effective amount of the drug in a tumor microenvironment, without the toxic side effects typically associated with systemic delivery of the drug.

Some aspects of the present disclosure provide a therapeutic conjugate comprising a therapeutic moiety linked through a FAPα-cleavable linker to a half-life extension moiety, wherein the circulating serum half-life of the therapeutic conjugate in vivo is at least 48 hours, and the conjugate does not comprise a cell-binding moiety that binds to a cell surface protein of a cell with a Kd of 1x10 ^-6 M or less.

Other aspects of the present disclosure provide a therapeutic conjugate comprising a therapeutic moiety linked through a FAPα-cleavable linker to a half-life extension moiety, wherein the circulating serum half-life of the therapeutic conjugate in vivo is at least 48 hours, and the conjugate does not comprise a cell-binding moiety that binds to a cell surface protein of a cell in a tumor microenvironment.

Yet other aspects of the present disclosure provide a therapeutic conjugate comprising a therapeutic moiety linked through a FAPα-cleavable linker to a half-life extension moiety, wherein the circulating serum half-life of the therapeutic conjugate in vivo is extended by at least 2-fold relative to circulating serum half-life of the therapeutic moiety not linked to the half-life extension moiety, and the conjugate does not comprise a cell-binding moiety that binds to a cell surface protein of a cell with a Kd of 1x10 ^-6 M or less.

Still other aspects of the present disclosure provide a therapeutic conjugate comprising a therapeutic moiety linked through a FAPα-cleavable linker to a half-life extension moiety, wherein the circulating serum half-life of the therapeutic conjugate in vivo is extended by at least 2-fold relative to circulating serum half-life of the therapeutic moiety not linked to the half-life extension moiety, and the conjugate does not comprise a cell- binding moiety that binds to a cell surface protein of a cell in a tumor microenvironment.

The therapeutic moiety not linked to the half-life extension moiety is referred to herein as a “free” therapeutic moiety (i.e., the parent molecule of the therapeutic moiety resulting from cleavage of the conjugate by FAPα ).

In some embodiments, the half-life extension moiety comprises a serum protein. For example, the serum protein may be selected from fibronectin, transferrin, and human serum albumin (HSA). In some embodiments, the half-life extension moiety comprises a molecule that binds to a serum protein. For example, the molecule that binds to a serum protein may be fibronectin, transferrin, or HSA.

In some embodiments, the molecule that binds to a serum protein the molecule that binds to a serum protein is an antibody. The antibody, for example, may be an Fab, F(ab)2, F(ab'), F(ab')2, F(ab')3, Fd, Fv, disulfide linked Fv, dAb or sdAb (or NANOBODY®), CDR, scFv, (scFv)2, di-scFv, bi-scFv, tascFv (tandem scFv), AVIBODY® (e.g., diabody, triabody, and tetrabody), T-cell engager (BiTE®), Fc, scFv-Fc, Fcab, mAb2, small modular immunopharmaceutical (SMIP), Genmab/unibody or duobody, V-NAR domain, IgNAR, minibody, IgGACH2, DVD- Ig, probody, intrabody, or a multispecificity antibody.

In some embodiments, the molecule that binds to a serum protein is a non-antibody molecule. The non-antibody molecule, for example, may be an affibody, an AFFIMER® polypeptide, an affilin, an anticalin, an atrimer, an avimer, a DARPin, an FN3 scaffold (e.g. Adnectins, Centyrins), a fynomer, a Kunitz domain, a nanofitin, a pronectins, a tribody, bicyclic peptides, or a Cys-knot. Other non-antibody molecules that bind to a serum protein are encompassed by the present disclosure.

In some embodiments, the half-life extension moiety comprises an HSA-binding recombinantly engineered variant of stefin polypeptide (i.e., AFFIMER® polypeptide).

In some embodiments, the recombinantly engineered variant of stefin polypeptide (i.e., AFFIMER® polypeptide) comprises an amino acid sequence that has at least 70%, at least 80%, at least 90%, or 100% identity to an amino acid sequence of any one of SEQ ID NOS: 110-132.

In some embodiments, the half-life extension moiety comprises an antibody Fc domain, optionally from IgA, IgD, IgE, IgG, or IgM or a subclass thereof.

In some embodiments, the half-life extension moiety comprises a biocompatible polymer, optionally selected from the group consisting of a poly(ethylene glycol) (PEG), a hydroxyethyl starch, an XTEN™ polymer, and a proline- alanine- serine polymer.

In some embodiments, the therapeutic conjugate is represented by one of the formula: X — L ¹ — SRS — L ² — TM, wherein X is the half-life extension moiety, L ¹ is a spacer or bond, SRS is a substrate recognition sequence cleavable by FAPα, L ² is a self-immolative linker (e.g., which is metabolized or otherwise eliminated after FAPα cleavage to release the free therapeutic moiety) or bond, and TM is the therapeutic moiety. In some embodiments, the therapeutic conjugate is represented by one of the formula: X— (L ¹ — SRS— L ² — TM) _n ; X— L ¹ — (SRS— L ² — TM) _n ; (X) _m — (L ¹ — SRS— L ² — TM) _n ; or (X) _m — L ¹ — (SRS — L ² — TM)n, wherein X is the half-life extension moiety, L ¹ is a spacer or bond, SRS is a substrate recognition sequence cleavable by FAPα, L ² is a self-immolative linker or bond, TM is the therapeutic moiety, m is an integer from 1 to 6, and n is an integer from 1 to 500, optionally 1 to 100, 1 to 10, or 1 to 5.

In some embodiments, the therapeutic conjugate comprises a moiety capable of chemically conjugating in vivo to albumin or other proteins in circulation in the serum of the patient. In some embodiments, the therapeutic conjugate is represented by one of the formula: Y— L ¹ — SRS— L ² — TM; Y— (L ¹ — SRS— L ² — TM) _n ; or Y— L ¹ — (SRS— L ² — TM) _n , wherein L ¹ , SRS, L ² and TM are as defined above, and Y is a reactive group capable of chemically cross-linking to a protein in vivo.

In some embodiments, Y is a reactive group capable of chemically cross-linking to free amines (such as the sidechain of lysines) present in a protein. A non-limiting example includes a linker including NHS (N-hydroxysuccinimide), such as shown in FIG. 6A.

In some embodiments, Y is a reactive group capable of chemically cross-linking to free thiol groups (such as the sidechain of cysteine) present in a protein. A non-limiting example includes a linker including maleimide, such as shown in FIG. 6B.

In some embodiments, the therapeutic conjugate is:

In some embodiments, the FAPα-cleavable linker is an oligopeptide. In some embodiments, the oligopeptide comprises a C-terminal proline covalently linked to the therapeutic moiety, optionally via a bond or a self-immolative linker, and/or an N-terminal blocking group. In some embodiments, the bond is a bond that can be cleaved by the proteolytic activity of FAP, e.g., an amide bond. In some embodiments, the FAPα-cleavable linker contributes to the Pl' specificity of FAP (is recognized by FAP as a Pl' residue).

In some embodiments, the bond can be cleaved by the proteolytic activity of FAPα, optionally wherein the bond is an amide bond.

In some embodiments, the self-immolative linker comprises a heterocyclic self- immolative moiety, optionally His-Ala, p-aminobenzyloxycarbonyl (PABC) or and 2,4- bis(hydroxymethyl)aniline.

In some embodiments, the FAPα-cleavable linker comprises a sequence selected from a D-Ala-Pro, PPGP (SEQ ID NO: 136), (D/E)-(R/K)-G-(E/D)-(T/S)-G-P (SEQ ID NO: 137),

DRGETGP (SEQ ID NO: 138), and GPAX (SEQ ID NO: 139), optionally a D-Ala-Pro sequence or a D-Ser sequence.

In some embodiments, the therapeutic conjugate of any one of the preceding claims wherein the substrate recognition sequence (SRS) of the FAPα-cleavable linker is represented by

wherein R ² is hydrogen or (C ₁ -C ₆ ) alkyl or hydrogen, R ³ is hydrogen or a branched or straight chain lower alkyl, e.g., a lower alkyl such as methyl (if (d) is an amino acid side chain, then R ³ is not hydrogen), R ⁴ is a branched or straight chain lower alkoxy, such as hydroxymethyl (e.g., for serine) or 1 -hydroxyethyl (e.g., for threonine) (in one embodiment, R ⁴ is hydroxymethyl), X ¹ is O or S, and/or X ² is O or S.

In some embodiments, the substrate recognition sequence of the FAPα-cleavable linker comprises a third amino position, optionally N-terminal to (d)-Ala (or other (d)-amino acid in that position and formed by R3), and optionally wherein the amino acid at the third amino acid position is serine or threonine.

In some embodiments, the FAPα-cleavable linker has a kcat/Km for cleavage by FAPα at least 10-fold, at least 100-fold, 1000-fold, 5000-fold, or 10,000-fold greater than a kcat/Km for cleavage by prolyl endopeptidase (EC 3.4.21.26; PREP). In some embodiments, the therapeutic moiety is a cytotoxic agent, i.e., which when released from the therapeutic conjugate causes cell death of the target cells at the concentration at which the therapeutic conjugate is administered.

In some embodiments, the therapeutic moiety is a cytostatic agent, i.e., which when released from the therapeutic conjugate causes mitotic arrest or quiescence of the target cells at the concentration at which the therapeutic conjugate is administered.

In some embodiments, the therapeutic moiety is an epigenetic agent, i.e., which when released from the therapeutic conjugate causes epigenetic alteration of the target cells at the concentration at which the therapeutic conjugate is administered, which may, for example, result in differentiation (or dedifferentiation) of the cell to another cellular phenotype.

In some embodiments, the therapeutic moiety is a chemotherapeutic drug moiety. For example, the chemotherapeutic drug moiety may be a taxane, a platinum-based agents, or a proteasome inhibitor. Other chemotherapeutic drug moieties are encompassed by the present disclosure.

In some embodiments, the taxane is selected from the group consisting of paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the taxane is paclitaxel. Other taxanes are encompassed by the present disclosure.

In some embodiments, the platinum-based agent is selected from the group consisting of oxaliplatin, cisplatin, carboplatin, nedaplatin, picoplatin, phenanthriplatin, triplatin, spiroplatin, satraplatin, iproplatin, and satraplatin. In some embodiments, the platinum-based agent is oxaliplatin. Other platinum-based agents are encompassed by the present disclosure.

In some embodiments, the proteasome inhibitor is selected from the group consisting of bortezomib, lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib (salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP- 18770), epoxomicin, and beta-hydroxy beta-methylbutyrate. Other proteasome inhibitors are encompassed by the present disclosure.

In some embodiments, the therapeutic moiety induces an innate immune (v. adaptive immune) response in vivo. In some embodiments, the therapeutic moiety is acutely toxic. Acute toxicity describes the adverse effects of a drug, for example, that result either from a single exposure or from multiple exposures in a short period of time (e.g., less than 24 hours). The adverse effects of acute toxicity typically occur within 14 days of the administration of the drug. In some embodiments, the therapeutic moiety is selected from TLR agonists, RIG-I agonists, iDASH inhibitors, and STING agonists. In some embodiments, the therapeutic moiety is Val-boroPro (Talabostat).

In some embodiments, the therapeutic moiety includes a ligand for a receptor, such as ligand that when released from the therapeutic conjugate is able to bind to an extracellular ligand binding domain of a cell surface receptor. Exemplary receptor ligands include somatostatin, cholecystokinin-2 (CCK2), folate, bombesin, gastrin-releasing peptide, neurotensin, substance P, glucagon-like peptide 1, neuropeptide Y and analogs of those ligands. The receptor ligand can itself have pharmacological activity or can be used to deliver a conjugated drug moiety, toxin or radioisotope.

In some embodiments, the therapeutic moiety is represented by the general formula wherein RBM is a receptor binding moiety, Z is cytotoxic, cytostatic or epigenetic moiety or a radioisotope containing moiety, and p is 0 (Z is absent) or an integer from 1 to 8. In some embodiments, p is 1.

In some embodiments, the therapeutic moiety is represented by the general formula

— RBM— L ³ — Z wherein RBM is a receptor binding moiety, Z is cytotoxic, cytostatic or epigenetic moiety or a radioisotope containing moiety, and L ³ is a bond or a cleavable or non-cleavable linker. For instance, L ³ can be a linker that is acid labile or enzyme sensitive (such as includes a cathepsin cleavage site) such that Z is released intracellularly on internalization of the moiety -RBM-L ³ -Z through cell binding dependent on the receptor binding moiety RBM.

In some embodiments, the therapeutic moiety has a circulating serum half-life that it is at least 5, 10, 25, 50, 100 or even 1000 times longer than the circulating serum half-life of the free therapeutic moiety. The circulating serum half-life of a therapeutic moiety is a pharmacokinetic parameter that is defined as the time it takes for the concentration of the therapeutic moiety in the serum to be reduced by 50%.

In some embodiments, the therapeutic moiety has a circulating serum half-life that it is at least 10, 25, 50, 100, 200, or 300 hours.

In some embodiments, for a period of at least 10, 24, 48, 72, 96, or 120 hours the therapeutic conjugate produces a concentration of free therapeutic moiety in target FAPα- expressing tissue that is at least 2, 5, 10, 20, 50, 75 or 100 times the concentration of free therapeutic moiety in systemic circulation over the same period of time. For instance, such differences in free therapeutic moiety concentrations can occur in subjects between FAPα- expressing tumors and serum. In some embodiments, when administered to a subject having an FAPα+ tumor, the therapeutic conjugate produces an intratumoral concentration of free therapeutic moiety that is at or above the EC ₅₀ for the antitumor activity of the free therapeutic moiety for a period at least 10, 24, 48, 72, 96, or 120 hours.

In some embodiments, when administered to a subject having an FAPα+ tumor, the therapeutic conjugate has a therapeutic index for antitumor activity of at least 2, 5, 10, 25, 50, 100, or 500. The therapeutic index (TI) is a quantitative measurement of the relative safety of a drug. It is a comparison of the amount of a therapeutic agent that causes the therapeutic effect to the amount that causes toxicity (TI also referred to as a therapeutic ratio).

In some embodiments, when administered to a subject having an FAPα+ tumor, the therapeutic conjugate has a therapeutic index for antitumor activity that is at least 2, 10, 50, 100, 250, 500, 1000, 5000, or 10,000 times greater than the therapeutic index of the free therapeutic moiety.

In some embodiments, a greater percentage of free therapeutic moiety is localized in a target tissue expressing FAPα, relative to free therapeutic moiety, when compared on an equivalent dose basis, optionally wherein the ratio of free therapeutic moiety localized in the target tissue relative to other tissue (such as blood, liver or heart) is at least 2, 5, 10, 100, or 1000 times greater for an equivalent dose of the therapeutic conjugate relative to the free therapeutic moiety.

In some embodiments, the maximum tolerated dose of the therapeutic conjugate is at least 2, 5, 10, 100, or 1000 times greater than the maximum tolerated dose of the free therapeutic moiety. The maximum tolerated dose is the highest dose of a therapeutic moiety (e.g., drug) that does not cause unacceptable side effects or overt toxicity in a specific period of time. The maximum tolerated dose may be determined in clinical trials by testing increasing doses on different groups of people until the highest dose with acceptable side effects is found.

In some embodiments, the cell permeability of the therapeutic conjugate is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% less than the cell permeability of free therapeutic moiety.

In some embodiments, the circulating half-life of the therapeutic conjugate is at least 25%, 50%, 75%, 100%, 150%, 200%, 500%, 750%, or 1000% longer than the circulating half-life of free therapeutic moiety. In some embodiments, the therapeutic conjugate has less than 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or 99.99% of the cytotoxic or cytolytic activity against tumor cells relative to free therapeutic moiety.

Also provided herein, in some aspects, is a composition comprising the therapeutic conjugate of any one of the preceding paragraphs and a pharmaceutically acceptable excipient.

Further provided herein, in some aspects, is a method comprising administering to a subject the therapeutic conjugate or composition of any one of the preceding paragraphs, wherein the subject has a diseased tissue, optionally a cancer.

In some embodiments, the therapeutic conjugate or composition is administered in an amount effective to increase lactate dehydrogenase (LDH) release in a tumor microenvironment the subject by at least 0.5-fold or at least 1-fold relative to an untreated control subject. In some embodiments, the therapeutic conjugate or composition is administered in an amount effective to increase LDH release in a tumor microenvironment the subject by about 0.5-fold, about 0.6-fold, about 0.7-fold, about 0.8-fold, about 0.9-fold, about 1-fold, about 1.5-fold, or about 2-fold. In some embodiments, the therapeutic conjugate or composition is administered in an amount effective to increase LDH release in a tumor microenvironment the subject by about 0.5-fold to about 1.5-fold, about 0.5-fold to about 2- fold, or about 1-fold to about 2-fold.

In some embodiments, the volume of the diseased tissue, optionally a tumor, is reduced by at least 50%, at least 60%, or at least 70% at about 2-3 weeks following administration of the therapeutic conjugate or composition. In some embodiments, the volume of the diseased tissue, optionally a tumor, is reduced by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80%. In some embodiments, the volume of the diseased tissue, optionally a tumor, is reduced by about 50% to about 60%, or about 50% to about 70%, or about 50% to about 80%.

Some aspects provide a therapeutic conjugate or composition of any one of the preceding paragraphs for use in a method for treating a diseased tissue, optionally a cancer.

Other aspects provide a use of the therapeutic conjugate of any one of the preceding paragraphs in the manufacture of a medicament for the treatment of a diseased tissue, optionally a cancer.

Some aspects of the present disclosure provide a therapeutic conjugate comprising a chemotherapeutic therapeutic moiety linked through a FAPα-cleavable linker to a half-life extension moiety, wherein the chemotherapeutic moiety induces an innate immune response in vivo and/or is acutely toxic, the circulating serum half-life of the therapeutic conjugate in vivo is at least 48 hours, and the therapeutic conjugate does not comprise a cell-binding moiety that binds to a cell surface protein of a cell with a Kd of 1x10 ^-8 M or less. In some embodiments, the chemotherapeutic moiety is selected from TLR agonists, RIG-I agonists, iDASH inhibitors, and STING agonists. In some embodiments, the chemotherapeutic moiety is Val-boroPro (Talabostat). In some embodiments, the half-life extension moiety is an antibody Fc domain. In some embodiments, the half-life extension moiety is HSA. In some embodiments, the half-life extension moiety is an HSA-binding recombinantly engineered variant of stefin polypeptide (i.e., AFFIMER® polypeptide), optionally comprising an amino acid sequence that has at least 70%, at least 80%, at least 90%, or 100% identity to an amino acid sequence of any one of SEQ ID NOS: 110-132.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating an embodiment of the extending half-life therapeutic conjugates described herein. In the diagram, “X” represents a serum half-like extension moiety, in this case a protein linked through a maleimide conjugate with the thiol sidechain of a cysteine residue, to a linker including an FAPα substrate sequence to a drug moiety (the I- DASH inhibitor valine-boroproline shown) that is released upon FAP cleavage of the linker.

FIG. 2 is a graph showing the anti-tumor activity of examples of the therapeutic conjugates described herein, as indicated by tumor volume in a mouse model of colon carcinoma. The “SQT-Gly V.2-6325” groups are therapeutic conjugates without cell- targeting moieties. The “AVA04-182” groups comprise cell-targeting moieties.

FIG. 3 is a graph showing the amount of free Val-boroPro (Talabostat) in tumors and in serum after administration of the therapeutic conjugates at the dosage levels shown.

FIGs. 4A-4B are graphs showing the EC ₅₀ of Talabostat administered alone (FIG.

4A) and administered with the therapeutic conjugates described herein (FIG. 4B) in mice and rats.

FIGs. 5A-5D are graphs depicting tumor volume and percent change in body weight in mice after being administered the vehicle (FIG. 5A), a conjugate comprising a cell-binding moiety (FIG. 5B), and a therapeutic conjugate described herein (the conjugate of FIG. 5B without the cell-binding moiety) (FIG. 5C). The percent change in body weight of the mice in FIG. 5C was also measured (FIG. 5D). FIG. 6A is the structure for “6325”, an exemplary FAP-activated I-DASH inhibitor including an NHS group for conjugation to lysine residues of proteins. FIG. 6B is the structure for “6323”, an exemplary FAP-activated I-DASH inhibitor including a maleimide group for conjugation to lysine residues of proteins. FIG. 6C is the structure for “6501”, an exemplary tetra-branched FAP-activated I-DASH inhibitor including a maleimide group for conjugation to lysine residues of proteins.

FIG. 7 is a graph showing the change in tumor volume over time in CT26-mFAP+ mice with either vehicle (control), MSA-6325 (mouse serum albumin conjugated with 6325) and PEG-6325 (6325 reacted with SUNBRIGHT PTE-200 PA, a 20KDa 4 arm functional PEG).

FIGs. 8A-8B are graphs showing the individual animal tumor volume growth curves over time in CT26-mFAP+ mice with either a human Fc fragment conjugated with 6325 (FIG 8A), or a full human IgG antibody conjugated with 6325 (FIG 8B).

FIG. 9 illustrates an exemplary generic structure for an FAP-activated somatostatin analog (octreotide)-based conjugate, wherein X is the half-life extension moiety, LI is a spacer or bond, XI and X2 are independently O or S, R2 is hydrogen or (C1-C6) alkyl or hydrogen, R3 is hydrogen or a branched or straight chain lower alkyl, e.g., a lower alkyl such as methyl, R4 is a branched or straight chain lower alkoxy, such as hydroxymethyl (e.g., for serine) or 1 -hydroxyethyl (e.g., for threonine) (in one embodiment, R4 is hydroxymethyl), L3 is a bond or a cleavable or non-cleavable linker, and Z is a cytotoxic, cytostatic or epigenetic moiety or a radioisotope containing moiety.

FIG. 10 illustrates an exemplary generic structure for an FAP-activated folic acid- based conjugate, wherein X is the half-life extension moiety, LI is a spacer or bond, XI and X2 are independently O or S, R2 is hydrogen or (C1-C6) alkyl or hydrogen, R3 is hydrogen or a branched or straight chain lower alkyl, e.g., a lower alkyl such as methyl, R4 is a branched or straight chain lower alkoxy, such as hydroxymethyl (e.g., for serine) or 1- hydroxyethyl (e.g., for threonine) (in one embodiment, R4 is hydroxymethyl), X3 is O or N(H), L3 is a bond or a cleavable or non-cleavable linker, and Z is a cytotoxic, cytostatic or epigenetic moiety or a radioisotope containing moiety.

FIG. 11 is an illustration of FAP-activated folic acid-based SMDC vintafolide, which is comprised of an FAP substrate recognition sequence (showing linker for protein conjugation), a folate targeting ligand, a peptide spacer, a self-immolative disulfide linker and the cytotoxic drug desacetyl vinblastine monohydrazine (DAVLBH). FIG. 12 is an illustration of a FAP-activated folate-taxoid conjugate which incorporates an FAP substrate recognition sequence (showing linker for protein conjugation), a folic acid targeting moiety, a self-immolative disulfide linker and hydrophilic PEGylated dipeptide spacer (solubilising spacer) and a taxoid SB-T-1214, which is a derivative of the chemotherapeutic drug Taxol (see Seitz et al. Bioorg. Med. Chem., 2015, 23, 2187-2194).

FIGs. 13A-13B illustrate the structures of FAP-activated folate-doxorubicin (FIG. 13A) and thioloate (FIG. 13B) HD AC inhibitors.

FIG. 14 shows a FAP-activated folate-cytotix maytansinoid conjugate which incorporates an FAP substrate recognition sequence (showing linker for protein conjugation), a folic acid targeting moiety, a self-immolative disulfide linker and an antitubulin cytotoxic maytansinoid drug moiety DM4.

FIG. 15 illustrates the structure of FAP-activated folate-MMAE.

FIG. 16 illustrates the structure of camptothecin.

FIG. 17 includes graphs showing the fold-increase in lactate dehydrogenase (LDH, a marker of pyroptosis) released into culture supernatants from J774A.1 mouse macrophage cell line cells cultured with one of various versions of the FAP-activated I-DASH inhibitor 6325 (Hu IgGl Fc-6325, Hu IgGl-6325, MSA-6325, SQT-Gly V.2-6325, SQT-Gly CF-6325, or SQT-Gly CG-6325) in the presence (open bar) or absence (grey filled bar) of rhFAPα for 24 hours. An untreated control was used for each set of experiments, and a Val-boroPro (VbP) control experiment was also performed (data not shown) All values are representative of the mean +/- SEM. Statistical analysis of No FAPα vs. + FAPα was performed via multiple T test in GraphPad Prism. *, P<0.05; **, P<0.01; ***, P<0.001.

FIGs. 18A-18B are graphs showing the conjugation of thiol (SH) groups determined by measurement of free SH of to compound maleimide linker prodrugs, 6323 (FIG. 18A) and 6501 (FIG. 18B).

FIGs. 19A-19B are graphs showing the pharmacokinetics of VbP released from Hu IgGl Fc-6325 in CT26-mFAP tumor-bearing mouse serum (FIG. 19A) and in tumor samples (FIG. 19B) in the presence or absence of 5057 , a FAPα-specific inhibitor. Each time point includes three mice per group.

FIGs. 20A-20B are graphs showing the efficacy of SQT-Gly conjugates (FIG. 20A) and Hu IgGl Fc conjugates (FIG. 20B), measured as tumor volume over time in a syngeneic murine colon cancer model (CT26-mFAP ⁺ mice). FIG. 21 includes graphs showing the pharmacokinetics and tissue distribution of VbP released from different FAP-activated prodrugs in mouse serum and tumor in a murine colon cancer model (CT26-mFAP ⁺ mice). Measurements were taking using liquid chromatography-mass spectrometry (LC-MS). Each dosing group consisted of a subset of 5 groups, representing the 1, 4, 6, 24, and 48 hour collection time points (n = 3/group) with the exception of 24 μg 3892, which only consisted of 1, 4, and 24 hours.

FIGs. 22A-22B include graphs showing the pharmacokinetics and tissue distribution of VbP released from SQT-Gly V.2-conjugates (FIG. 22A) and IgG Fc-conjugates (FIG. 22B) in mouse serum and tumor in a murine colon cancer model (CT26-mFAP ⁺ mice). Measurements were taking using LC-MS. Each dosing group consisted of a subset of 5 groups, representing the 1, 4, 6, 24, and 48 hour collection time points (n = 3/group).

FIG. 23 is a graph depicting FAP-activated prodrug G-CSF serum cytokine response in nontumor-bearing BALB/c mice levels. Levels of G-CSF in mouse serum following administration of vehicle (PBS), 42CQ-6501 and 42CQ-6501+MSA at 200ug/mouse are shown.

FIGs. 24A-24B are graphs showing the conjugation of a compound maleimide linker prodrug (6323) to the hinge region cysteine residues of recombinantly engineered variant of stefin polypeptide (AFFIMER®)-Fc proteins. Conjugations were performed with reaction ratios of 0, 10, 20 and 40 moles of 6323 per mole of reduced SH (4 SH per SQTGlyCF). The change in free SH groups vs. reaction ratio is shown in FIG. 24A. A timed conjugation reaction was done with a reaction ratio of 40 6323 per SH with the reaction stopped at 0, 5, 10, 15 and 20 minutes after addition of 6323. Kinetics of conjugation of 6323 to SQTGlyCF with a reaction ratio of 40 6323 per SH group are shown in FIG. 24B.

FIGs. 25A-25B are graphs showing the pharmacokinetics of VbP released following administration of 42CQ-6501 in CT26-mFAP tumor-bearing mice. Each dosing group consisted of a subset of 5 groups, representing the 1, 4, 6, 24 and 48 hr collection time points (n = 3/group). VbP was measured with LC-MS in serum (FIG. 25A) and tumor (FIG. 25B) samples.

FIGs. 26A-26B are graphs showing the efficacy of 42CQ-6323 conjugates (FIG. 26A) and 42CQ-6501 conjugates (FIG. 26B) ± MSA, measured as tumor volume over time in a syngeneic murine colon cancer model (CT26-mFAP ⁺ mice). FIGs. 27A-27B are graphs showing the pharmacokinetics of VbP released following administration of 42CQ-6323 conjugates (FIG. 27 A) and 42CQ-6501 conjugates (FIG. 27B) in wild-type and FAPα knockout mice (n=3/group).

FIG. 28 is the structure for “3892”, an exemplary prodrug of VbP.

DETAILED DESCRIPTION

The data provided herein demonstrates, unexpectedly, that the therapeutic conjugates of the present disclosure can be delivered systemically, without a cell-binding (e.g., cell targeting) moiety, yet are still capable of delivering a therapeutically effective amount of a therapeutic moiety (e.g., drug) to a target diseased tissue, while maintaining a therapeutic index that is superior to that of the therapeutic moiety delivered directly (e.g., by the same route of administration). Instead of a cell-binding moiety, the therapeutic conjugates described herein comprise a half-life extension moiety and an FAPα-cleavable linker, which when combined, enable local accumulation and release of the therapeutic moiety in a tumor microenvironment (or other target tissue expressing FAPα ) while reducing exposure of non- target tissue that expresses FAPα at lower levels of than the target tissue. Accordingly, the therapeutic conjugates provided herein demonstrate one or more of (1) superior efficacy and/or ability to reach a higher percentage of maximum effective concentrations of the released therapeutic moiety in the target tissue due to localized FAPα cleavage of the prodrug and extended circulating serum half-lives, and (2) an improved therapeutic index (TI) due to the ability to be administered at a lower systemic Cmax concentration and/or reduced systemic toxicity relative to the parent therapeutic moiety.

Described herein are therapeutic conjugates comprising a therapeutic moiety linked through a fibroblast activation protein, alpha (FAPα)-cleavable linker to a half-life extension moiety. Each component of these conjugates is described in detail below.

Half-life Extension Moieties

A therapeutic conjugate, in some embodiments, comprises a half-life extension moiety. The half-life extension moiety extends the circulating serum half-life of a therapeutic moiety in vivo. Circulating serum half-life is the amount of time it takes for the concentration or amount of the therapeutic moiety in the serum to be reduced by half (50%). Half-life times can be determined experimentally by measuring the concentration of the therapeutic moiety in the serum of a subject over time. Half-life may be calculated using the formula: 0.693 x (Vd/CL), where Vd represents the volume of distribution and CL represents clearance. The volume of distribution is the theoretical volume needed to contain the total amount of the therapeutic moiety at the same concentration that is observed in the blood plasma (e.g., the ratio of the amount of the therapeutic moiety in the body to the concentration of the therapeutic moiety measured in blood, plasma, and in free form in interstitial fluid). Clearance is the volume of plasma from which the therapeutic moiety is completely removed per unit time. Half-life may also be determined using high performance liquid chromatography (HPLC), fluorescence assays, radioassays, radioimmunoassays, and elemental mass spectrometric assays.

A half-life extension moiety may extend the circulating serum half-life of a molecule by at least 2-fold, relative to the circulating serum half-life of the molecule not linked to the half-life extension moiety. In some embodiments, a half-life extension moiety extends the circulating serum half-life of a molecule by at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, or at least 30-fold, relative to the circulating serum half-life of the molecule not linked to the half-life extension moiety. In some embodiments, a half-life extension moiety extends the circulating serum half-life of a molecule by 2-fold to 5-fold, 2-fold to 10-fold, 3 -fold to 5- fold, 3 -fold to 10-fold, 15-fold to 5-fold, 4-fold to 10-fold, or 5-fold to 10-fold, relative to the circulating serum half-life of the molecule not linked to the half-life extension moiety.

In some embodiments, a half-life extension moiety extends the circulating serum half- life of a therapeutic moiety by at least 10 hours, at least 12 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours, for example, at least 1 week after in vivo administration, relative to the circulating serum half-life of the therapeutic moiety not linked to the half-life extension moiety.

In some embodiments, the therapeutic moieties have a circulating serum half-life in human subjects of at least 10 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 24 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 48 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 72 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 96 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 120 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 144 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 168 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 192 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 216 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 240 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 264 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 288 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 312 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 336 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of at least 360 hours. In some embodiments, the therapeutic moieties have a serum half-life in human subjects of 24 to 360 hours, 48 to 360 hours, 72 to 360 hours, 96 to 360 hours, or 120 to 360 hours.

Serum proteins and antibody Fc domains are non-limiting examples of two major proteins that may be used as provided herein as half-life extension moieties. Both Fc and serum protein conjugates achieve extended half-lives not only by increasing the size of the therapeutic moiety, but both also take advantage of the body’s natural recycling mechanism: the neonatal Fc receptor, FcRn. The pH-dependent binding of these proteins to FcRn prevents degradation of the therapeutic conjugate in the endosome. Conjugates using these proteins can have half-lives in the range of 3-16 days. Conjugates that include an antibody Fc domain or a serum protein can improve the solubility and stability of the therapeutic moiety.

Serum Proteins

In some embodiments, the half-life extension moiety comprises a serum protein (e.g., a naturally-occurring or modified version of a protein in blood (e.g., serum) of a human). The serum protein may be, for example, fibronectin, transferrin, or albumin.

In some embodiments, the serum protein is fibronectin. Fibronectin is a high- molecular weight (~440kDa) glycoprotein of the extracellular matrix that binds to membrane- spanning receptor proteins called integrins. It exists as a protein dimer, including two nearly identical polypeptide chains linked by a pair of C-terminal disulfide bonds. Each fibronectin subunit has a molecular weight of 230-250 kDa and contains three types of modules: type I, II, and III. All three modules are composed of two anti-parallel β-sheets resulting in a Beta- sandwich; however, type I and type II are stabilized by intra-chain disulfide bonds, while type III modules do not contain any disulfide bonds. The absence of disulfide bonds in type III modules allows them to partially unfold under applied force. Fibronectin also binds to other extracellular matrix proteins such as collagen, fibrin, and heparan sulfate proteoglycans (e.g. syndecans). Fibronectin exists as a protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds. The fibronectin protein is produced from a single gene, but alternative splicing of its pre-mRNA leads to the creation of several isoforms.

In some embodiments, the serum protein is transferrin. Trasnferrin is a glycoprotein found in vertebrates that binds to and consequently mediates the transport of Iron (Fe) through blood plasma. It is produced in the liver and contains binding sites for two Fe ³⁺ atoms. Human transferrin is encoded by the TF gene and produced as a 76 kDa glycoprotein. Transferrin glycoproteins bind iron tightly, but reversibly. Although iron bound to transferrin is less than 0.1% (4 mg) of total body iron, it forms the most vital iron pool with the highest rate of turnover (25 mg/24 h). Transferrin has a molecular weight of around 80 kDa and contains two specific high-affinity Fe(III) binding sites. The affinity of transferrin for Fe(III) is extremely high (association constant is 1020 M ^-1 at pH 7.4) but decreases progressively with decreasing pH below neutrality. Transferrins are not limited to only binding to iron but also to different metal ions.

In some embodiments, the serum protein is human serum albumin (HSA). HSA is a protein encoded by the ALB gene. HSA is a 585 amino acid polypeptide (approx. 67 kDa) having a serum half-life of about 20 days and is primarily responsible for the maintenance of colloidal osmotic blood pressure, blood pH, and transport and distribution of numerous endogenous and exogenous ligands. HSA has three structurally homologous domains (domains I, II and III), is almost entirely in the alpha-helical conformation, and is highly stabilized by 17 disulfide bridges. A representative HSA sequence is provided by UniProtKB Primary accession number P02768 and may include other human isoforms thereof.

Serum Protein Binding Molecules

In some embodiments, the half-life extension moiety comprises a molecule that binds to a serum protein, such as proteins that bind non-covalently via a peptide or protein-binding domain to the serum protein, such as an antibody (including antigen-binding portions thereof). Examples of antibodies include, but are not limited to: Fab, F(ab) ₂ , F(ab'), F(ab') ₂ , F(ab')3, Fd, Fv, disulfide linked Fv, dAb or sdAb (or NANOBODY®), CDR, scFv, (scFv) ₂ , di-scFv, bi-scFv, tascFv (tandem scFv), AVIBODY (e.g., diabody, triabody, and tetrabody), T-cell engager (BiTE), Fc, scFv-Fc, Fcab, mAb2, small modular immunopharmaceutical (SMIP), Genmab/unibody or duobody, V-NAR domain, IgNAR, minibody, IgGACH2, DVD- Ig, probody, intrabody, and a multispecificity antibody. Other examples are provided in US 2005/0287153, incorporated herein by reference in its entirety.

In some embodiments, the serum protein-binding molecule is hydrophobic moiety that associates with serum albumin, such as acylated peptides (e.g., acylated heptapeptide in Zorzi et al., Nature Communications vol 8: 16092 (2017); the acyl group of Liraglutide).

In some embodiments, the molecule that binds to a serum protein is a non-antibody molecule, non-limiting examples of which include an affibody, an AFFIMER® polypeptide, an affilin, an anticalin, an atrimer, an avimer, a DARPin, an FN3 scaffold (e.g. Adnectins, Centyrins), a fynomer, a Kunitz domain, a nanofitin, a pronectins, a tribody, bicyclic peptides, and a Cys-knot.

In some embodiments, the molecule that binds a serum protein such as HSA comprises an AFFIMER® polypeptide. An AFFIMER® polypeptide is a small, highly stable polypeptide (e.g., protein) that is a recombinantly engineered variant of stefin polypeptides. Thus, the term “AFFIMER® polypeptide” may be used interchangeably herein with the term “recombinantly engineered variant of stefin polypeptide.” A stefin polypeptide is a subgroup of proteins in the cystatin superfamily - a family that encompasses proteins containing multiple cy statin-like sequences. The stefin subgroup of the cystatin family is relatively small (~ 100 amino acids) single domain proteins. They receive no known post-translational modification, and lack disulfide bonds, suggesting that they will be able to fold identically in a wide range of extracellular and intracellular environments. Stefin A is a monomeric, single chain, single domain protein of 98 amino acids. The structure of stefin A has been solved, facilitating the rational mutation of stefin A into the AFFIMER® polypeptide. The only known biological activity of cystatins is the inhibition of cathepsin activity, has enabled exhaustively testing for residual biological activity of the engineered proteins.

An AFFIMER® polypeptide displays two peptide loops and an N-terminal sequence that can all be randomized to bind to desired target proteins with high affinity and specificity, in a similar manner to monoclonal antibodies. Stabilization of the two peptides by the stefin A protein scaffold constrains the possible conformations that the peptides can take, increasing the binding affinity and specificity compared to libraries of free peptides. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. Variations to other parts of the stefin A polypeptide sequence can be carried out, with such variations improving the properties of these affinity reagents, such as increase stability, make them robust across a range of temperatures and pH, for example. In some embodiments, an AFFIMER® polypeptide includes a sequence derived from stefin A, sharing substantial identify with a stefin A wild type sequence, such as human stefin A. In some embodiments, an AFFIMER® polypeptide has an amino acid sequence that shares at least 25%, 35%, 45%, 55% or 60% identity to the sequences corresponding to human stefin A. For example, an AFFIMER® polypeptide may have an amino acid sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identity, e.g., where the sequence variations do not adversely affect the ability of the scaffold to bind to the desired target, and e.g., which do not restore or generate biological functions such as those that are possessed by wild type stefin A, but which are abolished in mutational changes described herein.

An anti-HSA AFFIMER® polypeptide comprises an AFFIMER® polypeptide in which at least one of the solvent accessible loops is from the wild-type stefin A protein having amino acid sequences to enable an AFFIMER® polypeptide to bind HSA, selectively, and in some embodiments, with Kd of 10’ ⁶ M or less.

In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-9 M to 1x10 ^-6 M at pH 7.4 to 7.6. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-6 M or less at pH 7.4 to 7.6. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-7 M or less at pH 7.4 to 7.6. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-8 M or less at pH 7.4 to 7.6. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-9 M or less at pH 7.4 to 7.6. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ¹⁰ M or less at pH 7.4 to 7.6. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ¹¹ M or less at pH 7.4 to 7.6. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-9 M to 1x10 ^-6 M at pH 7.4. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-6 M or less at pH 7.4. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-7 M or less at pH 7.4. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-8 M or less at pH 7.4. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ^-9 M or less at pH 7.4. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10 ¹⁰ M or less at pH 7.4. In some embodiments, the AFFIMER® polypeptide binds to HSA with a Kd of 1x10- ¹¹ M or less at pH 7.4.

In some embodiments, the anti-HSA AFFIMER® polypeptide is derived from the wild-type human stefin A protein having a backbone sequence and in which one or both of loop 2 (designated (Xaa) _n ) and loop 4 (designated (Xaa) _m ) are replaced with alternative loop sequences (Xaa) _n and (Xaa) _m , to have the general formula (I):

FRl-(Xaa)n-FR2-(Xaa)m-FR3 (I), wherein FR1 is an amino acid sequence having at least 70% identity to MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 1); FR2 is an amino acid sequence having at least 70% identity to GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2); FR3 is an amino acid sequence having at least 70% identity to EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3); Xaa, individually for each occurrence, is an amino acid; and n is an integer from 3 to 20, and m is an integer from 3 to 20.

In some embodiments, FR1 is a polypeptide sequence having 80%-98%, 82%-98%, 84%-98%, 86%-98%, 88%-98%, 90%-98%, 92%-98%, 94%-98%, or 96%-98% homology with SEQ ID NO: 1. In some embodiments, FR1 is a polypeptide sequence having 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or 95% homology with SEQ ID NO: 1. In some embodiments, FR1 is the polypeptide sequence of SEQ ID NO: 1. In some embodiments, FR2 is a polypeptide sequence having at least 80%-96%, 84%-96%, 88%- 96%, or 92%-96% homology with SEQ ID NO: 2. In some embodiments, FR2 is a polypeptide sequence having at least 80%, 84%, 88%, 92%, or 96% homology with SEQ ID NO: 2. In some embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% identity with SEQ ID NO: 2. In some embodiments, FR2 is the polypeptide sequence of SEQ ID NO: 2. In some embodiments, FR3 is a polypeptide sequence having at least 80%-95%, 85%-95%, or 90%-95% homology with SEQ ID No: 3. In some embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, or 95% homology with SEQ ID NO: 3. In some embodiments, FR3 is the polypeptide sequence of SEQ ID NO: 3.

In some embodiments, an anti-HSA AFFIMER® polypeptide comprises the amino acid sequence represented in general formula (II): MIP-Xaal-GLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA- (Xaa)ii-Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa) _m -Xaa6-D- Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4) (II), wherein Xaa, individually for each occurrence, is an amino acid; n is an integer from 3 to 20, and m is an integer from 3 to 20; Xaal is Gly, Ala, Val, Arg, Lys, Asp, or Glu; Xaa2 is Gly, Ala, Val, Ser or Thr; Xaa3 is Arg, Lys, Asn, Gin, Ser, Thr; Xaa4 is Gly, Ala, Val, Ser or Thr; Xaa5 is Ala, Val, Ile, Leu, Gly or Pro; Xaa6 is Gly, Ala, Val, Asp or Glu; and Xaa7 is Ala, Val, Ile, Leu, Arg or Lys.

In some embodiments, Xaal is Gly, Ala, Arg or Lys. In some embodiments, Xaal is Gly or Arg. In some embodiments, Xaa2 is Gly, Ala, Val, Ser or Thr. In some embodiments, Xaa2 is Gly or Ser. In some embodiments, Xaa3 is Arg, Lys, Asn, Gin, Ser, Thr. In some embodiments, Xaa3 is Arg, Lys, Asn or Gin. In some embodiments, Xaa3 is Lys or Asn. In some embodiments, Xaa4 is Gly, Ala, Val, Ser or Thr. In some embodiments, Xaa4 is Gly or Ser. In some embodiments, Xaa5 is Ala, Val, Ile, Leu, Gly or Pro. In some embodiments, Xaa5 is Ile, Leu or Pro. In some embodiments, Xaa5 is Leu or Pro. In some embodiments, Xaa6 is Gly, Ala, Val, Asp or Glu. In some embodiments, Xaa6 is Ala, Val, Asp or Glu. In some embodiments, Xaa6 is Ala or Glu. In some embodiments, Xaa7 is Ala, Val, Ile, Leu, Arg or Lys. In some embodiments, Xaa7 is Ile, Leu or Arg. In some embodiments, Xaa7 is Leu or Arg.

In some embodiments, an anti-HSA AFFIMER® comprises the amino acid sequence represented in general formula (III):

MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKT QVLA-(Xaa)n-STNYYIKVRAGDNKYMHLKVFNGP-(Xaa)m-ADR VLTGYQVDKNKDDELTGF (SEQ ID NO: 5) (III), wherein Xaa, individually for each occurrence, is an amino acid; n is an integer from 3 to 20, and m is an integer from 3 to 20. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, n is 8 to 10, 7 to 11, 6 to 12, 5 to

13, 4 to 14, or 3 to 15. In some embodiments, m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, m is 8 to 10, 7 to 11, 6 to 12, 5 to 13, 4 to 14, or 3 to 15.

In some embodiments, (Xaa) _n is represented by formula (IV): aal-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9 (IV), wherein aal is an amino acid with a neutral polar hydrophilic side chain; aa2 is an amino acid with a neutral nonpolar hydrophobic side chain; aa3 is an amino acid with a neutral nonpolar hydrophobic side chain; aa4 is an amino acid with a neutral polar hydrophilic side chain; aa5 is an amino acid with a positively charged polar hydrophilic side chain; aa6 is an amino acid with a positively charged polar hydrophilic side chain; aa7 is an amino acid with a neutral nonpolar hydrophobic side chain; aa8 is an amino acid with a neutral nonpolar hydrophobic side chain; and aa9 is an amino acid with a neutral nonpolar hydrophilic side chain.

In some embodiments, (Xaa) _m is represented by formula (V): aal-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9 (V), wherein aal is an amino acid with a neutral nonpolar hydrophobic side chain; aa2 is an amino acid with a positively charged polar hydrophilic side chain; aa3 is an amino acid with a neutral nonpolar hydrophobic side chain; aa4 is an amino acid with a positively charged polar hydrophilic side chain; aa5 is an amino acid with a neutral polar hydrophilic side chain; aa6 is an amino acid with a neutral polar hydrophilic side chain; aa7 is an amino acid with a negatively charged polar hydrophilic side chain; aa8 is an amino acid with a positively charged polar hydrophilic side chain; and aa9 is an amino acid with a neutral nonpolar hydrophilic side chain.

Examples of amino acids with a neutral nonpolar hydrophilic side chain include cysteine (Cys) and glycine (Gly). In some embodiments, the amino acid with a neutral nonpolar hydrophilic side chain is Cys. In some embodiments, the amino acid with a neutral nonpolar hydrophilic side chain is Gly.

Examples of amino acids with a neutral nonpolar hydrophobic side chain include alanine (Ala), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), tryptophan (Trp), and valine (Val). In some embodiments, the amino acid with a neutral nonpolar hydrophobic side chain is Ala. In some embodiments, the amino acid with a neutral nonpolar hydrophobic side chain is Ile. In some embodiments, the amino acid with a neutral nonpolar hydrophobic side chain is Leu. In some embodiments, the amino acid with a neutral nonpolar hydrophobic side chain is Met. In some embodiments, the amino acid with a neutral nonpolar hydrophobic side chain is Phe. In some embodiments, the amino acid with a neutral nonpolar hydrophobic side chain is Pro. In some embodiments, the amino acid with a neutral nonpolar hydrophobic side chain is Trp. In some embodiments, the amino acid with a neutral nonpolar hydrophobic side chain is Val. Examples of amino acids with a neutral polar hydrophilic side chain include asparagine (Asn), glutamine (Gin), serine (Ser), threonine (Thr), and tyrosine (Tyr). In some embodiments, the amino acid with a neutral polar hydrophilic side chain is Asn. In some embodiments, the amino acid with a neutral polar hydrophilic side chain is Gin. In some embodiments, the amino acid with a neutral polar hydrophilic side chain is Ser. In some embodiments, the amino acid with a neutral polar hydrophilic side chain is Thr. In some embodiments, the amino acid with a neutral polar hydrophilic side chain is Tyr.

Examples of amino acids with a positively charged polar hydrophilic side chain include arginine (Arg), histidine (His), and lysine (Lys). In some embodiments, the amino acid with a positively charged polar hydrophilic side is Arg. In some embodiments, the amino acid with a positively charged polar hydrophilic side is His. In some embodiments, the amino acid with a positively charged polar hydrophilic side is Lys.

Examples of amino acids with a negatively charged polar hydrophilic side chain include aspartate (Asp) and glutamate (Glu). In some embodiments, the amino acid with a negatively charged polar hydrophilic side chain is Asp. In some embodiments, the amino acid with a negatively charged polar hydrophilic side chain is Glu.

In some embodiments, (Xaa) _n is represented by formula (IV): aal-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9 (IV), wherein aal is an amino acid selected from Asp, Gly, Asn, and Val; aa2 is an amino acid selected from Trp, Tyr, His, and Phe; aa3 is an amino acid selected from Trp, Tyr, Gly, Trp, and Phe; aa4 is an amino acid selected from Gin, Ala, and Pro; aa5 is an amino acid selected from Ala, Gin, Glu, Arg, and Ser; aa6 is an amino acid selected from Lys, Arg, and Tyr; aa7 is an amino acid selected from Trp and Gin; aa8 is an amino acid selected from Pro and His; and/or aa9 is an amino acid selected from His, Gly, and Gin. In some embodiments, aal is Asp. In some embodiments, aal is Gly. In some embodiments, aal is Asn. In some embodiments, aa2 is Trp. In some embodiments, aa2 is Tyr. In some embodiments, aa2 is His. In some embodiments, aa2 is Phe. In some embodiments, aa3 is Trp. In some embodiments, aa3 is Tyr. In some embodiments, aa3 is Gly. In some embodiments, aa3 is Trp. In some embodiments, aa3 is Phe. In some embodiments, aa4 is Gin. In some embodiments, aa4 is Ala. In some embodiments, aa4 is Pro. In some embodiments, aa5 is Ala. In some embodiments, aa5 is Gin. In some embodiments, aa5 is Glu. In some embodiments, aa5 is Arg. In some embodiments, aa5 is Ser. In some embodiments, aa6 is Lys. In some embodiments, aa6 is Arg. In some embodiments, aa6 is Tyr. In some embodiments, aa7 is Trp. In some embodiments, aa7 is Gin. In some embodiments, aa8 is Pro. In some embodiments, aa8 is His. In some embodiments, aa9 is His. In some embodiments, aa9 is Gly. In some embodiments, aa9 is Gin.

In some embodiments, (Xaa) _m is represented by formula (IV): aal-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9 (IV), wherein aal is an amino acid selected from Tyr, Phe, Trp, and Asn; aa2 is an amino acid selected from Lys, Pro, His, Ala, and Thr; aa3 is an amino acid selected from Val, Asn, Gly, Gin, Ala, and Phe; aa4 is an amino acid selected from His, Thr, Lys, Trp, Lys, Val, and Arg; aa5 is an amino acid selected from Gin, Ser, Gly, Pro, and Asn; aa6 is an amino acid selected from Ser, Tyr, Glu, Leu, Lys, and Thr; aa7 is an amino acid selected from Ser, Asp, Val, and Lys; aa8 is an amino acid selected from Gly, Leu, Ser, Pro, His, Asp, and Arg; and/or aa9 is an amino acid selected from Gly, Gin, Glu, and Ala. In some embodiments, aal is Tyr. In some embodiments, aal is Phe. In some embodiments, aal is Trp. In some embodiments, aal is Asn. In some embodiments, aa2 is Lys. In some embodiments, aa2 is Pro. In some embodiments, aa2 is His. In some embodiments, aa2 is Ala. In some embodiments, aa2 is Thr. In some embodiments, aa3 is Val. In some embodiments, aa3 is Asn. In some embodiments, aa3 is Gly. In some embodiments, aa3 is Gin. In some embodiments, aa3 is Ala. In some embodiments, aa3 is Phe. In some embodiments, aa4 is His. In some embodiments, aa4 is Thr. In some embodiments, aa4 is Lys. In some embodiments, aa4 is Trp. In some embodiments, aa4 is Lys. In some embodiments, aa4 is Val. In some embodiments, aa4 is Arg. In some embodiments, aa5 is Gin. In some embodiments, aa5 is Ser. In some embodiments, aa5 is Gly. In some embodiments, aa5 is Pro. In some embodiments, aa5 is Asn. In some embodiments, aa6 is Ser. In some embodiments, aa6 is Tyr. In some embodiments, aa6 is Glu. In some embodiments, aa6 is Leu. In some embodiments, aa6 is Lys. In some embodiments, aa6 is Thr. In some embodiments, aa7 is Ser. In some embodiments, aa7 is Asp. In some embodiments, aa7 is Val. In some embodiments, aa7 is Lys. In some embodiments, aa8 is Gly. In some embodiments, aa8 is Leu. In some embodiments, aa8 is Ser. In some embodiments, aa8 is Pro. In some embodiments, aa8 is His. In some embodiments, aa8 is Asp. In some embodiments, aa8 is Arg. In some embodiments, aa9 is Gly. In some embodiments, aa9 is Gin. In some embodiments, aa9 is Glu. In some embodiments, aa9 is Ala.

In some embodiments, (Xaa) _n is represented by formula (V):

Asn-aal-aa2-Gln-Gln-Arg-Arg-Trp-Pro-Gly (V) (SEQ ID NO: 140), wherein aal is an amino acid selected from Trp and Phe; and aa2 is an amino acid selected from Tyr and Phe. In some embodiments, aal is Trp. In some embodiments, aal is Phe. In some embodiments, aa2 is Tyr. In some embodiments, aa2 it Phe.

In some embodiments, (Xaa) _n is represented by formula (VI): aal-aa2-Trp-aa3-aa4-Lys-Trp-Pro-aa5 (VI) (SEQ ID NO: 141), wherein aal is an amino acid selected from Asp and Gly; aa2 is an amino acid selected from Trp, Tyr, and Phe; aa3 is an amino acid selected from Gin and Ala; aa4 is an amino acid selected from Ala and Ser; and aa5 is an amino acid selected from His and Gly. In some embodiments, aal is Asp. In some embodiments, aal is Gly. In some embodiments, aa2 is Trp. In some embodiments, aa2 is Tyr. In some embodiments, aa2 is Phe. In some embodiments, aa3 is Gin. In some embodiments, aa3 is Ala. In some embodiments, aa4 is Ala. In some embodiments, aa4 is Ser. In some embodiments, aa5 is His. In some embodiments, aa5 is Gly.

In some embodiments, (Xaa) _n is represented by formula (VII): aal-aa2-aa3-aa4-aa5-aa6-Trp-Pro-Gly (VII), wherein aal is an amino acid selected from Gly and Asn; aa2 is an amino acid selected from Tyr, Phe, Trp, and His; aa3 is an amino acid selected from Trp, Tyr, and Phe; aa4 is an amino acid selected from Ala and Gin; aa5 is an amino acid selected from Ala, Ser, Gin, and Arg; and aa6 is an amino acid selected from Lys, Arg, and Tyr. In some embodiments, aal is Gly. In some embodiments, aal is Asn. In some embodiments, aa2 is Tyr. In some embodiments, aa2 is Phe. In some embodiments, aa2 is Trp. In some embodiments, aa2 is His. In some embodiments, aa3 is Trp. In some embodiments, aa3 is Tyr. In some embodiments, aa3 is Phe. In some embodiments, aa4 is Ala. In some embodiments, aa4 is Gin. In some embodiments, aa5 is Ala. In some embodiments, aa5 is Ser. In some embodiments, aa5 is Gin. In some embodiments, aa5 is Arg. In some embodiments, aa6 is Lys. In some embodiments, aa6 is Arg. In some embodiments, aa6 is Tyr.

In some embodiments, (Xaa) _n is represented by formula (IX): Gly-aal-aa2-Ala-aa3-aa4-Trp-Pro-Gly (IX) (SEQ ID NO: 142), wherein aal is an amino acid selected from Tyr, Phe, and His; aa2 is an amino acid selected from Trp and Tyr; aa3 is an amino acid selected from Ala, Ser, and Arg; and aa4 is an amino acid selected from Lys and Tyr. In some embodiments, aal is Tyr. In some embodiments, aal is Phe His. In some embodiments, aal is His. In some embodiments, aa2 is Trp. In some embodiments, aa2 is Tyr. In some embodiments, aa3 is Ala. In some embodiments, aa3 is Ser. In some embodiments, aa3 is Arg. In some embodiments, aa4 is Lys. In some embodiments, aa4 is Tyr.

In some embodiments, (Xaa) _n is represented by formula (X): aal-aa2-aa3-Gln-aa4-aa5-Trp-Pro-aa6 (X), wherein aal is an amino acid selected from Asp and Asn; aa2 is an amino acid selected from Trp and Phe; aa3 is an amino acid selected from Trp, Tyr, and Phe; aa4 is an amino acid selected from Ala, Gin, and Arg; aa5 is an amino acid selected from Lys and Arg; and aa6 is an amino acid selected from His and Gly. In some embodiments, aal is Asp. In some embodiments, aal is Asn. In some embodiments, aa2 is Trp. In some embodiments, aa2 is Phe. In some embodiments, aa3 is Trp. In some embodiments, aa3 is Tyr. In some embodiments, aa3 is Phe. In some embodiments, aa4 is Ala. In some embodiments, aa4 is Gin. In some embodiments, aa4 is Arg. In some embodiments, aa5 is Lys. In some embodiments, aa5 is Arg. In some embodiments, aa6 is His. In some embodiments, aa6 is Gly.

In some embodiments, an anti-HSA AFFIMER® polypeptide comprises a loop 2 amino acid sequence selected from any one of SEQ ID NOS: 6-56, 134-135 (Table 1). In some embodiments, an anti-HSA AFFIMER® polypeptide comprises a loop 4 amino acid sequence selected from any one of SEQ ID NOS: 57-109 (Table 1).

Table 1. Examples of HSA AFFIMER® Polypeptide Loop Sequences

In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 6-55, 134- 135. In some embodiments, (Xaa) _n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 6-55, 134-135. In some embodiments, (Xaa) _n comprises the amino acid sequence of any one of SEQ ID NOS: 6-55, 134-135.

In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 22, 24, 26, 35, 40, 41, and 45. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, (Xaa)n comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 41. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 45. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, (Xaa)n comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 41. In some embodiments, (Xaa) _n comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 45.

In some embodiments, (Xaa) _n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 22, 24, 26, 35, 40, 41, and 45. In some embodiments, (Xaa) _n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, (Xaa) _n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, (Xaa) _n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, (Xaa)n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, (Xaa) _n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, (Xaa) _n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 41. In some embodiments, (Xaa) _n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 45.

In some embodiments, (Xaa) _n comprises the amino acid sequence of any one of SEQ ID NOS: 22, 24, 26, 35, 40, 41, and 45. In some embodiments, (Xaa) _n comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, (Xaa) _n comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, (Xaa) _n comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, (Xaa) _n comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, (Xaa) _n comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, (Xaa) _n comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, (Xaa) _n comprises the amino acid sequence of SEQ ID NO: 45.

In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 57-108. In some embodiments, (Xaa) _m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 57-108. In some embodiments, (Xaa) _m comprises the amino acid sequence of any one of SEQ ID NOS: 57-108.

In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 75, 77, 79, 88, 93, 94, and 98. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 88. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 93. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 88. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 93. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, (Xaa) _m comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 98.

In some embodiments, (Xaa) _m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 75, 77, 79, 88, 93, 94, and 98. In some embodiments, (Xaa) _m comprises the amino acid sequence of any one of SEQ ID NOS: 75, 77, 79, 88, 93, 94, and 98. In some embodiments, (Xaa) _m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, (Xaa) _m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, (Xaa) _m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, (Xaa) _m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 88. In some embodiments, (Xaa) _m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 93. In some embodiments, (Xaa) _m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, (Xaa) _m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 98.

In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOS: 110-116 and 133 (Table 2). Table 2. Examples of HSA AFFIMER® Polypeptide Sequences

In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 110-116, and 133. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 111. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 113. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 114. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 115. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 116. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 133.

In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 111. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 113. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 114. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 115. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 116. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 133.

In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 110-116 and 133. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 110-116 and 133. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 110. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 111. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 112. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 113. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 114. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 115. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 116. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 133.

An anti-HSA AFFIMER® polypeptide provided herein, in some embodiments, is linked to another molecule and extend the half-life of that molecule (e.g., a therapeutic polypeptide). Provided herein is a range of anti-HSA AFFIMERS®, with a range of binding affinities, for example, that cross-react with other species such as mouse and cynomolgous (cyno) monkey. These anti-HSA AFFIMERS®, in some embodiments, make up what is referred to as the AFFIMER XT™ platform. These anti-HSA AFFIMERS® have been shown in in vivo pharmacokinetic (PK) studies to extend, in a controlled manner, the serum half-life of any other AFFIMER® polypeptide therapeutic to which it is conjugated in a single genetic fusion, for example, that can be made in E. Coli. AFFIMER XT™ can also be used to extend the half-life of other peptide or protein therapeutics.

The term half-life refers to the amount of time it takes for a substance, such as a therapeutic AFFIMER® polypeptide, to lose half of its pharmacologic or physiologic activity or concentration. Biological half-life can be affected by elimination, excretion, degradation (e.g.. enzymatic degradation) of the substance, or absorption and concentration in certain organs or tissues of the body. Biological half-life can be assessed, for example, by determining the time it takes for the blood plasma concentration of the substance to reach half its steady state level (“plasma half-life”).

In some embodiments, an anti-HSA AFFIMER® polypeptide extends the serum half- life of a molecule (e.g., a therapeutic polypeptide) in vivo. For example, an anti-HSA AFFIMER® polypeptide may extend the half-life of a molecule by at least 2-fold, relative to the half-life of the molecule not linked to an anti-HSA AFFIMER® polypeptide. In some embodiments, an anti-HSA AFFIMER® polypeptide extends the half-life of a molecule by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, or at least 30-fold, relative to the half-life of the molecule not linked to an anti-HSA AFFIMER® polypeptide. In some embodiments, an anti- HSA AFFIMER® polypeptide extends the half-life of a molecule by 2-fold to 5-fold, 2-fold to 10-fold, 3 -fold to 5-fold, 3 -fold to 10-fold, 15-fold to 5-fold, 4-fold to 10-fold, or 5-fold to 10-fold, relative to the half-life of the molecule not linked to an anti-HSA AFFIMER® polypeptide. In some embodiments, an anti-HSA AFFIMER® polypeptide extends the half- life of a molecule by at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, for example, at least 1 week after in vivo administration, relative to the half-life of the molecule not linked to an anti-HSA AFFIMER® polypeptide.

In some embodiments, an anti-HSA AFFIMER® polypeptide has an extended serum half-life and comprises an amino acid sequence selected from any one of SEQ ID NOS: 117- 127 (Table 3).

Table 3. Examples of half-life extension in-line fusion AFFIMER® Polypeptide sequences

In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 117-127. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 117. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 123. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 124. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 125. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 126. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 127.

In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 117. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 123. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 124. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 125. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 126. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 127.

In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOS: 117-127. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 117-127. In some embodiments, an anti- HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 117. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 121. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 122. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 123. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 124. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 125. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 126. In some embodiments, an anti-HSA AFFIMER® polypeptide comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of SEQ ID NO: 127.

Fc Domains

In some embodiments, the half-life extension moiety comprises an antibody Fc domain. The term “Fc domain” includes functional fragments of antibody Fc domains. A therapeutic conjugate may comprise, for example, the Fc region of an antibody (which facilitates effector functions and pharmacokinetics) linked to a therapeutic moiety through a FAP-cleavable linker (also referred to herein as an “Fc fusion”). In some embodiments, Fc fusions can be dimerized to form Fc fusion homodimers, or using non-identical Fc domains, to form Fc fusion heterodimers.

There are several reasons for choosing the Fc domain of human antibodies for use in generating the therapeutic conjugates of the present disclosure. The principle rationale is to produce a stable protein, large enough to demonstrate a similar pharmacokinetic profile compared with those of antibodies, and to take advantage of the properties imparted by the Fc domain; this includes the salvage neonatal FcRn receptor pathway involving FcRn-mediated recycling of the Fc fusion to the cell surface post endocytosis, avoiding lysosomal degradation and resulting in release back into the bloodstream, thus contributing to an extended serum half-life. Another obvious advantage is the Fc domain’s binding to Protein A, which can simplify downstream processing during production of the therapeutic conjugates and permit generation of highly pure preparation of the therapeutic conjugates.

In some embodiments, an Fc domain includes the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Oy2 and Cy3 and the hinge between Oyl and Oy2. Although the boundaries of the Fc domain may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc may refer to this region in isolation, or this region in the context of a whole antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions and are also included as Fc domains as used herein.

An Fc domain includes functional fragments of antibody Fc domains. A functional Fc fragment retains the ability to bind FcRn. A functional Fc fragment binds to FcRn but does not possess effector function. The ability of the Fc region or fragment thereof to bind to FcRn can be determined by standard binding assays known in the art. Exemplary effector functions include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions can be assessed using various assays known in the art for evaluating such antibody effector functions.

In an exemplary embodiment, the Fc domain is derived from an IgGl subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used. In some embodiments, the Fc domain is derived from an IgGl subclass and further comprises a LALA mutation.

Other non-limiting examples of Fc domains, functional Fc fragments and particular sequences are provided in WO 2019/236567, incorporated herein by reference.

Biocompatible Polymers

A wide variety of biocompatible macromolecular polymers and other molecules can be used as a half-life extension moiety. The molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da.

For the purpose of half-life extension, various methods including pegylation, polysialylation, HESylation, glycosylation, or recombinant PEG analogue fused to flexible and hydrophilic amino acid chain (500 to 600 amino acids) have been developed (See Chapman, (2002) Adv Drug Deliv Rev. 54. 531-545; Schlapschy et al., (2007) Prot Eng Des Sei. 20, 273-283; Contermann (2011) Curr Op Biotechnol. 22, 868-876; Jevsevar et al., (2012) Methods Mol Biol. 901, 233- 246).

Examples of biocompatible polymers that may be used as half-life extension moieties include but are not limited to polyalkyl ethers and alkoxy-capped analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogs thereof, especially polyoxyethylene glycol, the latter is also known as polyethylene glycol or PEG); discrete PEG (dPEG); polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide and derivatives thereof); polyhydroxyalkyl acrylates; polysialic acids and analogs thereof; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, e.g., carboxymethyldextran, dextran sulfates, aminodextran; cellulose and its derivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses; chitin and its derivatives, e.g., chitosan, succinyl chitosan, carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and its derivatives; starches; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyaminoacids and derivatives thereof, e.g., polyglutamic acids, polylysines, polyaspartic acids, polyaspartamides; maleic anhydride copolymers such as: styrene maleic anhydride copolymer, divinylethyl ether maleic anhydride copolymer; polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixtures thereof; and derivatives of the foregoing.

The polymer selected may be water soluble so that the therapeutic conjugate of which it is a component does not precipitate in an aqueous environment, such as a physiological environment. The water-soluble polymer may be any structural form including but not limited to linear, forked or branched. In some embodiments, the water soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water soluble polymers can also be used. By way of example, PEG is used in some embodiments of this disclosure, provided the properties of the particular PEG selected extends the circulating serum half-life of the therapeutic conjugate in accordance with the present disclosure.

In some embodiments, the biocompatible polymer is selected from the group consisting of poly(ethylene glycol), hydroxyethyl starch, XTEN, and a proline-alanine-serine polymer.

Other non-limiting examples of polymers are provided in WO 2019/236567, incorporated herein by reference.

Fibroblast Activation Protein, Alpha (FAPα)-cleavable Linkers A therapeutic conjugate, in some embodiments, comprises a fibroblast activation protein, alpha (FAPα)-cleavable linker, which links the half-life extension moiety to a therapeutic moiety. FAPα, a type II transmembrane serine protease, is a tumor-associated antigen that is expressed in stromal fibroblasts (Chai et al., Acta Pharmacologica Sinica, 2018, 39: 415-424). Expression of FAPα is almost exclusively limited to tumor masses, as it is nearly undetectable in normal healthy adult tissues. Fibroblasts undergo a morphological transformation in a tumor microenvironment characterized by substantial increases in the expression of FAP irrespective of tumor grade (Henry et al., Clin Cancer Res, 2007, 13(6): 1736).

FAP is a post-proline cleaving enzyme of the DASH family that selectively cleaves peptide substrates after proline residues. Without wishing to be bound by theory, it is thought that the inclusion of a FAPα-cleavable linker in a therapeutic conjugate will permit the therapeutic moiety to remain inactive until the therapeutic conjugate is in proximity to FAPα, at which point, the FAPα cleaves the linker, activating the therapeutic moiety. As FAPα is present in tumor tissues and not in healthy tissues, the therapeutic moiety will be activated primarily near/within tumor tissue.

In some embodiments, the linker comprises a substrate recognition sequence (SRS) cleavable by FAPα. For example, in some embodiments, the SRS is cleaved by FAPα and is represented by: wherein, R ² represents H or a (C ₁ -C ₆ ) alkyl, for example, is H; R ³ represents H or a (C ₁ -C ₆ ) alkyl, for example, is methyl, ethyl, propyl, or isopropyl; R ⁴ is absent or represents a (C ₁ -C ₆ ) alkyl, — OH, — NH2, or halogen; X represents O or S; and NH represents an amine that is part of L ² if L ² is a self-immolative linker or part of the therapeutic moiety if L ² is a bond.

In some embodiments, the FAPα-cleavable linker or the SRS is represented by wherein

R ² is hydrogen or (C ₁ -C ₆ ) alkyl. In some embodiments, R ² is hydrogen.

R ³ is hydrogen or a branched or straight chain lower alkyl, e.g., a lower alkyl such as methyl. If (d) is an amino acid side chain, then R ³ is not hydrogen.

R ⁴ is a branched or straight chain lower alkoxy, such as hydroxymethyl (e.g., for serine) or 1 -hydroxyethyl (e.g., for threonine). In one embodiment, R ⁴ is hydroxymethyl.

X ¹ is O or S.

X ² is O or S.

In some embodiments, the FAPα-cleavable linker or the SRS comprises a third amino position (e.g., N-terminal to (d)-Ala). This position, P3 from the cleavage site, in some embodiments is serine. In some embodiments, it is threonine.

In some embodiments, the linker comprises a substrate recognition sequence (SRS) cleavable by FAPα, such as D-Ala-Pro. In some embodiments, the SRS sequence cleavable by FAPα is PPGP (SEQ ID NO: 136), (D/E)-(R/K)-G-(E/D)-(T/S)-G-P (SEQ ID NO: 137), DRGETGP (SEQ ID NO: 138), GPAX (SEQ ID NO: 139) (where X is any amino acid) (Aggarwal et al., Biochemistry. 2008; 47(3): 1076-1086; Ji et al., Angew Chem Int Ed Engl. 2016; 55(3):1050-1055).

Spacers

In some embodiments, a therapeutic conjugate comprises a spacer or bond (L ¹ ) between the half-life extension moiety and the substrate recognition sequence (SRS) cleavable by FAPα.

The spacer may be any molecule, for example, one or more nucleotides, amino acids, chemical functional groups. In some embodiments, the spacer is a peptide linker (e.g., two or more amino acids). Spacers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. In some embodiments, spacers are not antigenic and do not elicit an immune response. An immune response includes a response from the innate immune system and/or the adaptive immune system. Thus, an immune response may be a cell-mediate response and/or a humoral immune response. The immune response may be, for example, a T cell response, a B cell response, a natural killer (NK) cell response, a monocyte response, and/or a macrophage response. Other cell response are contemplated herein. In some embodiments, linkers are non-protein-coding.

In some embodiments, L ¹ is a hydrocarbon (straight chain or cyclic) such as 6- maleimidocaproyl, maleimidopropanoyl and maleimidom ethyl cyclohexane- 1-carboxylate, or L ¹ is N-Succinimidyl 4-(2-pyridylthio) pentanoate, N- Succinimidyl 4-(N- maleimidomethyl) cyclohexane- 1 carboxylate, N-Succinimidyl (4-iodo-acetyl) aminobenzoate.

In some embodiments, L ¹ is a polyether such as a poly(ethylene glycol) or other hydrophilic linker. For instance, where the CBM includes a thiol (such as a cysteine residue), L ¹ can be a polyethylene glycol) coupled to the thiol group through a maleimide moiety. Non-limiting examples of linkers for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published December 12, 2019, incorporated by reference herein.

Self-Immolative Linkers

In some embodiments, a therapeutic conjugate comprises a self-immolative linker (L ² ) between the substrate recognition sequence (SRS) for FAPα and the therapeutic moiety, such as represented in the formula wherein, p represents an integer from 1 to 100, preferably 6 to 50, more preferably 6 to 12.

In other embodiments, where the CBM includes a thiol and L ¹ is a hydrocarbon moiety coupled to the thiol group through a maleimide moiety, L ¹ can be represented in the formula wherein, p represent an integer from 1 to 20, preferably 1 to 4.

A self-immolative moiety may be defined as a bifunctional chemical group that is capable of covalently linking together two spaced chemical moieties into a normally stable molecule, releasing one of the spaced chemical moieties from the molecule by means of enzymatic cleavage; and following enzymatic cleavage, spontaneously cleaving from the remainder of the bifunctional chemical group to release the other of said spaced chemical moieties. Therefore, in some embodiments, the self-immolative moiety is covalently linked at one of its ends, directly or indirectly through a spacer unit, to the ligand by an amide bond and covalently linked at its other end to a chemical reactive site (functional group) pending from the therapeutic moiety. The derivatization of the therapeutic moiety with the self- immolative moiety may render the drug less pharmacologically active (e.g. less toxic) or not active at all until the drug is cleaved.

A therapeutic conjugate is generally stable in circulation, or at least that should be the case in the absence of an enzyme capable of cleaving the amide bond between the substrate recognition sequence (FAPα-cleavable linker) and the self-immolative moiety. Upon exposure of a therapeutic conjugate to a suitable enzyme (FAPα), the amide bond is cleaved initiating a spontaneous self-immolative reaction resulting in the cleavage of the bond covalently linking the self-immolative moiety to the therapeutic moiety, to thereby effect release of the free therapeutic moiety in its underivatized or pharmacologically active form. The self-immolative moiety in conjugates either incorporate one or more heteroatoms and thereby provides improved solubility, improves the rate of cleavage and decreases propensity for aggregation of the conjugate.

In some embodiments, L ² is a benzyl oxy carbonyl group. In other embodiments, the self-immolative linker L ² is — NH — (CH ₂ )4 -C(=O)- or — NH-(CH ₂ )3-C(=O)-. In yet other embodiments, the self-immolative linker L ² is p-aminobenzyloxycarbonyl (PABC). In still other embodiments, the self-immolative linker L ² is 2,4-bis(hydroxymeihyl)aniline.

The therapeutic conjugates of the present disclosure can employ a heterocyclic self- immolative moiety covalently linked to the therapeutic moiety and the cleavable substrate recognition sequence. A self-immolative moiety may be defined as a bifunctional chemical group which is capable of covalently linking together two spaced chemical moieties into a normally stable molecule, releasing one of said spaced chemical moieties from the molecule by means of enzymatic cleavage; and following said enzymatic cleavage, spontaneously cleaving from the remainder of the bifunctional chemical group to release the other of said spaced chemical moieties. In accordance with the present present disclosure, the self- immolative moiety may be covalently linked at one of its ends, directly or indirectly through a spacer unit, to the ligand by an amide bond and covalently linked at its other end to a chemical reactive site (functional group) pending from the drug. The derivatization of the therapeutic moiety with the self-immolative moiety may render the drug less pharmacologically active (e.g. less toxic) or not active at all until the drug is cleaved.

The therapeutic conjugate is generally stable in circulation, or at least that should be the case in the absence of an enzyme capable of cleaving the amide bond between the substrate recognition sequence and the self-immolative moiety. However, upon exposure of the therapeutic conjugate to a suitable enzyme, the amide bond is cleaved initiating a spontaneous self-immolative reaction resulting in the cleavage of the bond covalently linking the self-immolative moiety to the drug, to thereby effect release of the free therapeutic moiety in its underivatized or pharmacologically active form.

The self-immolative moiety in conjugates of the present disclosure, in some embodiments, either incorporate one or more heteroatoms and thereby provides improved solubility, improves the rate of cleavage and decreases propensity for aggregation of the conjugate. These improvements of the heterocyclic self-immolative linker constructs of the present disclosure over non-heterocyclic, PAB-type linkers may result in surprising and unexpected biological properties such as increased efficacy, decreased toxicity, and more desirable pharmacokinetics.

In some embodiments, L ² is a benzyloxycarbonyl group.

In some embodiments, L ² is wherein R ¹ is hydrogen, unsubstituted or substituted C _{1 -3} alkyl, or unsubstituted or substituted heterocyclyl. In some embodiments, R ¹ is hydrogen. In some instances, R ¹ is methyl.

In some embodiments, L ² is selected from

In some embodiments, the self-immolative moiety L ₂ is selected from wherein

U is O, S or NR ⁶ ;

Q is CR ⁴ or N;

V ¹ , V ² and V ³ are independently CR ⁴ or N provided that for formula II and III at least one of Q, V ¹ and V ² is N;

T is NH, NR ⁶ , O or S pending from said therapeutic moiety;

R ¹ , R ² , R ³ and R ⁴ are independently selected from H, F, Cl, Br, I, OH, —N(R ⁵ ) ₂ , — N(R ^S )3 ⁺ , C ₁ -C ₈ alkylhalide, carboxylate, sulfate, sulfamate, sulfonate, —SO ₂ R ⁵ , — S(=O)R ⁵ , —SR ⁵ , -SO ₂ N(R ⁵ ) ₂ , -C(=O)R ⁵ , -CO ₂ R ⁵ , —C(= O)N(R ⁵ ) ₂ , —CN, N ₃ , —NO ₂ , C ₁ -C ₈ alkoxy, C ₁ -C ₈ halosubstituted alkyl, polyethyleneoxy, phosphonate, phosphate, C ₁ -C ₈ alkyl, C ₁ -C ₈ substituted alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ substituted alkenyl, C ₂ -C ₈ alkynyl, C ₂ -C ₈ substituted alkynyl, C ₆ -C ₂₀ aryl, C ₆ -C ₂₀ substituted aryl, C ₁ -C ₂₀ heterocycle, and C ₁ -C ₂₀ substituted heterocycle; or when taken together, R ² and Reform a carbonyl (=O ), or spiro carbocyclic ring of 3 to 7 carbon atoms: and

R ⁵ and R ⁶ are independently selected from H, C ₁ -C ₈ alkyl, C ₁ -C ₈ substituted alkyl, C ₂ - C ₈ alkenyl, C ₂ -C ₈ substituted alkenyl. C ₂ -C ₈ alkynyl, C ₁ -C ₈ substituted alkynyl, C ₆ - C ₂₀ aryl, C ₆ -C ₂₀ substituted aryl, C ₁ -C ₂₀ heterocycle, and C ₁ -C ₂₀ substituted heterocycle: where C ₁ -C ₈ substituted alkyl, C ₂ -C ₈ substituted alkenyl, C ₂ -C ₈ substiluted alkynyl, C ₆ - C ₂₀ substituted aryl, and C ₂ -C ₂₀ substituted heterocycle are independently substituted with one or more substituents selected from F, CI, Br, I, OH, —N(R ⁵ ) ₂ , — N(R ⁵ ) ₃ ⁺ , C ₁ -C ₈ alkylhalide, carboxylate, sulfate, sulfamate, sulfonate, C ₁ -C ₈ alkydsulfonate, C ₁ - C ₈ alkylamino, 4-dialkylaminopyridinium, C ₁ -C ₈ alkylhydroxyl, C ₁ -C ₈ alkylthiol. SO ₂ R ⁵ , —S(=O))R ⁵ , —SR ⁵ , — SO ₂ N(R ⁵ ) ₂ , — C(=O)R ⁵ , — CO ₂ R ⁵ , — C(==O)N(R ⁵ ) ₂ , —CN, —N ₃ , —NO ₂ , C ₁ -C ₈ alkoxy, C ₁ -C ₈ trifluoroalkyl, C ₁ -C ₈ alkyl. C ₃ - C ₁₂ carbocycle, C ₆ -C ₂₀ aryl, C ₂ -C ₂₀ heterocycle, polyethyleneoxy, phosphonate, and phosphate.

It will be understood that when T is NH, it is derived from a primary amine ( — NH2) pending from the therapeutic moiety (prior to coupling to the self-immolative moiety) and when T is N, it is derived from a secondary amine ( — NH — ) from the therapeutic moiety (prior to coupling to the self-immolative moiety). Similarly, when T is O or S, it is derived from a hydroxyl ( — OH) or sulfhydryl ( — SH) group respectively pending from the therapeutic moiety prior to coupling to the self-immolative moiety.

In some embodiments, the self-immolative linker L ² is —NH- (CH ₂ ) ₄ -C( =O)- or

— NH— (CH ₂ ) ₃ — C(=O)— .

In some embodiments, the self-immolative linker L ² is p- aminobenzyloxycarbonyl (PABC).

In some embodiments, the self-immolative linker L ² is 2,4-bis(hydroxymethyl)aniline e.

Other examples of self-immolative linkers that are readily adapted for use in therapeutic conjugates described herein are taught in, for example, US Patent 7,754,681; WO 2012/074693 Al; US 9,089,614; EP 1,732,607; WO 2015/038426A1 (all of which are incorporated by reference); Walther et al. “Prodrugs in medicinal chemistry and enzyme prodrug therapies” Adv Drug Deliv Rev. 2017 Sep 1; 118:65-77; and Tranoy-Opalinski et al. “Design of self- immolative linkers for tumour-activated prodrug therapy”, Anticancer Agents Med Chem. 2008 Aug;8(6):618-37; the teachings of each of which are incorporated by reference herein.

Yet other non-limiting examples of self-immolative linkers for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published December 12, 2019, incorporated by reference herein.

Therapeutic Moieties

In some embodiments, the therapeutic moiety (-TM) is an immune inducing agent, such as an agent that induces inflammation resulting in activation of innate and/or adaptive immune responses.

In some embodiments, the therapeutic moiety (-TM) is a cytotoxic agent, i.e., which when released from the therapeutic conjugate causes cell death of the target cells at the concentration at which the therapeutic conjugate is administered.

In some embodiments, the therapeutic moiety (-TM) is a cytostatic agent, i.e., which when released from the therapeutic conjugate causes mitotic arrest or quiescence of the target cells at the concentration at which the therapeutic conjugate is administered.

In some embodiments, the therapeutic moiety (-TM) is an epigenetic agent, i.e., which when released from the therapeutic conjugate causes epigenetic alteration of the target cells at the concentration at which the therapeutic conjugate is administered, which may, for example, result in differentiation (or dedifferentiation) of the cell to another cellular phenotype.

In some embodiments, the therapeutic moiety is represented by the general formula wherein RBM is a receptor binding moiety, Z is cytotoxic, cytostatic or epigenetic moiety or a radioisotope containing moiety, and p is 0 (Z is absent) or an integer from 1 to 8. In some embodiments, p is 1.

In some embodiments, the therapeutic moiety is represented by the general formula

— RBM— L ³ — Z wherein RBM is a receptor binding moiety, Z is cytotoxic, cytostatic or epigenetic moiety or a radioisotope containing moiety, and L ³ is a bond or a cleavable or non-cleavable linker. For instance, L ³ can be a linker that is acid labile or enzyme sensitive (such as includes a cathepsin cleavage site) such that Z is released intracellularly on internalization of the moiety -RBM-L ³ -Z through cell binding dependent on the receptor binding moiety RBM.

To further illustrate, the receptor binding moiety (RBM) can be a ligand for a receptor, such as ligand that when released from the therapeutic conjugate is able to bind to an extracellular ligand binding domain of a cell surface receptor. Exemplary receptor ligands include somatostatin, cholecystokinin-2 (CCK2), folate (folic acid), bombesin, gastrin- releasing peptide, calcitonin, oxytocin, EGF, α- melanocyte-stimulating hormone, minigastrin, neurotensin, substance P, glucagon-like peptide 1, neuropeptide Y and analogs of those ligands. The receptor ligand can itself have pharmacological activity in and of itself, or can be used to deliver a conjugated therapeutic moiety (e.g., drug moiety), toxin or radioisotope to (and preferably into) the cell expressing the receptor.

Additional examples include embodiments where the receptor binding moiety is a ligand or receptor agonist selected from the group consisting of: folate derivatives (proteins that bind to folate receptors and are overexpressed in ovarian cancer and other malignancies) (Low, PS et al 2008, acc. chem. res.41, 120-9); glutamate urea derivatives (binding to prostate specific membrane antigen, surface markers for prostate Cancer cells) (Hillier, SM et al, 2009, Cancer res.69, 6932-40); somatostatin (also known as Growth Hormone Inhibiting Hormone (GHIH) or growth hormone release inhibiting factor (SRIF) or growth hormone release inhibiting hormone) and its homologues, such as octreotide (Sandostatin) and lanreotide (Somatuline) (particularly for neuroendocrine tumors, GH-producing pituitary adenomas, paragangliomas, non-functional pituitary adenomas, pheochromocytomas (Ginj, M., et al, 2006, Proc. Natl. Acad. Sci. USA 103,16436-41), the somatostatin receptor subtypes in the following tumors (sstl, sst2, sst3, sst4 and sst5), GH-secreting pituitary adenomas (Reubi JC, L and olt, AM1984J. Clin. Endocrinol Metab 59: 1148-51; Re JC, L and olt AM 1987 Clin. Endocrinol Metab 65: 65. C.73; Moyse E, et al, J Clin Endocrinol Metab 61: 98-103), gastroenteropancreatic tumors (Reubi JC, et al, 1987J Clin Endocrinol Metab 65: 1127-34; Reubi, J.C, et al, 1990Cancer Res 50: 5969-77), pheochromocytoma (Epel-baum J, et al, 1995J Clin Endocrinol Metab80: 1837-44; Reubi J C, et al, 1992J Clin Endocrinol Metab 74: 1082-9), neuroblastoma (Prevost G,1996 neoendocrinolology 63: 188) 197; Mortel, Mill.L, et al, 1994 Amob J P102: 752-752), thyroid medullary carcinoma (Reubi, J.C, et al, 1991Lab 64: 7J, 1987J, 1987, et al, 1987J, 1987J, mouse colon receptor for colon Cancer, mouse colon receptor, mouse colon receptor, mouse colon receptor, mouse colon receptor, mouse colon receptor, mouse colon receptor, mouse colon receptor, mouseThe somatic subtype (CCK); bombesin (Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val- Gly-His-Leu-Met-NH)2) (SEQ ID NO: 143) Or Gastrin Releasing Peptide (GRP) and its receptor subclasses (BB1, GRP receptor subclasses (BB2), BB3 and BB4) (Ohlsson, b., et al, 1999, Sc and j. gastroenterology 34(12) 1224-9; weber, HC,2009, cur. opin. endocri. dib. obesityl6(l): 66-71, Gonzalez N, et al, 2008, cur. opin. endocri. dib. obesity 15(1), 58-64); neurotensin receptors and their receptor subtypes (NTR1, NTR2, NTR 3); substance P receptors and their receptor subtypes (e.g., NK1 receptor for glial tumors, Hennig I M, et al 1995int. J. cancer 61, 786-cake 792); neuropeptide Y (npy) receptor and its receptor subtype (Y1-Y6); homing peptides include RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (e.g., cRGDfV) (Laakkonen P, Vuorine K.2010, Integr Biol (Camb), sub.2 (7-8): 326-337; ChenK, Chen X.2011, Theranostics.l: 189-200; Garanger E, et al, Anti-Cancer Agents Med. 7(5): 552-558; Kerr, JS et al, Antincemer Research, 19(2A), 959-968; Thumshim, G, et al, 2003Chem. Eur.J.9, 7-2725), and TAASGVRSMH (SEQ ID NO: 144) or LTLRWVGLMS (SEQ ID NO: 145) (chondroitin sulfate proteoglycan NG2 receptor) and F3 (nucleolin receptor binding peptides) for cell surface expression (RG receptor) (amino acid receptor 31, BTE-Gly-26, WO 31, 92-26, Biodamen K.31, Biodamen K.326; And K.32, Biodamn. 31, Biodamn K.32; And K.32, Biodamn. 9, JP 31, 9, K, 9, K, 9, No. 35, No. 20, No. 35, No., proc.nat.acad.sci.usa 99(11), 7444-9); cell Penetrating Peptides (CPPs) (Nakase I, et al, 2012, J.Control Release.159(2), 181-188); peptide hormones, such as agonists and antagonists of Luteinizing Hormone Releasing Hormone (LHRH), gonadotropin releasing hormone (GnRH) agonists, acting by targeting Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) as well as testosterone production, such as buserelin (Pyr- His-Trp-Ser-Tyr-D-Ser (OtBu) -Leu-Arg-Pro-NHEt) (SEQ ID NO: 146), gonadorelin (Pyr- His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) (SEQ ID NO: 147), goserelin (Pyr-His-Trp- Ser-Tyr-D-Ser (OtBu) -Leu-Arg-Pro-AzGly-NH)2) (SEQ ID NO: 148) Himalarelin (Pyr-His- Trp-Ser-Tyr-D-His (N-Bn) -Leu-Arg-Pro-NHEt) (SEQ ID NO: 149), leuprolide (Pyr-His- Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt) (SEQ ID NO: 150), nafarelin (Pyr-His-Trp-Ser-Tyr- 2 Nal-Leu-Arg-Pro-Gly-NH)2) (SEQ ID NO: 151). Triptorelin (Pyr-His-Trp-Ser-Tyr-D- Trp-Leu-Arg-Pro-Gly-NH)2) (SEQ ID NO: 152) Nafarelin, desloralin, Aberelin (Ac-D-2Nal- D-4-chloroPhe-D-3- (3-pyridol) Ala-Ser- (N-Me) Tyr-D-Asn-Leu-isopyLLys-Pro-DAla- NH2) (SEQ ID NO: 153) Cetrorelix (Ac-D-2Nal-D-4-chloro-Phe-D-3- (3-pyridol) Ala-Ser- Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2) (SEQ ID NO: 154), degarelix (Ac-D-2Nal-D-4- chloroPhe-D-3- (3-pyridol) Ala-Ser-4-aminoPhe (L-hydroxyl) -D-4-aminoPhe (carba-moyl) - Leu-isoproylLys-Pro-D-Ala-NH)2) (SEQ ID NO: 155) And galangal (Ac-D-2Nal-D-4- chloroPhe-D-3- (3-pyridol) Ala-Ser-Tyr-D- (N9, NIO-diethyl) -homoArg-Leu- (N9, N10- diethyl) -homoArg-Pro-D-Ala-NH2) (SEQ ID NO: 156) (Thundimadathil, J., J. Aminoacids in Urology3(3): 127-140; Debruyne, F.,2006, Future Oncology, 2 (6); 677 696; Schally A.V; Nagy, A.1999Eur J Endocrinol 141: 1-14; Koppan M, et al 1999 Protate 38: 151-158);

Pattern Recognition Receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectins and nodular receptors (NLRs) (Fukata, M.et al, 2009, Semin. 21, Immunol 242-253; Maisoneune euonymus, C.et al, 2014, J.Ach.56, J.56.56, J.2011., J.56, J.2011.56, J.2011, 19870, 19856, USA, 14, III, V.56, III, V.l l, III. Short peptides, GRGDSPK (SEQ ID NO: 157) and cyclic RGD pentapeptides, such as cyclo (RGDfV) (SEQ ID NO: 160) (LI) and its derivatives (cyclo (-N (Me) R-GDfV) SEQ ID NO: 158), cyclo (R-Sar-DfV) SEQ ID NO: 159), cyclo- (RG-N (Me) D-fV) SEQ ID NO: 176), cyclo (RGD-N (Me) fV) SEQ ID NO: 177), cyclo (RGDf-N (Me) V-) (cilengitide)) SEQ ID NO: 178) have high affinity for integrin receptors (Dechantsreiter, MA et al, 1999J.Med.chem.42, 3033-40, Goodman, SL, et al, 2002J.Med.chem.45, 1045-51).

In some embodiments, the receptor binding moiety binds to a integrin αvβ3, a gastrin- releasing peptide receptor (GRPR), a somatostatin receptor (such as somatostatin receptor subtype 2), a melanocortin receptor, a cholecystokinin-2 receptor, a neuropeptide Y receptor or a neurotensin receptor.

In some embodiments, the receptor binding moiety binds to folate receptor α, and can be a folate receptor ligand, such as folic acid or folic acid analogs (such as etarfolatide, vintafolide, leucovorin and methotrexate).

In some embodiments, the receptor binding moiety binds to somatostatin receptor, and can be somatostatin or a somatostatin analogs, such as octreotate, octreotide or pentetreotide.

In some embodiments, the receptor binding moiety binds to αllbβ3, and can be an αllbβ3-targeted ligand, such as RGD or an RGD analog (i,e., dimer or multimeric analog), including illustrative cyclic RGD peptides like cyclo(-Arg-Gly-Asp-D-Phe Val-) [“c(RGDfV)”] (SEQ ID NO: 160), c(RGDfK) (SEQ ID NO: 161), c(RGDfC) (SEQ ID NO: 162), c(RADfC) (SEQ ID NO: 163), c(RADfK) (SEQ ID NO: 164), c(RGDfE) (SEQ ID NO: 165), c(RADfE) (SEQ ID NO: 166), RGDSK(SEQ ID NO: 167), RADSK(SEQ ID NO: 168), RGDS(SEQ ID NO: 169), c(RGDyC) (SEQ ID NO: 170), c(RADyC) (SEQ ID NO: 171), c(RGDyE) (SEQ ID NO: 172), c(RGDyK) (SEQ ID NO: 173), c(RADyK) (SEQ ID NO: 174) and H-E[c(RGDyK)] ₂ (SEQ ID NO: 175), EMD 12194, DMP728, DMP757 and SK&F107260.

Therapeutic moieties, in some embodiments, are selected based on their ability to induce an innate immune response in vivo. Innate immune responses include cellular responses to exogenous nucleic acids or proteins, typically of viral or bacterial origin, resulting in increased cytokine expression and release and cell death. Therefore, the innate immune response can be measured by the expression or activity level of cytokines (e.g., Type 1 interferons) or the expression levels of interferon-regulated genes (e.g., toll-like receptors). In some embodiments, the innate immune response is measured by the expression or activity level of granulocyte colony-stimulating factor (G-CSF). The response may also be measured by the level of cell death.

In some embodiments, a therapeutic conjugate induces a lower innate immune response compared to the therapeutic moiety alone. For example, in some embodiments, a therapeutic conjugate induces an innate immune response that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than the innate immune response induced when the therapeutic moiety is administered alone.

In some embodiments, a therapeutic moiety is an acutely toxic. As used herein, a moiety that is “acutely toxic” refers to a moiety that has adverse effects within 14 days of administration to a subject. Acute toxicity is compared to chronic toxicity or cumulative toxicity, in which the build-up of a therapeutic moiety over time results in the therapeutic moiety’s toxicity.

Toxicity may be measured using any method. In some embodiments, toxicity is measured by liver activity. In some embodiments, toxicity is measured by kidney activity. In some embodiments, toxicity is measured by pancreas activity.

In some embodiments, toxicity is measured by assessing circulating liver enzymes such as aspartate transaminase (AST) and/or alanine transaminase (ALT). AST and/or ALT levels at timepoints after the administration of therapeutic moiety may be compared to baseline values obtained prior to administration, for example, to those of control animals that did not receive the therapeutic moiety, or to values known to be associated with normal animals from previous experiments (historical controls) or from literature.

In some embodiments, toxicity is measured by assessing alkaline phosphatase (ALP) levels. ALP levels at timepoints after the administration of a therapeutic moiety may be compared to baseline values obtained prior to administration, for example, to those of control animals that did not receive the therapeutic moiety, or to values known to be associated with normal animals from previous experiments (historical controls) or from literature.

In some embodiments, toxicity is measured by assessing total bilirubin (TBIL) levels. TBIL levels at timepoints after the administration of a therapeutic moiety may be compared to baseline values obtained prior to administration, for example, to those of control animals that did not receive the therapeutic moiety, or to values known to be associated with normal animals from previous experiments (historical controls) or from literature.

In some embodiments, toxicity is measured by assessing albumin levels. Albumin levels at timepoints after the administration of a therapeutic moiety may be compared to baseline values obtained prior to administration, for example, to those of control animals that did not receive the therapeutic moiety, or to values known to be associated with normal animals from previous experiments (historical controls) or from literature.

In some embodiments, toxicity is measured by assessing serum glucose levels. Serum glucose levels at timepoints after the administration of a therapeutic moiety may be compared to baseline values obtained prior to administration, for example, to those of control animals that did not receive the therapeutic moiety, or to values known to be associated with normal animals from previous experiments (historical controls) or from literature.

In some embodiments, toxicity is measured by assessing lactate dehydrogenase (LDH) levels. Lactate dehydrogenase (LDH) levels at timepoints after the administration of a therapeutic moiety may be compared to baseline values obtained prior to administration, for example, to those of control animals that did not receive the therapeutic moiety, or to values known to be associated with normal animals from previous experiments (historical controls) or from literature.

Without wishing to be bound by theory, it is thought that a therapeutic conjugate holds the therapeutic moiety from being active until FAPα cleaves the linker. In this way, a therapeutic conjugate is not acutely toxic and has a higher therapeutic index (TI) than the free therapeutic moiety. TI is a quantitative measure of the relative safety of the therapeutic moiety, calculated by comparing the amount of the therapeutic moiety (or conjugate) that causes the therapeutic effect to the amount of the therapeutic moiety (or conjugate) that causes toxicity. In some embodiments, a therapeutic conjugate has a therapeutic index (TI) when delivered systemically that is at least 2-fold greater than the systemic delivery of the free therapeutic moiety, for example, at least 5, 10, 20, 30, 40, 50, 100, 250, 500 or even 1000 greater than the systemic delivery of the free therapeutic moiety. In some embodiments, the therapeutic index (TI) of a therapeutic conjugate is at least 5 times greater than the therapeutic index for the free therapeutic moiety when given systemically, for example at least 10, 20, 30, 40, 50, 75 or even 100 times greater.

In this way, therapeutic moieties (drugs) that were previously ineffective due to dose- limiting toxicities (e.g., talabostat), can be administered at therapeutic doses (e.g., intratumoral concentrations of 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, or more), as described herein.

Therapeutic moieties provided herein may be prodrugs, which are drugs (e.g., small molecules having a molecular weight of 10 kD or lower) that are inert in their prodrug form (e.g., have a reduced risk of systemic toxicities, have reduced cell permeability), but are activated by cleavage of the FAPα-cleavable linker. Upon cleavage, the therapeutic moiety is released into the extracellular space of the diseased tissue (e.g., tumor), where it may cause a local inflammatory response, promote immune cell infiltration, and in some embodiments, induce an innate immune response. When the diseased tissue is a tumor, the activated therapeutic moiety may also degrade the tumor stroma, and/or kill tumor cells.

In some embodiments, free therapeutic moiety interacts with an intracellular target and the pharmacological effect of the therapeutic moiety is dependent on the free therapeutic moiety being cell permeable, i.e., and able to interact with its intracellular target, whereas when part of the binder-drug conjugate the therapeutic moiety is substantially cell impermeable. For instance, the rate of accumulation of the binder-drug conjugate intracellularly is less than 50% of the rate for the free therapeutic moiety, for example, less than 25%, 10%, 5%, 1% or even less than 0.1% of the rate for the free therapeutic moiety. For instance, the EC50 for the pharmacological effect of the free therapeutic moiety is at least 2 fold less than (more potent than) the binder-drug conjugate, for example, at least 5, 10, 20, 30, 40, 50, 100, 250, 500 or even 1000 less than the binder-drug conjugate.

In some embodiments, the free therapeutic moiety interacts with an extracellular target and the pharmacological effect of the therapeutic moiety is substantially attenuated when covalently linked to L ¹ . For instance, the EC50 for the pharmacological effect of the free therapeutic moiety is at least 2 fold less than (more potent than) the binder-drug conjugate, for example, at least 5, 10, 20, 30, 40, 50, 100, 250, 500 or even 1000 less than the binder-drug conjugate.

In some embodiments, the binder-drug conjugate has a therapeutic index when delivered systemically that is at least 2-fold greater than the systemic delivery of the free therapeutic moiety, for example, at least 5, 10, 20, 30, 40, 50, 100, 250, 500 or even 1000 greater than the systemic delivery of the free therapeutic moiety.

In some embodiments, the free therapeutic moiety is an immunomodulator - which includes drug moieties acting as immune activating agents and/or inducers of an innate immunity pathway response. In some embodiments, the free therapeutic moiety induces the production of IFN-a. In some embodiments, the free therapeutic moiety induces the production of proinflammatory cytokines. In some embodiments, the free therapeutic moiety induces the production of IL-1β. In some embodiments, the free therapeutic moiety induces the production of IL- 18.

In some embodiments, the free therapeutic moiety promotes the expansion and survival of effector cells including NK, γδ T, and CD8+ T cells.

In some embodiments, the free therapeutic moiety is a damage-associated molecular pattern molecule. In some embodiments, the free therapeutic moiety is a pathogen-associated molecular pattern molecule.

Therapeutic moieties for use herein include, for example, those recognized in the official United States Pharmacopeia, official Homeopathic Pharmacopeia of the United States, official National Formulary, or any supplement thereof, and include, but are not limited, to small molecules chemicals/drugs, polynucleotides (e.g., RNA interference molecules, such as miRNA, siRNA, shRNA, and antisense RNA), and polypeptides (e.g., antibodies). Classes of therapeutic molecules that may be used as provided herein include, but are not limited to, recombinant proteins, antibodies, cytotoxic agents, anti-metabolites, alkylating agents, antibiotics, growth factors (e.g., erythropoietin, granulocyte colony- stimulating factor (G-CSF), granulocyte-macrophage colony- stimulating factor (GM-CSF), keratinocyte growth factor)), cytokines, chemokines, interferons (e.g., interferon-alpha, interferon-beta, interferon-gamma), blood factors (e.g., factor VIII, factor Vila, factor IX, thrombin, antithrombin), anti-mitotic agents, toxins, apoptotic agents, (e.g., DNA alkylating agents), topoisomerase inhibitors, endoplasmic reticulum stress inducing agents, platinum compounds, antimetabolites, vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, tyrosine kinase inhibitors, radiosensitizers, chemotherapeutic combination therapies, receptor traps, receptor ligands, angiogenic agents, anti-angiogenic agents, anti- coagulants and thrombolytics (e.g., tissue plasminogen activator, hirudin, protein C), neurotransmitters, erythropoiesis- stimulating agents, insulin, growth hormones (e.g., human growth hormone (hGH), follicle-stimulating hormone), metabolic hormones (e.g., incretins), recombinant IL-1 receptor antagonists, and bispecific T-cell engaging molecules (BITEs®).

In some embodiments, the free therapeutic moiety is a cyclic dinucleotide. In some embodiments, the free drug moiety is ADU-S100. In some embodiments, the free drug moiety is cytotoxic to cancer associated fibroblasts (CAFs).

In some embodiments, the free therapeutic moiety polarizes tumor associated macrophage populations towards Ml macrophage and/or inhibits M2 macrophage immunosuppressive activity.

In some embodiments, the free therapeutic moiety accelerates T-cell priming and/or dendritic cell trafficking.

In some embodiments, the free therapeutic moiety inhibits or depletes Treg cells, such as by blocking immunosuppressive function or migration to lymph nodes and/or the tumor microenvironment.

In some embodiments, the therapeutic moiety is a chemotherapeutic drug moiety. Examples of chemotherapeutic drug moieties include, but are not limited to, platinum-based drugs (e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin, satraplatin, etc.), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, and nitrosoureas), anti-metabolites (e.g., 5- fluorouracil (5-FU), azathioprine, methotrexate, leucovorin, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, pemetrexed, and raltitrexed), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel (taxol), and docetaxel), topoisomerase inhibitors (e.g., irinotecan (CPT-11; Camptosar), topotecan, amsacrine, etoposide (VP16), etoposide phosphate, and teniposide), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, and plicamycin), tyrosine kinase inhibitors (e.g., gefltinib (IRESSA®), sunitinib (SUTENT®; SU11248), erlotinib (TARCEVA®; OSI-1774), lapatinib (GW572016; GW2016), canertinib (Cl 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (GLEEVEC®; STI571), dasatinib (BMS-354825), leflunomide (SU101), vandetanib (ZACTIMA™; ZD6474), pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof.

Other non-limiting examples of therapeutic moieties are provided in WO 2019/236567, incorporated herein by reference. T axanes/T axoids

In some embodiments, the therapeutic moiety is a taxane. Taxanes are a class of diterpenes. The principal mechanism of action of the taxane class of drugs is the disruption of microtubule function. Microtubules are essential to cell division, and taxanes stabilize GDP- bound tubulin in the microtubule, thereby inhibiting the process of cell division as depolymerization is prevented. Thus, in essence, taxanes are mitotic inhibitors. In contrast to the taxanes, the vinca alkaloids prevent mitotic spindle formation through inhibition of tubulin polymerization. Both taxanes and vinca alkaloids are, therefore, named spindle poisons or mitosis poisons, but they act in different ways. Taxanes are also thought to be radiosensitizing. Non-limiting examples of taxanes include paclitaxel, docetaxel, and cabazitaxel.

Platinum-Based Agents

In some embodiments, the therapeutic moiety is a platinum-based agent. Platinum- based antineoplastic drugs (informally called platins) are chemotherapeutic agents typically used to treat cancer. Platinum-based antineoplastic agents cause crosslinking of DNA as monoadduct, interstrand crosslinks, intrastrand crosslinks or DNA protein crosslinks. Mostly they act on the adjacent N-7 position of guanine, forming a 1, 2 intrastrand crosslink. The resultant crosslinking inhibits DNA repair and/or DNA synthesis in cancer cells. Platinum- based antineoplastic agents are sometimes described as "alkylating-like" due to similar effects as alkylating antineoplastic agents, although they do not have an alkyl group. Non-limiting examples of platinum-based agents include oxaliplatin, cisplatin, carboplatin, nedaplatin, picoplatin, phenanthriplatin, triplatin, spiroplatin, satraplatin, iproplatin, and satraplatin.

Proteasome Inhibitors

In some embodiments, the therapeutic moiety is a proteasome inhibitor. Proteasome inhibitors are drugs that block the action of proteasomes, cellular complexes that break down proteins. Multiple mechanisms are likely to be involved, but proteasome inhibition may prevent degradation of pro-apoptotic factors such as the p53 protein, permitting activation of programmed cell death in neoplastic cells dependent upon suppression of pro-apoptotic pathways. For example, bortezomib causes a rapid and dramatic change in the levels of intracellular peptides. Non-limiting examples of proteasome inhibitors include bortezomib, lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib (salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP- 18770), epoxomicin, and beta-hydroxy beta- methylbutyrate. Exemplary proteasome inhibitors include

Toll-Like Receptor Agonists

In some embodiments, the free therapeutic moiety is a Toll-like receptor (TLR) agonist, such as a selected from the group consisting of a TLR1/2 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6/2 agonist, a TLR7 agonist, a TLR7/8 agonist, a TLR7/9 agonist, a TLR8 agonist, a TLR9 agonist, and a TLR11 agonist, for example, selected from the group consisting of a TLR3 agonist, a TLR7 agonist, a TLR7/8 agonist, and a TLR9 agonist. Toll-like receptor (TLR) agonists are involved in activating both innate and adaptive immune responses. Most TLR agonists activate professional antigen-presenting cells (APCs), such as dendritic cells and interact with T cells. As TLRs are central to innate immunity, TLR agonists have been used to enhance TLR activity and, relatedly, activate the innate immune response. TLR agonists are short, synthetic stretches of single-stranded DNA typically comprising a CpG dinucleotide motif.

Examples of toll-like receptor (TLR) agonists include, but are not limited to, TLR 1/2 agonists, TLR2 agonists, TLR3 agonists (e.g., PolyTC), TLR4 agonists (e.g., S-type lipopolysaccharide, paclitaxel, lipid A, and monophosphoryl lipid A), TLR5 agonists (e.g., flagellin), TLR6/2 agonists (e.g., MALP-2), TLR7 agonist, TLR7/8 agonists (e.g., gardiquimod, imiquimod, loxoribine, and resiquimod (R848)), TLR7/9 agonists (e.g., hydroxychloroquine sulfate), TLR8 agonists (e.g., motolimod (VTX-2337)), TLR9 agonists (e.g., CpG-ODN), and TLR11 agonists (e.g., profilin).

Examples of TLR agonists that can be used as the therapeutic moiety in the conjugates of include S-27609, CL307, UC-IV150, imiquimod, gardiquimod, resiquimod, motolimod, VTS-1463GS-9620, GSK2245035, TMX-101, TMX-201, TMX- 202, isatoribine, AZD8848, MED 19197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240, KU34B, or CL663, or as appropriate, analogs thereof with appropriate functional groups for directed linkage and release from the substrate recognition sequence or by linkage to a self-immolative linker.

Examples of agonists of TLRs, particularly TLR7 agonists, TLR8 agonists and TLR7/8 agonists include: isatoribine, loxoribine, bropirimine, imiquimod, resiquimod, gardiquimod, CL097, 3M-002, R848, SM360320, CL264, 3M-OO3, IMDQ, PF-04878691, motolimod (VTX-2337), and GSK2245035. See, e.g., International Publication No. WO 2019/236567, published December 12, 2019, for chemical structures of the foregoing TLR agonists.

In some embodiments, the therapeutic moiety is a TRL7/8 agonist represented in the general formula wherein X is CH ₂ , O, S or N, for example, CH ₂ , O or N; n is 0 (direct bond from N to O), or an integer from 1 to 5, for example 1 or 2; z is an integer from 1 to 5; m is an integer from 1 to 20, for example from 1 to 16; p is 0 (direct bond from ring to X), or an integer from 1 to 5, for example 1 or 2; and q is an integer from 1 to 5, for example 1 or 2. For instance, the TRL agonist is a TRL7/8 agonist such as one of:

International Publication Nos. WO 2008/135791 and WO 2016/141092 also describe classes of imidazoquinoline compounds having immuno-modulating properties which act via

TLR7.

Other examples of TRL agonists that be readily adapted for use as the therapeutic moiety of the conjugates are disclosed in, for example, Yoo et al. “Structure-activity relationships in Toll-like receptor 7 agonistic lH-imidazo[4,5- cjpyridines” Org. Biomol. Chem., 2013, 11, 6526-6545; Fletcher et al. “Masked oral prodrugs of Toll-like receptor 7 agonists: a new approach for the treatment of infectious disease”, 2006 Current opinion in investigational drugs (London, England). 7. 702-708; and Pryde et al. “The discovery of a novel prototype small molecule TLR7 agonist for the treatment of hepatitis C virus infection” Med. Chem. Commun., 2011, 2, 185-189.

Other non-limiting examples of TLR agonists for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published December 12, 2019, incorporated by reference herein.

It will also be appreciated by those skilled in the art that, particularly with the use of a self- immolative linker, the TRL agonists can be coupled to the linker though functional groups other than amines as shown above, such as through free hydroxyl groups for example.

Retinoic Acid-Inducible Gene I (RIG-I) Agonists In some embodiments, the free therapeutic moiety is a RIG-1 agonist. Retinoic acid- inducible gene I (RIG-1) agonists are used to induce innate immune responses. RIG-I is responsible for the type-1 interferon (IFNI) response. Upon its activation, RIG-I activates its RIG-I inflammasome, which leads to pyroptosis, an immunogenic mechanism of programmed cell death as well as the induction of pro-inflammatory cyotkines (Elion et al., Oncotarget. 2018; 9(48): 29007-29017). Pyroptosis results in the formation of pores in the plasma membrane, leading to hypotonic cell swelling and leakage of intracellular contents, including danger associated molecular patterns (DAMPs). The DAMPs and cytokines lead to a local acute inflammatory immune response.

Examples of RIG-I agonists include KIN700, KIN1148, KIN600, KIN500, KIN100, KIN101, KIN400, KIN2000, RGT100, and SD-9200.

Other non-limiting examples of RIG-I agonists for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published December 12, 2019, incorporated by reference herein. iDASH Inhibitors

In some embodiments, the free therapeutic moiety is an immuno-DASH inhibitor that inhibits the enzymatic activity of DPP8 and DPP9 and induces macrophage pyroptosis in vitro and/or in vivo. iDASH inhibitors are inhibitors of the of the DPP4, DPP8 and DPP9, post-proline cleaving enzymes, and act as checkpoint inhibitors of an immuno-checkpoint involving DASH enzymes. Inhibition of these target enzymes, which include both intracellular and extracellular targets, results in an increase in T cell cytotoxicity (release of cancer cell antigens, resulting in cancer cell death), an increase in dendritic cell trafficking (cancer antigen presentation), an increase in immuno stimulatory cytokines (priming and activation of APCs and T cells), an increase in intratumoral CXCL10 half-life (trafficking of T cells to tumors), infiltration of T cells into tumors, recognition of cancer cells by T cells, and myeloid-derived suppressor cell (MDSC) depletion and impairment of MDSC immunosuppressive activity. Non-limiting examples of iDASH inhibitors are described in WO 2019/236567, the entire contents of which are incorporated herein by reference.

In some embodiments, the iDASH inhibitor is a boronic acid inhibitor of the DASH enzymes DPP8 and DPP9 (and in some embodiments also DPP-4 and/or FAP).

In some embodiments, the iDASH inhibitor is a dipeptide boronic acid inhibitor of the DASH enzymes DPP8 and DPP9 (and in some embodiments also DPP-4 and/or FAP). In some embodiments, the iDASH inhibitor the dipeptide boronic acid has a proline or proline analog in the Pl position. The subject iDASH inhibitors can mediate tumor regression by immune-mediated mechanisms. The subject iDASH inhibitors induce macrophage pyroptosis, and directly or indirectly have such activities as immunogenic modulation, sensitize tumor cells to antigen- specific CTL killing, alter immune-cell subsets and function, accelerate T cell priming via modulation of dendritic cell trafficking, and invoke a general T- cell mediated antitumor activity.

Examples of iDASH inhibitors include saxagliptin, sitagliptin, linagliptin, alogliptin, Valine-boroProline (PT 100), vildagliptin, gemigliptin, anagliptin, teneligliptin, trelagliptin, omarigliptin, evogliptin, gosogliptin, dutogliptin.

In some embodiments, the immuno-DASH inhibitor for use in the method of the present disclosure are represented by the general formula; wherein

A represents a 4-8 membered heterocycle including the N and the Ca carbon; Z represents C or N;

R’ 1 represents a C-terminally linked amino acid residue or amino acid analog, or a C-terminally linked peptide or peptide analog, the amine terminus of which forms a covalent with LI, or if LI is a bond then with the substrate recognition sequence;

R’2 is absent or represents one or more substitutions to the ring A, each of which can independently be a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, — (CH ₂ ) _m — R7, — (CH ₂ ) _m — OH, — (CH ₂ ) _m — O-lower alkyl, — (CH ₂ ) _m — O-lower alkenyl, — (CH2) _n — O— (CH ₂ ) _m — R7, — (CH ₂ ) _m — SH, — (CH ₂ ) _m — S -lower alkyl, — (CH ₂ ) _m — S-lower alkenyl, — (CH ₂ ) _n — S— (CH ₂ ) _m — R7; if X is N, R’3 represents hydrogen, if X is C, R’3 represents hydrogen or a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, — (CH ₂ ) _m — R7, — (CH ₂ ) _m — OH, — (CH ₂ ) _m — O-lower alkyl, — (CH ₂ ) _m — O-lower alkenyl, — (CH ₂ ) _n — O— (CH2) _m — R7, — (CH ₂ ) _m — SH, — (CH ₂ ) _m — S-lower alkyl, — (CH ₂ ) _m — S-lower alkenyl, — (CH ₂ ) _n — S— (CH ₂ ) _m — R7;

R5 represents H, an alkyl, an alkenyl, an alkynyl, — C(X1)(X2)X3, — (CH ₂ ) _m — R7, — (CH ₂ ) _n -OH, — (CH ₂ ) _n -O-alkyl, — (CH ₂ ) _n -O-alkenyl, — (CH ₂ ) _n -O- alkynyl, — (CH ₂ ) _n -O— (CH ₂ ) _m -R7, — (CH ₂ ) _n -SH, — (CH ₂ ) _n -S -alkyl, — (CH ₂ ) _n -S- alkenyl, — (CH ₂ ) _n -S -alkynyl, — (CH ₂ ) _n -S— (CH ₂ ) _m -R7, — C(O)C(O) _n H2, — C(O)C(O)OR'7;

R6 represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, an aryl, — (CH2) _m — R7, — (CH ₂ ) _m — OH, — (CH ₂ ) _m — O-lower alkyl, — (CH ₂ ) _m — O-lower alkenyl, — (CH ₂ ) _n — O— (CH ₂ ) _m — R7, — (CH ₂ ) _m — SH, — (CH ₂ ) _m — S-lower alkyl, — (CH ₂ ) _m — S-lower alkenyl, — (CH ₂ ) _n — S— (CH ₂ ) _m — R7,

R7 represents, for each occurrence, a substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R'7 represents, for each occurrence, hydrogen, or a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; and

Y1 and Y2 can independently or together be OH, or a group capable of being hydrolyzed to a hydroxyl group, including cyclic derivatives where Y 1 and Y2 are connected via a ring having from 5 to 8 atoms in the ring structure (such as pinacol or the like),

R50 represents O or S;

R51 represents N ₃ , SH ₂ , NH ₂ , NO ₂ or O-R'7;

R52 represents hydrogen, a lower alkyl, an amine, OR'7, or a pharmaceutically acceptable salt, or R51 and R52 taken together with the phosphorous atom to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure

XI represents a halogen;

X2 and X3 each represent a hydrogen or a halogen m is zero or an integer in the range of 1 to 8; and n is an integer in the range of 1 to 8.

In preferred embodiments, the ring A is a 5, 6 or 7 membered ring, e.g., represented by the formula and more preferably a 5 or 6 membered ring (e.g., n is 1 or 2, though n may also be 3 or 4). The ring may, optionally, be further substituted.

In preferred embodiments, W represents

In preferred embodiments, R’ 1 is wherein R36 is a small hydrophobic group, e.g., a lower alkyl or a halogen and R38 is hydrogen, or R36 and R37 together form a 4-7 membered heterocycle including the N and the Ca carbon, as defined for A above.

In preferred embodiments, R’2 is absent, or represents a small hydrophobic group such as a lower alkyl or a halogen.

In preferred embodiments, R’3 is a hydrogen, or a small hydrophobic group such as a lower alkyl or a halogen.

In preferred embodiments, R’5 is a hydrogen, or a halogenated lower alkyl.

In preferred embodiments, XI is a fluorine, and X2 and X3, if halogens, are fluorine. Also deemed as equivalents are any compounds which can be hydrolytically converted into any of the aforementioned compounds including boronic acid esters and halides, and carbonyl equivalents including acetals, hemiacetals, ketals, and hemiketals, and cyclic dipeptide analogs.

In some preferred embodiments, the subject method utilizes, as a immuno-DASH inhibitor, a boronic acid analogs of an amino acid. For example, the present disclosure contemplates the use of boro-prolyl derivatives in the subject method. Exemplary boronic acid derived inhibitors of the present disclosure are represented by the general formula: wherein

R’ 1 represents a C-terminally linked amino acid residue or amino acid analog, or a C-terminally linked peptide or peptide analog, the amine terminus of which forms a covalent with LI, or if LI is a bond then with the substrate recognition sequence; and

R11 and R12 each independently represent hydrogen, a alkyl, or a pharmaceutically acceptable salt, or R11 and R12 taken together with the O — B — O atoms to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure.

In some embodiments, the immuno-DASH inhibitor is a peptide or peptidomimetic including a prolyl group or analog thereof in the Pl specificity position, and a nonpolar (and preferably hydrophobic) amino acid in the P2 specificity position, e.g., a nonpolar amino acid such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan or methionine, or an analog thereof. In other embodiments, the P2 position an amino acid with charged sidechain, such as Arginine, Lysine, Aspartic acid or Glutamic Acid. For example, the immuno-DASH inhibitor may include an Ala- Pro or Val-Pro dipeptide sequence or equivalent thereof, and be represented in the general formulas:

In preferred embodiments, the ring A is a 5, 6 or 7 membered ring, e.g., represented by the formula

In some preferred embodiments, R32 is a small hydrophobic group, e.g., a lower alkyl or a halogen.

In some preferred embodiments, R32 is - lower alkyl-guanidine, -lower-alkyl-amine, lower-alkyl-C(O)OH, such as -(CH ₂ ) _m -NH-C(=N)(NH ₂ ), -(CH ₂ ) _m -NH2 or -(CH ₂ ) _m -COOH, where m is 1-6, and preferably 1-3.

In preferred embodiments, R’2 is absent, or represents a small hydrophobic group such as a lower alkyl or a halogen.

In preferred embodiments, R’3 is a hydrogen, or a small hydrophobic group such as a lower alkyl or a halogen.

Another aspect of the disclosure relates to the immuno-DASH inhibitor represented by formula III, or a pharmaceutical salt thereof: wherein ring Z represents a 4-10 membered heterocycle including the N and the Cα carbon;

W represents -CN, — CH=NR4, a functional group which reacts with an active site residue of the target, or

X is O or S;

X2 is H, a halogen, or a lower alkyl;

Y 1 and Y2 are independently OH, or together with the boron atom to which they are attached represent a group that is hydrolysable to a boronic acid, or together with the boron atom to which they are attached form a 5-8 membered ring that is hydrolysable to a boronic acid;

R1 represents, independently for each occurrence, a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl, a thiocarbonyl, an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, — CF3, — (CH ₂ ) _m — R3, — (CH ₂ ) _m OH, — (CH ₂ ) _m — O-lower alkyl, — (CH ₂ ) _m — O-lower alkenyl, — (CH ₂ )n— O— (CH ₂ ) _m — R3, — (CH ₂ ) _m — SH, — (CH ₂ ) _m — S-lower alkyl, — (CH ₂ ) _m — S-lower alkenyl, or — (CH ₂ ) _n — S — (CH ₂ ) _m — R3;

R2 represents, for each occurrence, hydrogen, lower alkyl, lower alkynyl, — (CH ₂ ) _m — R3, — C(=O)-alkyl, — C(=O)-alkenyl, — C(=O)-alkynyl, or — C(=O)— (CH ₂ ) _m — R3;

R3 represents, for each occurrence, hydrogen, or a substituted or unsubstituted lower alkyl, lower alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R4 represents a hydrogen, a lower alkyl, a lower alkenyl, a lower alkynyl, — (CH ₂ ) _m — R3, — (CH ₂ ) _n — OH, — (CH ₂ )n— O-lower alkyl, — (CH ₂ ) _n — O-alkenyl, — (CH ₂ ) _n — O-alkynyl, — (CH ₂ ) _n — O— (CH ₂ ) _m — R7, — (CH ₂ ) _n — SH, — (CH ₂ ) _n — S- lower alkyl, — (CH ₂ ) _n — S-lower alkenyl, — (CH ₂ ) _n — S-lower alkynyl, — (CH ₂ ) _n — S — (CH ₂ ) _m — R3, — C(O)C(O)NH ₂ , or — C(O)C(O)OR8;

R5 represents O or S;

R6 represents N3, SH, NH2, NO2 or OR8;

R7 represents hydrogen, a lower alkyl, an amine, OR8, or a pharmaceutically acceptable salt, or R5 and R6 taken together with the phosphorous atom to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure;

R8 represents, hydrogen, a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocyclyl;

RIO is absent or represents one to three substitutions to the ring Z to which they are appended, each of which can independently be a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, an isocyano, a thiocyanato, an isothiocyanato, a cyanato, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, lower alkyl-C(O)OH, -O-lower alkyl-C(O)OH, -guanidinyl; — (CH ₂ ) _m — R7, — (CH ₂ ) _m — OH, — (CH ₂ ) _m — O-lower alkyl, — (CH ₂ ) _m — O-lower alkenyl, — (CH ₂ ) _n — O— (CH ₂ ) _m — R3, — (CH ₂ ) _m — SH, — (CH ₂ ) _m — S -lower alkyl, — (CH ₂ ) _m — S -lower alkenyl, — (CH ₂ ) _n — S— (CH ₂ ) _m — R3; n is 0, 1, 2, or 3; and m is 0, 1, 2, or 3.

Another aspect of the disclosure relates to the immuno-DASH inhibitor represented by formula IV, or a pharmaceutical salt thereof: wherein ring A represents a 3-10 membered ring structure including the N; ring Z represents a 4-10 membered heterocycle including the N and the Ca carbon;

W represents -CN, — CH=NR4, a functional group which reacts with an active site residue of the target, or X is O or S;

XI represents a halogen;

Y 1 and Y2 are independently OH, or together with the boron atom to which they are attached represent a group that is hydrolysable to a boronic acid, or together with the boron atom to which they are attached form a 5-8 membered ring that is hydrolysable to a boronic acid;

R1 represents a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl, a thiocarbonyl, an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, — CF3, — (CH2)m — R3, — (CH2)mOH, — (CH2)m — O-lower alkyl, — (CH2)m— O-lower alkenyl, — (CH2)n— O— (CH2)m— R3, — (CH2)m— SH, — (CH2)m — S -lower alkyl, — (CH2)m — S -lower alkenyl, or — (CH2)n — S — (CH2)m — R3;

R2 represents, for each occurrence, hydrogen, lower alkyl, lower alkynyl, — (CH2)m— R3, — C(=O)-alkyl, — C(=O)-alkenyl, — C(=O)-alkynyl, or — C(=O)— (CH2)m— R3;

R3 represents, for each occurrence, hydrogen, or a substituted or unsubstituted lower alkyl, lower alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R4 represents a hydrogen, a lower alkyl, a lower alkenyl, a lower alkynyl, — (CH2)m— R3, — (CH2)n— OH, — (CH2)n— O-lower alkyl, — (CH2)n— O-alkenyl, — (CH2)n— O-alkynyl, — (CH2)n— O— (CH2)m— R7, — (CH2)n— SH, — (CH2)n— S -lower alkyl, — (CH2)n — S-lower alkenyl, — (CH2)n — S-lower alkynyl, — (CH2)n — S — (CH2)m — R3, — C(O)C(O)NH2, or — C(O)C(O)OR8;

R5 represents O or S;

R6 represents N3, SH, NH2, NO2 or OR8;

R7 represents hydrogen, a lower alkyl, an amine, OR8, or a pharmaceutically acceptable salt, or R5 and R6 taken together with the phosphorous atom to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure;

R8 represents, hydrogen, a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocyclyl;

R9 and RIO, each independently, are absent or represents one to three substitutions to the ring A or to the ring Z to which they are appended, each of which can independently be a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, an isocyano, a thiocyanato, an isothiocyanato, a cyanato, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, — (CH2)m — R7, — (CH2)m — OH, — (CH2)m — O-lower alkyl, — (CH2)m — O-lower alkenyl, — (CH2)n— O— (CH2)m— R3, — (CH2)m— SH, — (CH2)m— S -lower alkyl, — (CH2)m— S- lower alkenyl, — (CH2)n — S — (CH2)m — R3; n is 0, 1, 2, or 3; and m is 0, 1, 2, or 3.

In some preferred embodiments, the immuno-DASH inhibitor is a boronic acid inhibitor of the DASH enzymes DPP8 and DPP9 (and optionally also DPP-4 and/or FAP).

In some preferred embodiments, the immuno-DASH inhibitor is a dipeptide boronic acid inhibitor of the DASH enzymes DPP8 and DPP9 (and optionally also DPP-4 and/or FAP). In some preferred embodiments, the immuno-DASH inhibitor the dipeptide boronic acid has a proline or proline analog in the Pl position. The subject immuno-DASH inhibitors can mediate tumor regression by immune-mediated mechanisms. The subject immuno-DASH inhibitors induce macrophage pyroptosis, and directly or indirectly have such activities as immunogenic modulation, sensitize tumor cells to antigen-specific CTL killing, alter immune-cell subsets and function, accelerate T cell priming via modulation of dendritic cell trafficking, and invoke a general T-cell mediated antitumor activity.

In some embodiments, the subject combination of immuno-DASH inhibitor and PD-1 inhibitor can be administered as part of a therapy involving one or more other chemotherapeutic agents, immuno-oncology agents or radiation. It can also be used a part of therapy including tumor vaccines, adoptive cell therapy, gene therapy, oncolytic viral therapies and the like.

In some embodiments, the immuno-DASH inhibitor of the present methods is represented by formula I, or a pharmaceutical salt thereof: wherein ring A represents a 3-10 membered ring structure; ring Z represents a 4-10 membered heterocycle including the N and the Cα carbon;

W represents -CN, —CH=NR4, a functional group which reacts with an active site residue of the target, or

X is O or S;

X1 represents a halogen;

Y 1 and Y2 are independently OH, or together with the boron atom to which they are attached represent a group that is hydrolysable to a boronic acid, or together with the boron atom to which they are attached form a 5-8 membered ring that is hydrolysable to a boronic acid;

R1 represents a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl, a thiocarbonyl, an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, — CF3, — (CH2)m — R3, — (CH2)mOH, — (CH2)m — O-lower alkyl, — (CH2)m— O-lower alkenyl, — (CH2)n— O— (CH2)m— R3, — (CH2)m— SH, — (CH2)m — S -lower alkyl, — (CH2)m — S -lower alkenyl, or — (CH2)n — S — (CH2)m — R3;

R2 represents, for each occurrence, hydrogen, lower alkyl, lower alkynyl, — (CH2)m— R3, — C(=O)-alkyl, — C(=O)-alkenyl, — C(=O)-alkynyl, or — C(=O)— (CH2)m— R3;

R3 represents, for each occurrence, hydrogen, or a substituted or unsubstituted lower alkyl, lower alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R4 represents a hydrogen, a lower alkyl, a lower alkenyl, a lower alkynyl, — (CH2)m— R3, — (CH2)n— OH, — (CH2)n— O-lower alkyl, — (CH2)n— O-alkenyl, — (CH2)n— O-alkynyl, — (CH2)n— O— (CH2)m— R7, — (CH2)n— SH, — (CH2)n— S -lower alkyl, — (CH2)n — S-lower alkenyl, — (CH2)n — S-lower alkynyl, — (CH2)n — S — (CH2)m — R3, — C(O)C(O)NH2, or — C(O)C(O)OR8;

R5 represents O or S;

R6 represents N3, SH, NH2, NO2 or OR8;

R7 represents hydrogen, a lower alkyl, an amine, OR8, or a pharmaceutically acceptable salt, or R5 and R6 taken together with the phosphorous atom to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure; R8 represents, hydrogen, a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocyclyl;

R9 and RIO, each independently, are absent or represents one, two, or three substitutions to the ring A or to the ring Z to which they are appended, each of which can independently be a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, an isocyano, a thiocyanato, an isothiocyanato, a cyanato, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, lower alkyl-C(O)OH, -O-lower alkyl-C(O)OH, -guanidinyl; — (CH2)m — R7, — (CH2)m — OH, — (CH2)m — O-lower alkyl, — (CH2)m — O-lower alkenyl, — (CH2)n — O — (CH2)m — R3, — (CH2)m — SH, — (CH2)m — S-lower alkyl, — (CH2)m — S-lower alkenyl, — (CH2)n— S— (CH2)m— R3 ; n is 0, 1, 2, or 3; and m is 0, 1, 2, or 3.

In some embodiments, the immuno-DASH inhibitor of Formula I is represented in Formula la, or is a pharmaceutical salt thereof: wherein X, W, Z, Rl, R2, R9 and RIO are as defined above for Formula I, and p is 1, 2 or 3.

In some preferred embodiments of la: Rl is a lower alkyl; R9 is absent, or independently for each occurrence, is a lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, - O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R2 is hydrogen, RIO is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; and W is -B(OH)2 or - CN (and more preferably -B(OH)2).

In some embodiments, the immuno-DASH inhibitor of Formula I is represented in Formula lb, or is a pharmaceutical salt thereof:

wherein X, W, Rl, R2, R9 and RIO are as defined above for Formula I, and p is 1, 2 or 3.

In some preferred embodiments of lb: Rl is a lower alkyl; R9 is absent, or independently for each occurrence, is a lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, - O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R2 is hydrogen, RIO is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; and W is -B(OH)2 or - CN (and more preferably -B(OH)2).

In some embodiments, the immuno-DASH inhibitor of Formula I is represented in Formula Ic, or is a pharmaceutical salt thereof: wherein X, W, Rl, R2, R9 and RIO are as defined above for Formula I, and p is 1, 2 or 3.

In some preferred embodiments of Ic: Rl is a lower alkyl; R9 is absent, or independently for each occurrence, is a lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, - O-lower alkyl, -O-lower alkyl-C(O)OH, -guanidinyl; X is O; each R2 is hydrogen, RIO is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; and W is -B(OH)2 or - CN (and more preferably -B(OH)2).

In some embodiments, the immuno-DASH inhibitor is represented by:

Another aspect of the disclosure relates to the immuno-DASH inhibitor represented by formula II, or a pharmaceutical salt thereof: wherein ring A, along with each occurrence of Ria, represents a 7-12 membered polycyclic ring structure; ring Z represents a 4-10 membered heterocycle including the N and the Ca carbon;

W represents -CN, — CH=NR4, a functional group which reacts with an active site residue of the target, or

X is O or S;

XI represents a halogen; Y is C or N;

Y 1 and Y2 are independently OH, or together with the boron atom to which they are attached represent a group that is hydrolysable to a boronic acid, or together with the boron atom to which they are attached form a 5-8 membered ring that is hydrolysable to a boronic acid;

Ria represents a lower alkyl, — (CH2)m — , — (CH2)m — O — (CH2)m — ; — (CH2)m— N— (CH2)m— ; or — (CH2)m— S— (CH2)m— ;

R2 represents, for each occurrence, hydrogen, lower alkyl, lower alkynyl, — (CH2)m— R3, — C(=O)-alkyl, — C(=O)-alkenyl, — C(=O)-alkynyl, or — C(=O)— (CH2)m— R3;

R3 represents, for each occurrence, hydrogen, or a substituted or unsubstituted lower alkyl, lower alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R4 represents a hydrogen, a lower alkyl, a lower alkenyl, a lower alkynyl, — (CH2)m— R3, — (CH2)n— OH, — (CH2)n— O-lower alkyl, — (CH2)n— O-alkenyl, — (CH2)n— O-alkynyl, — (CH2)n— O— (CH2)m— R7, — (CH2)n— SH, — (CH2)n— S -lower alkyl, — (CH2)n — S-lower alkenyl, — (CH2)n — S-lower alkynyl, — (CH2)n — S — (CH2)m — R3, — C(O)C(O)NH2, or — C(O)C(O)OR8;

R5 represents O or S;

R6 represents N3, SH, NH2, NO2 or OR8;

R7 represents hydrogen, a lower alkyl, an amine, OR8, or a pharmaceutically acceptable salt, or R5 and R6 taken together with the phosphorous atom to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure;

R8 represents, hydrogen, a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocyclyl;

R9 and RIO, each independently, are absent or represents one, two, or three substitutions to the ring A or to the ring Z to which they are appended, each of which can independently be a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, an isocyano, a thiocyanato, an isothiocyanato, a cyanato, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, lower alkyl-C(O)OH, -O-lower alkyl-C(O)OH, -guanidinyl; — (CH2)m — R7, — (CH2)m — OH, — (CH2)m — O-lower alkyl, — (CH2)m — O-lower alkenyl, — (CH2)n — O — (CH2)m — R3, — (CH2)m — SH, — (CH2)m — S-lower alkyl, — (CH2)m — S-lower alkenyl, — (CH2)n— S— (CH2)m— R3 ; n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; and p is 1, 2, or 3.

In some embodiments, the immuno-DASH inhibitor of Formula II is represented in

Formula Ila, or is a pharmaceutical salt thereof: wherein X, W, Z, R2, R9 and R10 are as defined above for Formula II.

In some preferred embodiments of Ila: R9, independently for each occurrence, is a lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl- C(O)OH, -guanidinyl; X is O; each R2 is hydrogen, R10 is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; and W is -B(OH)2 or -CN (and more preferably - B(OH)2).

In some embodiments, the immuno-DASH inhibitor of Formula II is represented in Formula lib, or is a pharmaceutical salt thereof: wherein X, W, R2, R9 and R10 are as defined above for Formula II.

In some preferred embodiments of lib: R9, independently for each occurrence, is a lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl- C(O)OH, -guanidinyl; X is O; each R2 is hydrogen, R10 is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; and W is -B(OH)2 or -CN (and more preferably - B(OH)2).

In some embodiments, the immuno-DASH inhibitor of Formula II is represented in Formula lie, or is a pharmaceutical salt thereof: wherein X, W, R2, R9 and RIO are as defined above for Formula II.

In some preferred embodiments of lie: R9, independently for each occurrence, is a lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl- C(O)OH, -guanidinyl; X is O; each R2 is hydrogen, RIO is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; and W is -B(OH)2 or -CN (and more preferably - B(OH)2).

In some embodiments, the immuno-DASH inhibitor of Formula II is represented in Formula lid, or is a pharmaceutical salt thereof: wherein X, W, R2, R9 and R10 are as defined above for Formula II.

In some preferred embodiments of lid: R9, independently for each occurrence, is a lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl- C(O)OH, -guanidinyl; X is O; each R2 is hydrogen, RIO is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; and W is -B(OH)2 or -CN (and more preferably - B(OH)2). In some embodiments, the immuno-DASH inhibitor of Formula II is represented in Formula lie, or is a pharmaceutical salt thereof: wherein X, W, Z, R2, R9 and RIO are as defined above for Formula II.

In some preferred embodiments of lie: R9, independently for each occurrence, is a lower alkyl, -OH, -NH2, -N3, -lower alkyl-C(O)OH, -O-lower alkyl, -O-lower alkyl- C(O)OH, -guanidinyl; X is O; each R2 is hydrogen, RIO is absent, or represents a single substitution of -OH, -NH2, -CN or -N3; Z is a pyrrolidine or piperidine ring (and more preferably a pyrrolidine ring); and W is -B(0H)2 or -CN (and more preferably -B(OH)2).

In some embodiments, the immuno-DASH inhibitor is one of the following:

Other non-limiting examples of iDASH inhibitors for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published December 12, 2019, incorporated by reference herein.

Stimulator of Interferon Genes Protein (STING) Agonists

In some embodiments, the free therapeutic moiety is a STING agonist. Stimulator of interferon genes protein (STING) agonists bind to STING, activating the STING pathway, which promotes iKK-related kinase TANK-binding kinase 1 (TBK1) signaling. TBK1 signaling activates nuclear factor-kappa B (NF-kB) and interferon regulatory factor 3 (IRF3) in immune cells in the tumor microenvironment. Their activation leads to the production of pro-inflammatory cytokines, including interferons (IFNs). Expression of IFN-beta promotes the cross-presentation of tumor-associated antigens by CD8a+ and CD 103+ dendritic cells to cytotoxic T lymphocytes (CTLs). This results in a CTL-mediated immune response against tumor cells and causes tumor cell lysis.

STING agonists include cyclic dinucleotides and derivatives thereof, such as modified cyclic dinucleotides including, for example, modified nucleobases, modified ribose or modified phosphate linkages. In some embodiments, the modified cyclic dinucleotide comprises a modified phosphate linkage, e.g., thiophosphate. In some embodiment, the STING agonist comprises a cyclic dinucleotide (e.g., a modified cyclic dinucleotide) having 2 ', 5' or 3 ', 5' phosphate linkages. In some embodiments, STING agonists include cyclic dinucleotides that have Rp or Sp stereochemistry around the phosphate bond.

In some embodiments, the STING agonist is Rp, Rp dithio 2 ', 3' c-di-AMP (eg, Rp, Rp-dithio c- [A (2 ', 5') pA (3 ', 5') p]) or its cyclic dinucleotide analog. In some embodiments, the STING agonist is a compound described in US Patent Publication US 2015/0056224. In some embodiments, the STING agonist is c- [G (2 ', 5 ') pG (3 ', 5 ') p], a dithioribose O-substituted derivative thereof. In some embodiments, the STING agonist is c- [A (2 ', 5 ') pA (3 ', 5 ') p] or a dithioribose O-substituted derivative thereof. In some embodiments, the STING agonist is c- [G (2 ', 5 ') pA (3 ', 5 ') p] or a dithioribose O- substituted derivative thereof. In some embodiments, the STING agonist is 2'-0-propargyl- cyclic- [A (2 ', 5') pA (3 ', 5') p] (2'-0-propargyl-ML-CDA).

STING agonists also include xanthenone and derivatives thereof, including flavone acetic acid (FAA), xanthene-acetic acid (XAA), dimethylxanthenone-4-acetic acid (DMXAA), and derivatives thereof. Examples of STING agonists include ADU-S100 (ML- RR-S2-CDA or MIW815), MK-1454, vadimezan (5,6-dimethylxanthenone-4-acetic acid (DMXAA)), 2'3'-cGAMP, c-di-GMP, 3'3'-cGAMP, and ML-RR-CDA.

Non-limiting examples of STING agonists include agonists represented in the one of the general formulas

wherein

X ₁ and X ₂ are, independently, O or S, and preferably are the same (O,O or S,S);

X ₃ and X ₄ are, independently, a purine, such as a guanine or guanine analog, or a pymridine, and wherein the wavy lines indicate covalent attachment site to L1 , or where L1 is a bond, to the substrate recognition sequence:

R ₁ and R ₂ are, independently, H, hydroxyl, a halogen (preferably F or CI) or an optionally substituted straight chain alkyl of from 1 to 18 carbons and from 0 to 3 heteroatoms, an optionally substituted alkenyl of from 1-9 carbons, an optionally substituted alkynyl of from 1 -9 carbons, or an optionally substituted aryl, wherein substitution(s), when present, may be independently selected from the group consisting of C _1-6 alkyl straight or branched chain, benzyl, halogen, trihalomethyl, C _{1 -6} alkoxy. -NO _{2 ,} —NH _{2 ,} OH, =O, —COOR' or -OR’, wherein R ₁ and R ₂ are not both H,

R' is H or lower alkyl, —CH ₂ OH, or —CONH ₂ .

In some embodiments, the STING agonist is represented in one of the formula: In the STING agonist structures above, X ₃ and X ₄ may each independently be, for example, 9-purine, 9-adenine, 9-guanine, 9-hypoxanthine, 9-xanthine, 9-uric acid, or 9- isoguanine, provided that one of X3 or X4 includes a functional group with which L ² shares a bond if L ² is a self immolative linker, or a funcational group with which DM shares a bond if L ² is (that) a bond.

X ₃ and X ₄ may be identical or different.

In some embodiments, the STING agonists may be provided in the form of predominantly Rp,Rp or Rp,Sp stereoisomers. In some embodiments, the STING agonists may be provided in the form of predominantly Rp,Rp stereoisomers.

Exemplary STING agonists include:

In some embodiments, the STING agonist is represented in one of the following structures”

Still another STING agonist that can be used as Drug Moiety in the present binder conjugates is

It will also be appreciated by those skilled in the art that, particularly with the use of a self-immolative linker, the STING agonist can be coupled to the linker though functional groups other than amines as shown above, such as through free hydroxyl groups for example.

Non-limiting examples of STING agonists for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published December 12, 2019, incorporated by reference herein. Additional examples of STING agonists are described in International Publication Nos. WO 2017/123669 and WO 2015/077354 as well as US Patent Publication No. US 2015/0056224, each of which is hereby incorporated by reference.

It will also be appreciated by those skilled in the art that, particularly with the use of a self-immolative linker, the STING agonist can be coupled to the linker though functional groups other than amines as shown above, such as through free hydroxyl groups for example.

Anthracyclines In some embodiments, the drug moiety is an anthracycline or derivative thereof, preferably doxorubicin or other analogs that are able to induce immunogenic cell death of tumor cells.

Anthracyclines and analogs thereof specifically include, without limitation, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, aclarubicin, mitoxantrone, actinomycin, bleomycin, plicamycin, and mitomycin. For example, the anthracycline moiety can be represented by the formula wherein,

R ^c represents (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ )hydroxyalkyl, or (C ₁ -C ₆ )alkanoyloxy(C ₁ -C ₆ )alkyl, in particular methyl, hydroxymethyl, diethoxyacetoxymethyl, or butyryl oxy methyl;

R ^d represents hydrogen, hydroxyl, or (C ₁ -C ₆ )alkoxy, in particular methoxy; one of R ^e and R ^f represents a hydrogen atom: and the other represents a hydrogen atom or a hydroxy or tetrahydropyrany-2-yloxy (OTHP) group.

Radiopharmaceuticals

In some embodiments, the therapeutic moiety is a radiopharmaceutical. For example, the therapeutic moiety can include a chelator for a radionuclide useful for radiotherapy or imaging procedures. Radionuclides useful within the present disclosure include gamma- emitters, positron-emitters, Auger electron-emitters, X-ray emitters and fluorescence- emitters, with beta- or alpha-emitters for therapeutic use. Examples of radionuclides useful as toxins in radiation therapy include: ⁴³ K, ⁴⁷ Sc, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁷ Cu, ⁶⁸ Ga, ⁷¹ Ge, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ⁷⁷ As, ⁸¹ Rb, ⁹⁰ Y, ⁹⁷ Ru, ^99m Tc, ¹⁰⁰ Pd, ¹⁰¹ Rh, ¹⁰³ Pb, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹¹ In, ¹¹³ In, ¹¹⁹ Sb ¹²¹ Sn, ¹²³ I, ¹²⁵ I, ¹²⁷ Cs, ¹²⁸ Ba, ¹²⁹ Cs, ¹³¹ I, ¹³¹ Cs, ¹⁴³ Pr, ¹⁵³ Sm, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁶⁹ Eu, ¹⁷⁷ LU, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁸⁹ Re, ¹⁹¹ Os, ¹⁹³ Pt, ¹⁹⁴ Ir, ¹⁹⁷ Hg, ¹⁹⁹ Au, ²⁰³ Pb, ²¹¹ At, ²¹² Pb, ²¹² Bi and ²¹³ Bi. Conditions under which a chelator will coordinate a metal are described, for example, by Gansow et al., U.S. Pat. Nos. 4,831,175, 4,454,106 and 4,472,509. Examples of chelators includes, 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA) 1,4,7,10- tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) 1 ,4,8,11- tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA).

Therapeutic Conjugate Compositions and Methods of Use

In some embodiments, a therapeutic conjugate is formulated with a pharmaceutically acceptable excipient to form a composition. In some embodiments, a FAPα cleavable linker or an SRS is formulated with a pharmaceutically acceptable excipient to form a composition. A molecule or other substance/agent is considered “pharmaceutically acceptable” if it is approved or approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. An excipient may be any inert (inactive), non-toxic agent, administered in combination with a therapeutic conjugate. Pharmaceutically acceptable excipients comprise a variety of materials known in the art, including but not limited to saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers.

In some embodiments, a therapeutic conjugate or composition is administered to a subject. A subject may be any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, and rodents. A “subject” refers to a human subject. In some embodiments, the subject has a diseased tissue, such as a cancer.

Therefore, a therapeutic conjugate or composition may be administered to a subject to treat a diseased tissue, such as cancer. In some embodiments, a therapeutic conjugate is used to manufacture a medicament for the treatment of a diseased tissue (e.g., cancer). Non- limiting examples of cancers include skin cancer (e.g., melanoma or non-melanoma, such as basal cell or squamous cell), lung cancer, prostate cancer, breast cancer, colorectal cancer, kidney (renal) cancer, bladder cancer, non-Hodgkin’s lymphoma, thyroid cancer, endometrial cancer, exocrine cancer, and pancreatic cancer. Other cancers are contemplated herein. The term treat, as known in the art, refers to the process of alleviating at least one symptom associated with a disease (e.g., cancer). A symptom may be a physical, mental, or pathological manifestation of a disease. Symptoms associated with various diseases are known. To treat or prevent a particular condition, a therapeutic conjugate as provided herein should be administered in an effective amount, which can be any amount used to treat or prevent the condition. Thus, in some embodiments, an effective amount is an amount used to alleviate a symptom associated with the particular disease being treated. Methods are known for determining effective amounts of various therapeutic molecules, for example.

Routes of administration include intravenous, intramuscular, intratumoral, intraperitoneal, intranasal, and subcutaneous. Other routes of administration are encompassed by the present disclosure.

Thus, the therapeutic conjugates of the present disclosure may be formulated for intravenous, intramuscular, intratumoral, intraperitoneal, intranasal, or subcutaneous administration.

EXAMPLES

Example 1 - Anti-tumor Activity of Therapeutic Conjugates

Therapeutic conjugates comprising Fc conjugated to talabostat (Val-boroPro) via a FAPα-cleavable linker were generated and injected into a syngeneic model, CT26-mFAP+ mice, which are used for mouse colon cancer and have a FAPα gene knock-in. The tumor volume in the mice was measured nine times over 20 days. As can be seen in FIG. 2, the mice injected with the therapeutic conjugates (“SQT-Gly” groups) had lower tumor volumes than some of their counterpart mice injected with therapeutic conjugates comprising a cell- binding moiety (the “AVA04-182” groups) and the controls. Without wishing to be bound by theory, it is thought that the AVA04-182 groups did not work as well as the SQT-Gly groups because they were cleared by anti-drug antibodies.

A pharmacokinetic study was also undertaken. The therapeutic conjugates were also injected in different dose and the amount of free talabostat was measured over 50 hours. As can be seen in FIG. 3, the therapeutic conjugates show preferential intratumoral exposure to the FAP-released iDASH inhibitor.

Example 2 - EC ₅₀ Studies

Half-maximal effective concentration (EC ₅₀ ) studies were undertaken to examine the difference between mice and rats administered talabostat alone (FIG. 4A) and mice and rats administered the therapeutic conjugates comprising talabostat (FIG. 4B). When talabostat is administered alone, the talabostat maximum tolerated dose (MTD) is about 3 mg/m ² in a mouse tumor model and 0.18 mg/m ² in rats. That is, the talabostat MTD is 1/17 the tumor model EC ₅₀ (FIG. 4A). In contrast, when the talabostat is administered as part of the therapeutic conjugate, the MTD is 160 mg/m ² in the mouse model and the EC 50 for rats is greater than 500 mg/m ² (FIG. 4B). Talabostat, as part of the therapeutic conjugate, can be administered to rats and mice at therapeutically effective doses.

Example 3 - In Vivo Tumor Studies

CT26-mFAP+ mice were injected with vehicle (FIG. 5A), a therapeutic conjugate comprising a cell-binding moiety (FIG. 5B), or Fc conjugated to talabostat (Val-boroPro) via a FAPα-cleavable linker (FIG. 5C), and tumor volume was measured over 22 days. In the vehicle group, zero of the 10 mice showed a reduction in tumor volume and all mice were sacrificed before the end of the study (around day 20). In the group that received the therapeutic conjugate comprising a cell-binding moiety, three of the 10 mice had tumor volumes that decreased back to baseline. In contrast, the therapeutic conjugates described herein (without a cell binding moiety) resulted in six out of ten mice having tumor volumes that decreased back to baseline during the study. Further, the body weights of the mice were measured during the study and the percent change in body weight was calculated (FIG. 5D). None of the mice measured had a percent change in body weight of greater than 15%.

In another set of experiments, CT26-mFAP+ mice were injected with vehicle (control), 200 μg or 400 μg MSA-6325 (mouse serum albumin conjugated with 6325), or 740 μg PEG-6325 (6325 reacted with SUNBRIGHT PTE-200 PA, a 20KDa 4 arm functional PEG), and change in tumor volume was measured over time (FIG. 7). “6325” (FIG. 6A) is an exemplary FAP-activated I-DASH inhibitor including an NHS group for conjugation to lysine residues of proteins.

In yet another set of experiments, CT26-mFAP+ mice were injected with either a human Fc fragment conjugated with 6325 (FIG 8A), or a full human IgG antibody conjugated with 6325 (FIG 8B) and change in tumor volume was measured over time.

Example 4 - In vitro Studies

In further in vitro studies, several conjugates were tested for their ability to induce pyroptosis in J774 cells in the absence and presence of FAPα. Briefly, J774A.1 mouse macrophage cell line cells were cultured in triplicate wells with serial 10-fold dilutions of either VbP (data not shown) or one of the FAP-activated I-DASH inhibitors (Hu IgGl Fc- 6325, Hu IgGl-6325, MSA-6325, SQT-Gly V.2-6325, SQT-Gly CF-6325 or SQT-Gly CG- 6325) in the presence or absence of rhFAPα at a final concentration of 25nM for 24 hours. Each experiment included an untreated control. . Following the incubation, the levels of lactate dehydrogenase (LDH, a marker of pyroptosis) released into culture supernatants were measured using the CytoTox 96 Non-radioactive Cytotoxicity Assay according to the manufacturer’s instructions. The results are shown in FIG. 17 as fold-increase in LDH release. All conjugates tested exhibited a clear differentiation with or without FAPα that was statistically significant. This was not the case for VbP alone (data not shown), indicating that the varied responses were due to activation of the prodrugs by FAPα.

In an additional study, the conjugation of two prodrugs (6323 and 6501 (FIG. 6C)) to the hinge region cysteine residues of huIgG1 Fc was examined. Briefly, compound maleimide linker prodrugs (6323 and 6501) were dissolved in DMSO at a concentration of 10OmM. 6323 or 6501 were added to reduced Fc and the samples were incubated at room temperature. Unreacted 6323 or 6501 was removed with a Zeba spin column. Before measuring free thiol (SH) groups, the samples were reduced again and dithiothreitol (DTT) removed by passing samples over the Zeba spin desalting column again. Conjugation of the thiol groups was determined by measuring free SH groups using Ellman’s reagent. The concentration of the reduced Fc samples was determined from the absorbance at 280nm, using an extinction coefficient of 66500 M ^-1 cm ^_1 . Conjugations were done with reaction ratios of 0, 10, 20, and 40 moles of 6323 or 6501 per mole of reduced SH (4 SH per Fc). The decreases in the number of free SH groups on Fc by conjugation to 6323 (FIG. 18 A) or 6501 (FIG. 18B) is represented as the change in free SH groups vs. reaction ratio.

A study relating to the conjugation of 6323 to AFFIMER®-Fc proteins was undertaken. A compound maleimide linker prodrug (6323) was dissolved in DMSO at a concentration of 10OmM. The concentration of the reduced SQTGlyCF samples was determined from the absorbance at 280nm. 6323 was added to the reduced SQTGlyCF and the samples were incubated at room temperature. Unreacted 6323 was removed with a Zeba spin column. Before measuring free SH groups, samples were reduced again as described above to ensure that any unreacted cysteines were reduced. Conjugation of the thiol groups was determined by measurement of free SH groups with using Ellman’s reagent. The concentration of thiols was determined from the absorbance at 412nm using an extinction coefficient of 14,150 M ^-1 cm ^-1 .

The number of thiols per SQTGlyCF was calculated as the ratio of the thiol concentration to the protein concentration ([SH]/[SQTGlyCF]). Conjugations were done with reaction ratios of 0, 10, 20 and 40 moles of 6323 per mole of reduced SH (4 SH per SQTGlyCF). The change in free SH groups vs. reaction ratio is shown in FIG. 24A. A timed conjugation reaction was done with a reaction ratio of 40 6323 per SH with the reaction stopped at 0, 5, 10, 15 and 20 minutes after addition of 6323. Kinetics of conjugation of 6323 to SQTGlyCF with a reaction ratio of 40 6323 per SH group are shown in FIG. 24B.

Example 5 - Pharmacokinetic Studies (Tumor Mouse Models)

The pharmacokinetics of different compounds were examined in murine models of cancer. Mice (BALB/c) were inoculated subcutaneously in the right flank with 5x10 ⁵ CT26- mFAP cells. Tumors were allowed to become established, after which, animals were treated with or without 5057 (FAPα inhibitor) for 24 hours. Then, animals were treated with hu IgGl Fc-6325 (Fc-6325, 200 μg/animal) for 1, 4, 6, 24 and 48 hours. At the designated time points, blood and tumor samples were collected to measure Val-boroPro (VbP) on LC-MS. Treatment with Fc-6325 resulted in higher concentrations of VbP in the tumor than in the serum at all time points. The FAPα inhibitor significantly reduced the levels of VbP, the values of VbP in the tumor at 24 and 48 hours were greater than at 1-6 hours. It is thought that this was due to FAP inhibition waning during the 6-24 hour period. Concentrations of VbP (nM) over time (hours) for serum (FIG. 19A) or tumor (FIG. 19B) samples is shown. Each time point includes 3 mice per group.

Next, conjugates of different compounds were tested. Mice (n=10/group, BALB/c) were inoculated subcutaneously in the right flank with 5x10 ⁵ CT26-mFAP cells. Conjugates were injected intraperitoneally once tumor volumes averaged approximately 50-100 mm ³ . Treatments were administered twice a week for 3 weeks. The change in tumor volume over time in CT26-mFAP ⁺ mice with either vehicle (control) or SQT-Gly conjugates (FIG. 20A) or Hu IgGl Fc conjugates (FIG. 20B) is shown. In FIGs. 20A-20B, the dotted lines represent twice-weekly (BIW) dosing. The SQT-Gly conjugates tested were as follows: SQT- Gly V.2 (IgGl Fc) conjugated with either 6325 (NHS) or 6323 (MAL) and SQT-Gly CF (IgGl LALA Fc) conjugated with either 6325 or 6323. The hu IgGl Fc conjugates tested were as follows: Hu IgGl Fc fragment conjugated with either 6325 or 6323 or 6501 (a tetrameric prodrug; FIG. 6C). All mice treated with conjugates had a reduced tumor size compared to the vehicle control on Day 20. The efficacy of NHS (6325) or MAL (6323 or 6501) conjugates was comparable between the half-life extended versions of SQT-Gly and hu IgGl Fc. In addition, the pharmacokinetics and tissue distribution of VbP released from different FAP-activated prodrugs were examined. Mice (BALB/c) were inoculated subcutaneously in the right flank with 5x10 ⁵ CT26-mFAP cells. Tumors were allowed to become established, after which, animals were dosed via a 200 uL intraperitoneal injection of 24 μg 3892 (the structure of 3892 is shown in FIG. 28), 1138 μg 3892, 52 μg 6323, 56 μg 6325, or 1113 μg 6435 (N-Ac-Lys-6325). Each dosing group consisted of a subset of 5 groups, representing the 1, 4, 6, 24 and 48 hour collection time points (n = 3/group) with the exception of 24 ug 3892, which only consisted of 1, 4 and 24 hours. At the collection time points, blood and tumor samples were collected to measure VbP levels on LC-MS. VbP concentration vs. time in serum and tumor following prodrug administration is represented in FIG. 21. The 1138 ug dose of 3892 and 1113 ug dose of 6435 (N-Ac-Lys-6325) represented 25x and 10x equivalents of released VbP in comparison to 20 ug VbP alone, respectively. Both exhibited serum and tumor distribution profiles similar to VbP alone, with peak concentrations occurring 1 hour after dosing, followed by a steady decline over time. The lower prodrug doses of 24 μg 3892, 52 μg 6325 and 56 μg 6325 were selected to represent the approximate contained VbP concentrations associated with the conjugates. Both 6323 and 6325 resulted in concentrations of approximately 200 nM tumor VbP that remained steady in the early time points, followed by an increase from 24 to 48 hours to 400 nM.

Next, the pharmacokinetics and tissue distribution of VbP released from SQT-Gly V.2-conjugates and IgG Fc-conjugates were examined. Mice (BALB/c) were inoculated subcutaneously in the right flank with 5x10 ⁵ CT26-mFAP cells. Tumors were allowed to become established, after which, animals were dosed via a (200μg/200 uL) intraperitoneal injection of SQT-Gly V.2-6323, SQT-Gly V.2-6325, Mouse IgG2a Fc-6325, or Hu IgGl Fc- 6325. Each dosing group consisted of a subset of 5 groups, representing the 1, 4, 6, 24 and 48 hour collection time points (n = 3/group). At the collection time points, blood and tumor samples were collected to measure released VbP levels using LC-MS. VbP concentration vs. time in serum and tumor following administration of SQT-Gly V.2-conjugates (FIG. 22A) or IgG Fc-conjugates (FIG. 22B) is shown.

The serum and tumor PK/TD profile of SQT-Gly V.2-6323 was assessed for comparison to SQT-Gly V.2-6325. While serum VbP levels for SQT-Gly V.2-6325 remained low (<10 nM) at all time points tested, SQT-Gly V.2-6323 resulted in a spike >10-fold higher than SQT-Gly V.2-6325 at 1 hour (37 vs. 3 nM), potentially indicating that the MAL conjugation sites increase accessibility for prodrug cleavage by serum FAP. Tumor VbP levels were also higher for SQT-Gly V.2-6323, which may further support increased prodrug activation of the MAL conjugate.

Mu IgG2a Fc-6325 was assessed for comparison to Hu IgGl Fc-6325. There is a possibility that efficacy of the latter is not due solely to its design as an extended half-life prodrug, but also as the results of a secondary immune response triggered by the presence of a foreign antigen (Hu IgGl Fc). As the Mu IgG2a Fc is of mouse origin, it was designed to bypass this issue. The PK/TD profiles of the two were quite similar. There was a reduction of VbP levels in the tumor observed for Mu IgG2a Fc-6325 between 24-48 hours that was not observed for Hu IgGl Fc-6325; however, which may indicate that the half-life of the mouse conjugate is shorter than the human conjugate.

Example 6 - 42CQ-Based Conjugates

The FAP-activated prodrug G-CSF serum cytokine response in nontumor-bearing BALB/c mice was examined. Female BALB/c mice of 11-12 weeks of age were injected intraperitonially with vehicle (PBS), 42CQ-6501, or 42CQ-6501+MSA at 200μg/mouse, in groups of 5 mice per treatment.

VbP alone is known to elicit a large serum G-CSF response. While cytokine recruitment is believed to play an important role in tumor immunity, its presence in the serum represents a systemic response, which can lead to unwanted adverse effects. A panel of 42CQ-based conjugates were screened to assess whether they resulted in attenuated serum G- CSF responses in comparison to VbP. 42CQ is an AFFIMER® polypeptide designed to bind to serum albumin (SEQ ID NO: 133). Consequently, the 42CQ-based conjugates (with 6501) were prepared with or without prebinding to MSA to determine whether there was an evident difference between the two formulations.

Six hours after dosing, blood was collected to measure G-CSF in the serum using a mouse G-CSF Quantikine ELISA kit. At the dose tested, 42CQ-6501 induced a significant increase in serum concentration of G-CSF compared to vehicle-treated mice, while prebinding 42CQ-6501 with MSA resulted in significant attenuation of serum concentration of G-CSF compared to 42CQ-6501 (FIG. 23).

Next, the pharmacokinetics of the 42CQ-6501 conjugate in CT26-mFAP tumor- bearing mouse was examined. Briefly, BALB/c mice were inoculated subcutaneously in the right flank with 5x10 ⁵ CT26-mFAP cells. Tumors were allowed to become established, after which, animals were dosed via a intraperitoneal injection of 100 or 200 μg/animal of 42CQ- 6501. Each dosing group consisted of a subset of 5 groups, representing the 1, 4, 6, 24 and 48 hour collection time points (n = 3/group). At the collection time points, blood and tumor samples were collected to measure VbP levels on LC-MS. VbP concentration vs. time in serum (FIG. 25A) and tumor (FIG. 25B) following administration of 100 or 200 μg/animal of 42CQ-6501 are shown. Mean serum levels of VbP were between 4 and 15 times lower than tumor levels at all time points for both doses of 42CQ-6501.

In a further experiment, the effect of 42CQ-based conjugates ± MSA on tumor growth in a syngeneic murine colon cancer (CT26) model was examined. Mice (n=10/group, BALB/c) were inoculated subcutaneously in the right flank with 5x10 ⁵ CT26-mFAP cells. MSA prebinding may attenuate the systemic release of VbP, so the efficacy of both 42CQ- 6323 and 42CQ-6501 conjugates was tested with and without MSA prebinding. The 42CQ- based conjugates ± MSA were injected intraperitoneally once tumor volumes averaged approximately 50-100 mm ³ . Tumor growth inhibition for 42CQ-6323+ MSA and 42CQ- 6501+ MSA is shown in FIGs. 26A and 26B, respectively. Dotted lines represent BIW dosing. All mice treated with 42CQ-based conjugates, except 42CQ-6501 without MSA, had a reduced tumor size compared to the control.

In an additional experiment, the pharmacokinetics of VbP released from FAPα- activated AFFIMER® prodrugs (42CQ-6323 and 42CQ-6501) in wild-type (WT) and FAPα knockout (KO) mice was examined. Non-tumor bearing WT or FAPα KO mice (n=3/group) were dosed via an intraperitoneal injection of 200 μg/animal of 42CQ-6323, 42CQ-6323- MSA, 42CQ-6501, or 42CQ-6501-MSA. Blood was collected at 0 (pre-dose), 15, 30, 60, 120, and 240 minutes, and at 24 hours post-dose to measure the concentration of VbP in plasma on LC-MS. VbP concentration vs. time in plasma following administration of 200 μg/animal of 42CQ-6323+ MSA or 42CQ-6501+ MSA is represented in FIGs. 27A and 27B, respectively.

No VbP was detected in the FAPα knockout mice at any timepoint. VbP levels in plasma were greater for 6501 conjugates than for 6323 conjugates, consistent with the greater effective drug- AFFIMER® protein ratio (DAR) for the 6501 conjugates. Pre-binding to albumin before injection appears to limit the initial spike of VbP in the blood (compare MSA groups to non-MSA groups). All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

Where a range of values is provided, each value between the upper and lower ends of the range are specifically contemplated and described herein.