Conjugates and uses thereof

Abstract

A conjugate comprises (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen, (b) a drug, and (c) a functional linker that links the drug to the sdAb. The conjugate is used for treating cancer. A method for preparing the conjugate comprises conjugating the functional linker and the drug to the sdAb.

Description

[0001] Related Applications

[0002] This application claims priority to International Application No. PCT / CN2022 / 141998, filed on December 26, 2022, the disclosure of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to a novel conjugate comprising (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen, (b) a drug, and (c) a functional linker that links the drug to the single domain antibody; and to the use of the conjugate for treating cancer. A method for preparing the conjugate is also provided. Background Art

[0004] Antibody-drug conjugates are one of the most promising therapeutic approaches in the field of oncology. As a targeted therapy, antibody-drug conjugates (ADCs) use antibodies to selectively deliver cytotoxic drugs to targets such as tumor-associated antigens and have achieved significant clinical and commercial success. There are several factors that contribute to the overall efficacy of ADC therapies, including tumor penetration and accumulation, target binding and cellular uptake, release of active catabolites within target cells, and the pharmacokinetic (PK) profile of the conjugate. Similar to ADCs, radionuclide-drug conjugates (RDCs) use antibodies or small molecules to precisely deliver cytotoxic / imaging radionuclide payloads to targets, which may increase efficacy and prevent cytotoxicity due to systemic exposure to the radionuclide. Previous studies have shown that full-length antibodies that specifically bind to tumor antigens are capable of carrying radionuclides to tumor tissues. However, due to their large size, RDCs carried by full-length antibodies can still result in long-term systemic retention of the radionuclide, thereby causing undesirable toxicity.

[0005] Single domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. Like full-length antibodies, they are capable of selectively binding to specific antigens but have a smaller molecular weight (only 12 - 15 kDa). However, the small size causes sdAbs to be easily retained in the kidneys, which may cause renal toxicity and insufficient efficacy against target tissues (e.g., tumors) in therapeutic applications. Therefore, there is a great need for improved targeted therapies in this field. Summary of the Invention

[0006] In one aspect, there is provided a conjugate comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the sdAb, wherein when the conjugate is administered to a subject, the functional linker increases the tumor uptake, accumulation, and / or retention of the drug compared to a conjugate without the functional moiety.

[0007] In one aspect, there is provided a conjugate comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the sdAb, wherein when the conjugate is administered to a subject, the functional linker decreases the renal uptake, accumulation, and / or retention of the drug compared to a conjugate without the functional moiety.

[0008] In another aspect, there is provided a conjugate comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the sdAb, wherein when the conjugate is administered to a subject, the functional linker increases the tumor / renal ratio of the drug compared to a conjugate without the functional moiety.

[0009] In another aspect, there is provided a conjugate comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the sdAb, wherein when the conjugate is administered to a subject, the functional linker prolongs the blood circulation and / or half-life of the drug compared to a conjugate without the functional moiety.

[0010] In another aspect, there is provided a conjugate comprising: (a) an antibody mimetic that specifically binds to a tumor antigen, wherein the antibody mimetic is selected from Affibody, DARPin, Anticalin, Avimer, Versabody, or Duocali; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the antibody mimetic, wherein when the conjugate is administered to a subject, the functional linker is capable of: (i) increasing the tumor uptake, accumulation, and / or retention of the drug, (ii) decreasing the renal uptake, accumulation, and / or retention of the drug, (iii) increasing the tumor / renal ratio of the drug, (iv) prolonging the blood circulation of the drug, and / or (v) prolonging the half-life of the drug compared to a conjugate without the functional moiety.

[0011] In some embodiments, the binding affinity of the conjugate to the tumor antigen is between about 10 -12 and about 10 -8 M. In some embodiments, the functional moiety comprises one or more components selected from the group consisting of: polyethylene glycol (PEG) groups, renin-cleavable groups, blood protein-binding groups, spacers, and any combination thereof.

[0012] In some embodiments, the PEG group comprises structure, and wherein n is any integer in the range from about 1 to about 25. In some embodiments, the functional moiety comprises the blood protein-binding group.

[0013] In some embodiments, the blood protein is selected from albumin, fetuin, transferrin, and IgG.

[0014] In some embodiments, the blood protein-binding group includes an albumin-binding group. In some embodiments, the albumin-binding group comprises structure, where n is any integer ranging from 1 to 20, and R m is H, CH3, or COOH. In some embodiments, the albumin-binding group comprises structure, where R x is selected from: , , , , , , , , , , , , and wherein n is any integer in the range from 1 to 6, and R x1 , R x2 , R x3 , R x4 , R x5 , R x6 , and R x7 each independently selected from H, N, S, O, Se, P, halogen, C 5-20 aryl group, C 1-20 alkyl group, C 2-20 alkenyl group, and C 2-20 alkynyl group, and wherein: (1) the C 5-20 aryl group, the C 1-20 alkyl group, the C 2-20 alkenyl group, or the C 2-20The alkynyl group is unsubstituted or substituted by one or more substituents selected from N, S, O, Se, P, and halogen atoms, (2) 0, 1, or 2 carbon atoms in the 5-20 aryl group, C 1-20 alkyl group, the C 2-20 alkenyl group, or the C 2-20 alkynyl group are replaced by a group selected from C 6-10 arylene, 5- to 10-membered heteroarylene group, C 3-7 carbocyclene group, 5- to 10-membered heterocyclene group, and wherein the arylene, heteroarylene, carbocyclene, and heterocyclene groups are unsubstituted or substituted by one or more substituents selected from N, S, O, Se, P, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthiol, -N(C 1-6 alkyl)(C 1-6 alkyl), nitro, and sulfonic acid groups, and / or (3) 0, 1, or 2 -CH- or -CH2- groups in the C 5-20 aryl group, the C 1-20 alkyl group, the C 2-20 alkenyl group, or the C 2-20 alkynyl group are replaced by a group selected from -O-, -S-, -S-S-, -C(O)-, and -N(C 1-6 alkyl)- groups. In some embodiments, the albumin-binding group comprises the structure of, wherein R y is selected from H, F, Cl, Br, I, -CH3, -OCH3, COOH, -CF3, and wherein n is any integer ranging from 1 to 6.

[0015] In some embodiments, the functional moiety comprises the renin-cleavable group. In some embodiments, the renin-cleavable group is selected from brush border enzyme-cleavable groups, lysosomal enzyme-cleavable groups, and combinations thereof. In some embodiments, the renin-cleavable group includes a dipeptide or oligopeptide. In some embodiments, the dipeptide or oligopeptide comprises a structure selected from: methionine-isoleucine, glycine-lysine, glycine-phenylalanine-lysine, methionine-valine-lysine, glycine-tyrosine, glycine-lysine-lysine, glycine-arginine-lysine, aspartic acid-glycine-lysine, methionine-glycine-lysine, methionine-isoleucine-lysine, glycine-tyrosine-lysine, glycine-valine, glycine-isoleucine, methionine-phenylalanine-lysine, glycine-(3-(2-naphthyl)alanine)-lysine, glycine-diphenylalanine-lysine, methionine-glycine-lysine, and any derivatives thereof.

[0016] In some embodiments, the functional linker includes a spacer. In some embodiments, the spacer includes a structure selected from the group consisting of natural amino acid residues, unnatural amino acid residues, C 5-20 aryl groups, C 1-20 alkyl groups, C 2-20 alkenyl groups, and C 2-20 alkynyl groups, and wherein: (i) the C 5-20 aryl group, the C 1-20 alkyl group, the C 2-20 alkenyl group, or the C 2-20 alkynyl group is unsubstituted or substituted with one or more substituents selected from N, S, O, Se, P, and halogen atoms, (ii) 0, 1, or 2 carbon atoms in the C 5-20 aryl group, the C 1-20 alkyl group, the C 2-20 alkenyl group, or the C 2-20 alkynyl group are replaced with a group selected from C 6-10 arylene, 5- to 10-membered heteroarylene groups, C 3-7 carbocyclylene, 5- to 10-membered heterocyclylene groups, and wherein the arylene, heteroarylene, carbocyclylene, and heterocyclylene groups are unsubstituted or substituted with one or more substituents selected from N, S, O, Se, P, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthiol, -N(C 1-6 alkyl)(C 1-6 alkyl), nitro, and sulfonic acid groups, and / or (iii) 0, 1, or 2 -CH- or -CH2- groups in the C 1-20 alkyl group, the C 2-20 alkenyl group, or the C 2-20 alkynyl group are replaced with a group selected from -O-, -S-, -S-S-, -C(O)-, and -N(C 1-6 alkyl)- groups. In some embodiments, the amino acid residues are selected from lysine, aspartate, asparagine, diaminobutyric acid, phenylalanine, tyrosine, threonine, serine, proline, leucine, isoleucine, valine, arginine, histidine, glutamate, glutamine, and alanine.

[0017] In some embodiments, the functional linker further comprises a chelating factor group. In some embodiments, the chelating factor group comprises a structure selected from the following: NODASA, NODAGA, TETA, TRITA, TRAP, DTPA, CHX-DTPA, EDTA, CDTA, CPTA, DOTP, DOTPI, EGTA, HBED, TTHA, DTPA, DOTA, DOTAGA, NOTA, HP-DOA3, CBTE2a, TE2A, TMT, DPDP, HYNIC, DFO, HEDTA, NOPO, MAG3, NCS-MP-NODA, NH2-MPAA-NODA, H2DEDPA, H4octapa, Macropa, Pypa, Py4pa, THP, and SarAr.

[0018] In some embodiments, the functional linker is conjugated to the sdAb or the antibody mimetic via an amino acid of the sdAb or the antibody mimetic. In some embodiments, the amino acid of the sdAb or the antibody mimetic for conjugation is selected from cysteine, lysine, histidine, aspartate, glutamate, glutamine, arginine, tyrosine, tryptophan, threonine, and serine. In some embodiments, the functional linker comprises a conjugation group capable of conjugating to the sdAb or the antibody mimetic. In some embodiments, the conjugation group is selected from amine, thiol, alcohol, ketone, aldehyde, nitrile, carboxylic acid, ester, alkene, alkyne, anhydride, succinimide, maleimide, phosphine, disulfide, alkoxyamine, azide, alkyl halide, isothiocyanate (NCS), epoxide, isocyanate, hydrazine, and acyl halide.

[0019] In some embodiments, the tumor antigen is selected from prostate-specific membrane antigen (PSMA), fibroblast activation protein α (FAP-α, FAP), folate receptor, luteinizing hormone-releasing hormone (LHRH), norepinephrine transporter (NAT), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-2 (HER-2), vascular endothelial growth factor (VGFR), mucin-1 (MUC-1), mucin-4 (MUC-4), urokinase-type plasminogen activator receptor (uPAR), tumor-associated glycoprotein 72 (TAG-72), Claudin 18.2 (CLDN18.2), vascular endothelial growth factor receptor (VEGFR), C-X-C chemokine receptor type 4 (CXCR4), Hepsin, TMPRSS2, and cMET.

[0020] In some embodiments, the tumor antigen is CLDN18.2. In some embodiments, the sdAb comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO. 1. In some embodiments, the sdAb comprises: (1) CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2 and an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 2, (2) CDR2 comprising the amino acid sequence shown in SEQ ID NO. 3 and an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 3, and (3) CDR3 comprising the amino acid sequence shown in SEQ ID NO. 4 and an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 4.

[0021] In some embodiments, the tumor antigen is FAP. In some embodiments, the sdAb comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO. 5. In some embodiments, the sdAb comprises: (1) CDR1 comprising the amino acid sequence shown in SEQ ID NO. 6 and an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 6, (2) CDR2 comprising the amino acid sequence shown in SEQ ID NO. 7 and an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 7, and (3) CDR3 comprising the amino acid sequence shown in SEQ ID NO. 8 and an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 8.

[0022] In some embodiments, the drug is selected from chemotherapeutic agents, toxins, cytokines, enzymes, immunomodulators, chelating complexes, diagnostic agents, nanoparticles, and radioisotopes. In some embodiments, the drug is a radioisotope selected from the following: bismuth-213, cesium-131, cesium-137, c cesium-131, cobalt-60, holmium-166, iodine-125, iodine-131, iridium-192, lead-212, lutetium-177, palladium-103, phosphorus-32, potassium-42, radium-223, rhenium-186, rhenium-188, samarium-153, scandium-47, selenium-75, sodium-24, strontium-89, technetium-99m, thorium-227, xenon-133, ytterbium-169, ytterbium-177, yttrium-90, actinium-225, astatine-211, bismuth-213, carbon-11, nitrogen-13, oxygen-15, fluorine-18, cobalt-57, copper-64, copper-67, gallium-67, gallium-68, indium-111, iodine-123, iodine-124, krypton-81m, rubidium-82, strontium-82, thallium-201, and zirconium-89.

[0023] In another aspect, there is provided a method for treating a disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the conjugate of the present application. In some embodiments, the disorder is cancer.

[0024] In another aspect, there is provided a method for preparing the conjugate of the present application, the method comprising conjugating the functional linker and the drug to the sdAb. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 An exemplary synthetic scheme of the functional linker of the present application is depicted.

[0026] Figure 2 The serum concentration of the CLDN18.2 sdAb-linker conjugate within 48 hours is depicted.

[0027] Figure 3 The serum concentration of the FAP sdAb-linker conjugate within 48 hours is depicted.

[0028] Figure 4 The in vivo SPECT imaging within 48 hours after injection of the CLDN18.2-linker conjugate is depicted.

[0029] Figure 5 The in vivo SPECT imaging within 168 hours after injection of the FAP-linker conjugate is depicted. DETAILED DESCRIPTION

[0030] DEFINITIONS

[0031] Unless otherwise specifically stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any methods or materials similar or equivalent to those described herein can be used when implementing this application. The following terms are defined for this application.

[0032] It should be understood that the embodiments of the present application described herein include "consisting of the embodiments" and / or "consisting essentially of the embodiments".

[0033] The reference herein to "about" a value or parameter includes (and describes) variations that relate to that value or parameter itself. For example, the description of "about X" includes the description of "X".

[0034] The term "about X-Y" used herein has the same meaning as "about X to about Y". The expression "about X, Y, and / or Z" used herein has the same meaning as "about X, about Y, and / or about Z".

[0035] As used herein, the terms "a", "an", or "the" include aspects having not only one member but also aspects having more than one member. For example, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, a reference to "a cell" includes a plurality of such cells, and a reference to "the agent" includes a reference to one or more agents known to those skilled in the art, and so forth.

[0036] As used herein, the term "effective amount" refers to the amount of a compound, composition, or conjugate sufficient to treat a specified disorder, condition, or disease in a subject (such as to ameliorate, mitigate, reduce, and / or delay one or more of its symptoms).

[0037] As understood in the art, an "effective amount" can be one or more doses. For example, a single dose or multiple doses may be required to achieve the desired therapeutic endpoint.

[0038] As used herein, "therapeutically effective amount" means the amount administered to a subject that elicits the desired pharmacological and / or physiological effect for a condition in the subject. The effect in the subject can be prophylactic (in terms of completely or partially preventing a condition or its symptoms) and / or can be therapeutic (in terms of partial or complete cure of a condition and / or an adverse effect attributable to the condition).

[0039] As used herein, the term "subject" refers to a living being to be treated by the methods of the present invention. Such a living being is preferably a mammal (e.g., mouse, ape, horse, cow, pig, dog, cat, etc.), and more preferably a human.

[0040] As described herein, "tumor antigen" refers to an antigenic substance produced at the tumor site.

[0041] As described herein, "single photon emission computed tomography" (or SPECT) is a nuclear imaging modality used in diagnostic medicine. SPECT produces a three-dimensional image of the distribution of a radioactive tracer (or probe) injected into the bloodstream and subsequently taken up by specific tissues. This is accomplished via the use of a specialized nuclear medicine camera. Thus, SPECT allows for the assessment of perfusion and function of specific tissues.

[0042] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.

[0043] Full-length antibodies contain two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. The variable regions in both chains typically contain three highly variable loops, called complementarity-determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the main antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0044] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, diabodies, Fab, Fab’, F(ab’)2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabodies (dsdiabody), single-chain Fv (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, single-domain antibodies (e.g., camelized single-domain antibodies), nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds an antigen but does not contain the full antibody structure. An antigen-binding fragment is capable of binding the same antigen as the parent antibody or parent antibody fragment (e.g., parent scFv). In some embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody that are grafted into the framework regions from one or more different human antibodies.

[0045] As used herein, the term "single domain antibody (sdAb, also known as nanobody)" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Like a full antibody, it is capable of selectively binding to a specific antigen. The molecular weight of a single domain antibody is only 12 - 15 kDa, much smaller than that of a common antibody composed of two protein heavy chains and two light chains (150 - 160 kDa), and even smaller than the Fab fragment (about 50 kDa, one light chain and half a heavy chain) and the single-chain variable fragment (about 25 kDa, two variable domains, one from the light chain and one from the heavy chain).

[0046] The first single domain antibodies were engineered from the heavy chain antibodies found in camelids; these are called VHH fragments. Cartilaginous fish also have heavy chain antibodies (IgNAR, immunoglobulin new antigen receptor) from which single domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains of common immunoglobulin G (IgG) from humans or mice into monomers. Although most current research on single domain antibodies is based on heavy chain variable domains, nanobodies derived from light chains have also shown specific binding to target epitopes.

[0047] Camelid nanobodies have been shown to be as specific as antibodies and in some cases they are more robust. They are easily isolated using the same phage display selection procedures used for antibodies, allowing them to be cultured at high concentrations in vitro. Their smaller size and single domain make these antibodies easier to transform into bacterial cells for mass production, making them ideal for research purposes.

[0048] As used herein, the term "CDR" or "complementary determining region" is intended to mean the non - contiguous antigen - binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described by Kabat, et al. (1997) J. Biol. Chem. 252:6609 - 6616; Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901 - 917 (1987); Al - Lazikani B. et al., J. Mol. Biol., 273:927 - 948 (1997); MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832 - 3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55 - 77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657 - 670 (2001), where these definitions include overlapping or subsets of amino acid residues when compared to one another. However, application of any of these definitions to refer to the CDRs of an antibody or engineered antibody or its variants is intended to fall within the scope of the term as defined and used herein. For comparison, the amino acid residues that comprise the CDRs as defined by each of the references cited above are listed in Table 1 below. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832 - 3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301 - D307 (2010); and Adolf - Bryfogle J. et al., Nucleic Acids Res., 43: D432 - D438 (2015). The content of the references cited in this paragraph is incorporated herein by reference in its entirety for the purposes of this application and may be included in one or more claims herein.

[0049] Table 1: CDR Definitions

[0050]

[0051] 1. Residue numbering following the nomenclature of Kabat et al. as above

[0052] 2. Residue numbering following the nomenclature of Chothia et al. as above

[0053] 3. Residue numbering following the nomenclature of MacCallum et al. as above

[0054] 4. Residue numbering following the nomenclature of Lefranc et al. as above

[0055] 5. Residue numbering following the nomenclature of Honegger and Plückthun as above

[0056] In some embodiments, the CDRs of an antibody can be determined according to the IMGT numbering system. For the IMGT numbering system, (i) VH CDR1 is typically present at amino acid positions 25 to 35 of the heavy chain, (ii) VH CDR2 is typically present at amino acid positions 51 to 57 of the heavy chain, and (iii) VH CDR2 is typically present at amino acid positions 93 to 102 of the heavy chain. For the IMGT numbering system, (i) VL CDR1 is typically present at amino acid positions 27 to 32 of the light chain, (ii) VL CDR2 is typically present at amino acid positions 50 to 52 of the light chain, and (iii) VL CDR3 is typically present at amino acid positions 89 to 97 of the light chain.

[0057] "Framework" or "FR" residues are those variable domain residues other than the CDR residues defined herein.

[0058] For the polypeptide and antibody sequences identified herein, "percent amino acid sequence identity (%)" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide or antibody being compared, after a sequence alignment in which any conservative substitutions are considered part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes of this application, the amino acid sequence identity % values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R. C., BMCBioinformatics 5(1):113, 2004).

[0059] As used herein, the term "antibody mimetic" refers to a molecule that is capable of mimicking the ability of an antibody to bind an antigen, but is not limited to a native antibody structure. Examples of such antibody mimetics include, but are not limited to, affibodies, DARPins, Anticalins, Avimers, and Versabodies.

[0060] Affibody molecules represent a new class of affinity proteins based on a protein domain of 58 amino acid residues, derived from one of the IgG-binding domains of staphylococcal protein A. This three-helix bundle domain has been used as a scaffold for constructing combinatorial phagemid libraries from which affibody variants targeting desired molecules can be selected using phage display technology (Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA, Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain, Nat Biotechnol 1997; 15:772-7. Ronmark J, Gronlund H, Uhlen M, Nygren PA, Human immunoglobulin A (IgA) -specific ligands from combinatorial engineering of protein A, Eur J Biochem 2002;269:2647-55). The simple and robust structure of affibody molecules combined with their low molecular weight (6 kDa) makes them suitable for a variety of applications, such as, for example, as detection reagents (Ronmark J, Hansson M, Nguyen T, et al., Construction and characterization of affibody-Fc chimeras produced in Escherichia coli, J Immunol Methods 2002;261:199-211) and for inhibiting receptor interactions (Sandstorm K, Xu Z, Forsberg G, Nygren PA, Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding Affibody ligand developed by combinatorial protein engineering, Protein Eng 2003;16:691-7). Further details of affibodies and their production methods can be obtained by reference to US Patent No. 5,831,012, which is incorporated herein by reference in its entirety.

[0061] DARPin (Designed Ankyrin Repeat Protein) is an example of an antibody-mimicking DRP (Designed Repeat Protein) technology that has been developed to exploit the binding capabilities of non-antibody polypeptides. Different from antibodies, repeat proteins such as ankyrin repeat proteins or leucine-rich repeat proteins are ubiquitous binding molecules that occur both intracellularly and extracellularly. Its unique modular architecture is characterized by repeating structural units (repeats) that stack together to form an elongated repeat domain that presents a variable and modular target-binding surface. Based on this modularity, combinatorial libraries of polypeptides with highly diverse binding specificities can be generated. The strategy involves the consensus design of self-compatible repeats that display variable surface residues and their random assembly into repeat domains. Additional information on DARPin and other DRP technologies can be found in U.S. Patent Application Publication No. 2004 / 0132028 and International Patent Application Publication No. WO 02 / 20565, both of which are hereby incorporated by reference in their entirety.

[0062] Anticalin is another antibody-mimicking technology, however in this case, the binding specificity is derived from lipocalins, which are a family of low-molecular-weight proteins that are naturally and abundantly expressed in human tissues and body fluids. Anticalin can also be engineered as a dual-targeting protein (so-called Duocalin). Duocalin binds two separate therapeutic targets in a monomeric protein that is easily produced using standard manufacturing processes, while retaining target specificity and affinity regardless of the structural orientation of its two binding domains. Additional information on Anticalin can be found in U.S. Patent No. 7,250,297 and International Patent Application Publication No. WO 99 / 16873, both of which are hereby incorporated by reference in their entirety.

[0063] Another antibody mimetic technology that can be used in the present application is Avimer. Avimer evolved from a large family of human extracellular receptor domains through in vitro exon shuffling and phage display, resulting in multi-domain proteins with binding and inhibitory properties. Linking multiple independent binding domains has been shown to generate avidity and confer improved affinity and specificity compared to conventional single-epitope binding proteins. Other potential advantages include the facile and efficient production of multi-target specific molecules in Escherichia coli (E. coli), improved thermal stability, and resistance to proteases. Avimers with sub-nanomolar affinities have been obtained for a variety of targets. Additional information about Avimer can be found in U.S. Patent Application Publication Nos. 2006 / 0286603, 2006 / 0234299, 2006 / 0223114, 2006 / 0177831, 2006 / 0008844, 2005 / 0221384, 2005 / 0164301, 2005 / 0089932, 2005 / 0053973, 2005 / 0048512, 2004 / 0175756, all of which are hereby incorporated by reference in their entirety.

[0064] Versabody is another antibody mimetic technology that can be used in the present application. Versabody is a small protein of 3 - 5 kDa with > 15% cysteine, and these cysteines form a scaffold with a high disulfide bond density, replacing the hydrophobic core of a normal protein. Given the structure of Versabody, these antibody mimetics offer versatile forms including multivalency, multispecificity, various half-life mechanisms, tissue targeting modules, and the absence of the antibody Fc region. Additionally, Versabody is produced in high yield in E. coli, and due to its hydrophilicity and small size, Versabody is highly soluble and can be formulated at high concentrations. Versabody has extremely high thermal stability (it can be boiled) and offers an extended shelf life. Additional information about Versabody can be found in U.S. Patent Application Publication No. 2007 / 0191272, which is hereby incorporated by reference in its entirety.

[0065] As used herein, the term "epitope" refers to a specific set of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they can bind to the same epitope within that antigen.

[0066] As used herein, the terms "specifically bind", "specifically recognize", and "is specific for" refer to a measurable and reproducible interaction, such as the binding between an antibody and its antigen, which determines the presence of a target or antigen in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically recognizes an antigen is an antibody that binds to that antigen with greater affinity, avidity, more readily, and / or for a longer duration compared to binding to other targets or antigens. In some embodiments, for example, as measured by radioimmunoassay (RIA), the degree of binding of an antibody to an unrelated target or antigen is less than about 10% of the binding of the antibody to its antigen. In some embodiments, an antibody that specifically binds its antigen has a dissociation constant (K -8 ) of ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 D M. In some embodiments, the specific binding can include, but does not require, exclusive binding. The binding specificity of an antibody can be determined experimentally by methods known in the art. Such methods include, but are not limited to, for example, Western blotting, ELISA assays, RIA assays, ECL assays, IRMA assays, EIA assays, BIACORE TM assays, and peptide scanning.

[0067] As used herein, the term "conjugate" refers to a molecule that contains more than one functional moiety, wherein the first functional moiety and the second functional moiety are linked (e.g., covalently linked) to each other via a linker, spacer, or reactive linking group, as described herein.

[0068] As used herein, the term "PEG (polyethylene glycol)" refers to water-soluble poly(ethylene oxide) having the structure "-(OCH2CH2) n -". In some embodiments, n ranges from about 1 to about 25. As used herein, PEG also includes "-CH2CH2-O(CH2CH2O) n -CH2CH2-" and "-(OCH2CH2) n O-", depending on whether the terminal oxygen has been removed. Throughout this application, it should be understood that the term "PEG" includes structures having various terminal groups, etc. The term "PEG" also means a polymer containing a majority (i.e., greater than 50%) of -OCH2CH2- repeating subunits. In specific forms, PEG encompasses those branched, linear, and dendritic structures.

[0069] As used herein, a group having two or more attachment points (i.e., divalent, trivalent, or polyvalent) within a compound of the technology of the present invention is denoted by using the suffix "ene" (prefix "sub-"). For example, a divalent alkyl group is an alkylene group, a divalent aryl group is an arylene group, a divalent heteroaryl group is a divalent heteroarylene group, and so on.

[0070] As used herein, the term "aromatic" or "aryl" refers to a closed-ring structure having at least one ring with a conjugated π-electron system, and includes both carbocyclic aryl and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups. The carbocyclic or heterocyclic aromatic group may contain 5 to 20 ring atoms. The term includes covalently linked monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) groups. The aromatic group may be unsubstituted or substituted. Non-limiting examples of the "aromatic" or "aryl" group include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthryl, and phenanthryl. Substituents of each of the above aryl and heteroaryl ring systems are selected from the acceptable substituents described herein. The term "arylene" can be interpreted accordingly.

[0071] As used herein, the term "alkyl" includes both saturated linear and branched alkyl groups. In some embodiments, the alkyl group includes, but is not limited to, C 1-20 alkyl group, C 1-15 , C 1-12 alkyl group, C 1-6 alkyl group, and C 1-4 alkyl group. In some embodiments, the alkyl group includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and hexyl. The term "alkylene" can be interpreted accordingly.

[0072] As used herein, the term "alkenyl" refers to a group containing one or more carbon-carbon double bonds, which may be linear or branched. In some embodiments, the alkenyl group includes, but is not limited to, C 2-20 alkenyl group, C 2-15 alkenyl group, C 2-12 alkenyl group, C 2-6 alkenyl group, and C 2-4 alkenyl group. The term "alkenylene" can be interpreted accordingly.

[0073] As used herein, the term "alkynyl" refers to a carbon chain containing one or more triple bonds, which may be linear or branched. In some embodiments, the alkynyl group includes, but is not limited to, C 2-20 alkynyl group, C 2-15 alkynyl group, C 2-12 alkynyl group, C 2-6 alkynyl group, and C 2-4 alkynyl group. The term "alkynylene" can be interpreted accordingly.

[0074] In some embodiments, the alkyl, alkenyl, or alkynyl group is unsubstituted. In some embodiments, the alkyl, alkenyl, or alkynyl group is substituted. In some embodiments, the substituted alkyl, alkenyl, or alkynyl group has 1 to 10 substituents, 1 to 5 substituents, or 1, 2, or 3 substituents. In some embodiments, the substituted alkyl, alkenyl, or alkynyl group bears a sulfonic acid substituent and / or a halogen substituent.

[0075] In some embodiments, the alkyl, alkenyl, alkynyl group or arylene, alkylene, alkenylene, alkynylene group contains (a) 0, 1, or 2 carbon atoms that can be substituted with a group selected from: C 6-10 arylene, 5- to 10-membered heteroarylene, C 3-7 carbocyclene, and 5- to 10-membered heterocyclene groups, and (b) 0, 1, or 2 -CH2- groups that can be replaced with a group selected from: -O-, -S-, -S-S-, -C(O)-, and -N(C 1-6 alkyl)- groups.

[0076] As used herein, a C 6-10 aryl group is a monocyclic or polycyclic 6- to 10-membered aromatic hydrocarbon ring system having 6 to 10 carbon atoms, such as phenyl. As used herein, a 5- to 10-membered heteroaryl group is a monocyclic or polycyclic 5- to 10-membered aromatic ring system (such as a 5- or 6-membered ring) containing at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms, such as O, S, and N). The term "heteroarylene" shall be construed accordingly.

[0077] Examples of monocyclic heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and tetrazolyl groups. Examples of polycyclic heteroaryl groups include, but are not limited to, benzothienyl, benzofuryl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, benzotriazolyl, indolyl, isoindolyl, and indazolyl groups. Preferred polycyclic groups include indolyl, isoindolyl, benzimidazolyl, indazolyl, benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, and benzisothiazolyl groups, more preferably benzimidazolyl, benzoxazolyl, and benzothiazolyl, and most preferably benzothiazolyl. However, monocyclic heteroaryl groups are preferred. In some embodiments, the heteroaryl group is a 5- to 6-membered heteroaryl group. Particularly preferred heteroaryl groups are thienyl, pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, oxazolyl, isoxazolyl, triazolyl, pyridyl, pyridazinyl, pyrimidinyl, and pyrazinyl groups. In some embodiments, the heteroaryl group includes, but is not limited to, thienyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, and triazinyl, and most preferably pyridyl.

[0078] As used herein, a 5- to 10-membered heterocyclic group is a non-aromatic saturated or unsaturated monocyclic or polycyclic C 5-10 carbocyclic ring system in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms are replaced by moieties selected from N, O, S, S(O), and S(O)2. In some embodiments, the 5- to 10-membered heterocyclic group is a 5- to 6-membered ring. The term "heterocyclylene" shall be construed accordingly.

[0079] Examples of heterocyclic groups include azetidinyl, oxetidinyl, thietidinyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl, pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, methylenedioxyphenyl, ethylenedioxyphenyl, thiomorpholinyl, S-oxo-thiomorpholinyl, S,S-dioxo-thiomorpholinyl, morpholinyl, 1,3-dioxolanyl, 1,4-dioxolanyl, trioxolanyl, trithianyl, imidazolinyl, pyranyl, pyrazolinyl, thioxocyclopentyl, thioxothiazolyl, 1H-pyrazol-5-(4H)-one group, 1,3,4-thiadiazole-2(3H)-thione group, oxopyrrolidinyl, oxothiazolyl, oxopyrazolyl, succinimidyl and maleimidyl groups and moieties. Preferred heterocyclic groups are pyrrolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl, pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, thiomorpholinyl and morpholinyl groups and moieties. More preferred heterocyclic groups are tetrahydropyranyl, tetrahydrothiopyranyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, morpholinyl and pyrrolidinyl groups.

[0080] As used herein, C 3-7 A carbocyclic group is a non-aromatic saturated or unsaturated hydrocarbon ring having 3 to 7 carbon atoms. In some embodiments, it is a saturated or monounsaturated hydrocarbon ring (i.e., a cycloalkyl moiety or a cycloalkenyl moiety) having 3 to 7 carbon atoms (more preferably 5 to 6 carbon atoms). Examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and their monounsaturated variants. Particularly preferred carbocyclic groups are cyclopentyl and cyclohexyl. The term "carbocyclylene" should be construed accordingly.

[0081] As used herein, the terms "halogen" or "halide" may be used interchangeably and include, but are not limited to, F, Cl, Br and I.

[0082] As used herein, the term "reactive linking group" is a reactive group capable of joining two or more chemical groups, moieties or units by covalent bonds. Reactive linking groups can be used to attach one component selected from the group consisting of PEG groups, renin-cleavable groups, blood protein-binding groups, spacers and chelating factors to another component. In some embodiments, reactive linking groups include, but are not limited to, amines, imines, carboxylic esters, alcohols, thiols, selenols, phenols, esters, acetones, aldehydes, carbenes, sulfonyl halides, imidoesters, anhydrides, disulfides, maleimides, phosphines, disulfides, alkoxyamines, azides, alkynes, strained alkynes, strained alkenes, halogens, sulfonates, haloacetyls, hydrazides, diazirines, phosphines, tetrazines, isothiocyanates and oxaziridines.

[0083] As used herein, the term "protecting group" refers to a moiety that masks, reduces or prevents the reactivity of a reactive group when attached to the reactive group in a molecule. Examples of protecting groups can be found in T. W. Greene and P. G M. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York, 1999 and Harrison and Harrison et al., Compendium of Synthetic Organic Methods, Volumes 1-8 (John Wiley and Sons, 1971-1996), which are incorporated herein by reference in their entirety. Representative hydroxyl protecting groups include, but are not limited to, acyl groups, benzyl and trityl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers. Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boc), trimethylsilyl (TMS), 2-trimethylsilylethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethoxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC), etc.

[0084] As used herein, the term "chelating factor" or "chelating factor group" refers to a moiety capable of coordinating (or binding) to a metal ion in a multidentate manner (e.g., via two or more atoms in the moiety). In some embodiments, the chelating factor can include donor atoms that bind to the metal, such as S, N or O. For example, the chelating factor can be a tridentate ligand having three donor atoms. In some embodiments, the chelating factor group is capable of coordinating to a radioisotope as described herein.

[0085] As used herein, the term "pharmacokinetic curve" or "PK curve" refers to the drug concentration curve in blood or plasma. Such a curve can be the relationship of drug concentration versus time (i.e., "concentration-time PK curve") or the relationship of drug concentration versus the number of administered doses (i.e., "concentration-dose PK curve"). The PK curve is characterized by PK parameters.

[0086] As used herein, the term "pharmacokinetic parameter" or "PK parameter" refers to a measure of drug concentration in blood or plasma, such as: (1) "drug Cmax", the maximum drug concentration reached in blood or plasma; (2) "drug Tmax", the time taken to reach Cmax after administration; and (3) "drug exposure", the total concentration of drug present in blood or plasma over a selected time period, which can be measured using the area under the curve (AUC) of the drug release profile over the selected time period (t). Modification of one or more PK parameters provides a modified PK curve.

[0087] As used herein, the term "pharmacodynamic (PD) curve" or "PD curve" refers to the drug efficacy curve in a patient (or subject), which is characterized by PD parameters.

[0088] As used herein, the term "PK parameter" or "PD parameter" includes "drug Emax" (maximum drug efficacy), "drug EC 50 50%" (drug concentration at 50% of Emax), and side effects.

[0089] As used herein, "treatment / treating" is a method for obtaining a beneficial or desired result, including a clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, providing remission (partial or complete) of the disease, reducing the dose of one or more other medications required to treat the disease, delaying disease progression, increasing or enhancing the quality of life, increasing weight gain, and / or extending survival. "Treatment" also encompasses alleviation of the pathological outcome of cancer (e.g., reduction in tumor volume). The methods of this application contemplate any one or more of these aspects of treatment.

[0090] In the context of cancer, the term "treatment" includes any one or all of the following: inhibiting cancer cell growth, inhibiting cancer cell replication, reducing the overall tumor burden, and alleviating one or more symptoms associated with the disease.

[0091] All publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety.

[0092] Conjugate

[0093] In one aspect, the present invention provides a conjugate comprising: (1) an sdAb that specifically binds to a tumor antigen, (2) a drug, and (3) a functional linker comprising a functional moiety that links the drug to the sdAb.

[0094] In some embodiments of the conjugate, when the conjugate is administered to a subject, compared to a conjugate without the functional moiety, the functional linker can: (i) modify the PK profile of the conjugate, (ii) modify the PD profile of the conjugate, (iii) modify the biodistribution of the conjugate, (iv) increase the efficacy of the conjugate, (v) cause a decrease in nephrotoxicity and an increase in therapeutic efficacy in the subject. In some embodiments, when the conjugate is administered to a subject, compared to a conjugate without the functional moiety, the functional linker can achieve at least one of (i)-(v) as described above. In some embodiments, when the conjugate is administered to a subject, compared to a conjugate without the functional moiety, the functional linker can achieve at least two of (i)-(v) as described above. In some embodiments, when the conjugate is administered to a subject, compared to a conjugate without the functional moiety, the functional linker can achieve at least three of (i)-(v) as described above. In some embodiments, when the conjugate is administered to a subject, compared to a conjugate without the functional moiety, the functional linker can achieve at least four of (i)-(v) as described above. In some embodiments, when the conjugate is administered to a subject, compared to a conjugate without the functional moiety, the functional linker can achieve all of (i)-(v) as described above.

[0095] In another aspect, the present application provides a conjugate comprising: (a) a single-domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety as described herein, wherein when the conjugate is administered to a subject, compared to a conjugate without the functional moiety, the functional linker increases the tumor uptake, accumulation, and / or retention of the drug.

[0096] In another aspect, there is provided a conjugate comprising: (a) a single-domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety as described herein, wherein when the conjugate is administered to a subject, compared to a conjugate without the functional moiety, the functional linker reduces the renal uptake, accumulation, and / or retention of the drug.

[0097] In another aspect, there is provided a conjugate comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety as described herein, wherein when the conjugate is administered to a subject, the functional linker increases the tumor / kidney ratio of the drug as compared to a conjugate without the functional linker.

[0098] In another aspect, there is provided a conjugate comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker as described herein, wherein when the conjugate is administered to a subject, the functional linker prolongs the blood circulation of the drug as compared to a conjugate without the functional linker.

[0099] In another aspect, there is provided a conjugate comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker as described herein, wherein when the conjugate is administered to a subject, the functional linker prolongs the half-life of the drug as compared to a conjugate without the functional linker.

[0100] In another aspect, there is provided a conjugate comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker as described herein, wherein the functional linker comprises at least one (such as at least two or at least three) functional moiety selected from the group consisting of a PEG group, a renin-cleavable group, a spacer, and a blood protein-binding group.

[0101] In some embodiments, the functional linker in the conjugate is capable of achieving at least one, at least two, at least three, at least four, or all of the following functions: (i) increasing the tumor uptake, accumulation, and / or retention of the drug as compared to a conjugate without the functional linker when the conjugate is administered to a subject; (ii) decreasing the kidney uptake, accumulation, and / or retention of the drug as compared to a conjugate without the functional linker when the conjugate is administered to a subject; (iii) increasing the tumor / kidney ratio of the drug as compared to a conjugate without the functional linker when the conjugate is administered to a subject; (iv) prolonging the blood circulation of the drug as compared to a conjugate without the functional linker when the conjugate is administered to a subject; and (v) prolonging the half-life of the drug as compared to a conjugate without the functional linker when the conjugate is administered to a subject.

[0102] On the other hand, the present disclosure provides a conjugate comprising: (a) an antibody mimetic that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety as described herein. In some embodiments, the antibody mimetic is selected from the group consisting of affibody, DARPin, Anticalin, Avimer, Versabody, and Duocali. In some embodiments, the conjugate has a molecular weight of no more than about 60 KDa, such as no more than about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, about 5, or about 3 KDa.

[0103] Functional linker

[0104] On the other hand, the present disclosure provides a functional linker for use in a conjugate as described herein, wherein the functional linker comprises a functional moiety. In some embodiments, the functional linker is capable of performing at least one of the functions including the following: (i) increasing the tumor uptake, accumulation, and / or retention of the conjugate compared to a conjugate without the functional linker when the conjugate is administered to a subject; (ii) decreasing the renal uptake, accumulation, and / or retention of the conjugate compared to a conjugate without the functional linker when the conjugate is administered to a subject; (iii) increasing the tumor / renal ratio of the conjugate compared to a conjugate without the functional linker when the conjugate is administered to a subject; (iv) prolonging the blood circulation of the conjugate compared to a conjugate without the functional linker when the conjugate is administered to a subject; and (v) prolonging the half-life of the conjugate compared to a conjugate without the functional moiety when the conjugate is administered to a subject.

[0105] In some embodiments, the functional linker of the present application comprises one or more components selected from the following: polyethylene glycol (PEG) groups, renin-cleavable groups, blood protein-binding groups, spacers, and any derivatives thereof.

[0106] In some embodiments where the functional linker comprises a PEG group, the PEG group can increase the hydrophilicity of the conjugate for better clearance and / or can act as a spacer for separating different functional moieties. The PEG group can also prolong the circulation time of the drug and reduce its renal uptake, accumulation, and / or retention. In some embodiments, the functional linker comprises one PEG group. In some embodiments, the functional moiety comprises more than one PEG group. In some embodiments, the functional linker comprises 2, 3, 4, or 5 PEG groups as described herein.

[0107] In some embodiments, the PEG group comprises from about 1 to about 25 monomer units. In some embodiments, the PEG group comprises from about 1 to about 5 monomer units. In some embodiments, the PEG group comprises from about 5 to about 10 monomer units. In some embodiments, the PEG group comprises from about 10 to about 15 monomer units. In some embodiments, the PEG group comprises from about 15 to about 20 monomer units. In some embodiments, the PEG group comprises from about 20 to about 25 monomer units. In some embodiments, the PEG group comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 monomer units.

[0108] In some embodiments, the PEG group comprises the structure of, where n is any integer in the range from about 1 to about 25. In some embodiments, the PEG group comprises the structure of, where n is any integer in the range from about 1 to about 5. In some embodiments, the PEG group comprises the structure of, where n is any integer in the range from about 5 to about 10. In some embodiments, the PEG group comprises the structure of, where n is any integer in the range from about 10 to about 15. In some embodiments, the PEG group comprises the structure of, where n is any integer in the range from about 15 to about 20. In some embodiments, the PEG group comprises the structure of, where n is any integer in the range from about 20 to about 25. In some embodiments, the PEG group comprises the structure of, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

[0109] In some embodiments, the functional linker comprises a blood protein binding group. In some embodiments, the blood protein is selected from albumin, fetuin, transferrin, and IgG. In some embodiments, the blood protein binding group comprises an albumin binding group or an albumin binder.

[0110] Albumin is a protein produced by the liver that serves as a key lipid delivery mediator for tissues. Fatty acids of short to medium length (6 to 12 carbons) bind to albumin with an affinity between approximately 0.5 μM and approximately 60 μM, while longer fatty acids (14 to 18 carbons) have an affinity 10-fold higher (e.g., below 50 nM). Any suitable albumin-binding group known in the art can be used in the functional linkers of the present application. In some embodiments where the functional linker comprises an albumin-binding group, the binding affinity of the conjugate to human serum albumin is between about 10 -7 and about 10 -4 M. In some embodiments where the functional linker comprises an albumin-binding group, the binding affinity of the conjugate to human serum albumin ranges between about 10 -6 M and about 10 -4 M. In some embodiments where the functional linker comprises an albumin-binding group, the binding affinity of the conjugate to human serum albumin ranges between about 10 -5 M and about 10 -4 M. In some embodiments where the functional linker comprises an albumin-binding group, the binding affinity of the conjugate to human serum albumin ranges between about 10 -7 M and about 10 -5 M. In some embodiments where the functional linker comprises an albumin-binding group, the binding affinity of the conjugate to human serum albumin ranges between about 10 -7 M and about 10 -6 M. In some embodiments where the functional linker comprises an albumin-binding group, the binding affinity of the conjugate to human serum albumin ranges between about 10 -6 M and about 10 -5 M.

[0111] Exemplary albumin-binding groups that can be used in the functional linkers of the present application include, but are not limited to, short-chain fatty acids, medium-chain fatty acids, long-chain fatty acids, myristic acid, substituted or unsubstituted indole-2-carboxylic acid, substituted or unsubstituted thioamides, substituted or unsubstituted 4-oxo-4-(5,6,7,8-tetrahydronaphthalen-2-yl)butyric acid, substituted or unsubstituted naphthoyl sulfonamides, substituted or unsubstituted diphenylcyclohexyl phosphate, substituted or unsubstituted 4-iodophenylalkanoic acids, substituted or unsubstituted 3-(4-iodophenyl)propionic acid, substituted or unsubstituted 2-(4-iodophenyl)acetic acid, or substituted or unsubstituted 4-(4-iodophenyl)butyric acid. In some embodiments, the blood protein-binding agent group comprises a fatty acid, butyric acid, or a derivative thereof. In some embodiments, the blood protein-binding agent group comprises: 3-iodo-tyrosine, short-chain or medium-chain fatty acids, long-chain fatty acids, 4-(p-iodophenyl)butyric acid, or 4-(p-methyl)butyric acid.

[0112] In some embodiments, the albumin-binding group that can be used in the functional linker of the present application comprises a structure of, where n is any integer ranging from 1 to 20, and R m is H, CH3, or COOH.

[0113] In some embodiments, the albumin-binding group that can be used in the functional linker of the present application comprises a structure of, where R x is selected from

[0114] , , , ,

[0115] , , , ,

[0116] , , , , and

[0117] where n is any integer ranging from 1 to 6,

[0118] R x1 , R x2 , R x3 , R x4 , R x5 , R x6 and R x7 each independently is selected from H, N, S, O, Se, P, halogen, C 5-20 aryl group, C 1-20 alkyl group, C 2-20 alkenyl group, and C 2-20 alkynyl group. In some embodiments, the C 5-20 aryl group, C 1-20 alkyl group, C 2-20 alkenyl group, or C 2-20 alkynyl group may be unsubstituted or substituted with one or more substituents selected from N, S, O, Se, P, and halogen atoms. In some embodiments, 0, 1, or 2 carbon atoms in the C 5-20 aryl group, C 1-20 alkyl group, C 2-20 alkenyl group, or C 2-20 alkynyl group are selected from C 6-10 arylene, 5- to 10-membered heteroarylene group, C 3-7Group substitution of a carbocyclic group or a 5- to 10-membered heterocyclic group, and wherein the aryl group, heteroaryl group, carbocyclic group, and heterocyclic group are unsubstituted or substituted with one or more substituents selected from N, S, O, Se, P, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthiol, -N(C 1-6 alkyl)(C 1-6 alkyl), nitro, and sulfonic acid groups. In some embodiments, C 5-20 aryl group, C 1-20 alkyl group, C 2-20 alkenyl group, or C 2-20 alkynyl group, 0, 1, or 2 -CH- or -CH2- groups are substituted with a group selected from -O-, -S-, -S-S-, -C(O)-, and -N(C 1-6 alkyl)- groups.

[0119] In some embodiments, the albumin-binding group that can be used in the functional linker of the present application comprises a structure, wherein R y is selected from H, F, Cl, Br, I, -CH3, -OCH3, COOH, -CF3, and n is any integer ranging from 1 to 6.

[0120] In some embodiments, the albumin-binding group comprises a structure selected from the following:

[0121] , , , , , , and .

[0122] In some embodiments, the functional linker comprises a renal enzyme-cleavable group that promotes the excretion of radioactive metabolites into the urine of a subject. In some embodiments, the renal enzyme-cleavable group is selected from brush border enzyme-cleavable groups, lysosomal enzyme-cleavable groups, and combinations thereof.

[0123] In some embodiments, the renal enzyme cleavable group includes oligopeptides. In some embodiments, the oligopeptides include dipeptides or derivatives thereof. In some embodiments, the oligopeptides include tripeptides or derivatives thereof. In some embodiments, the oligopeptides include structures selected from the following: methionine-isoleucine, glycine-lysine, glycine-phenylalanine-lysine, methionine-valine-lysine, glycine-tyrosine, glycine-lysine-lysine, glycine-arginine-lysine, aspartic acid-glycine-lysine, methionine-glycine-lysine, methionine-isoleucine-lysine, glycine-tyrosine-lysine, glycine-valine, glycine-isoleucine, methionine-phenylalanine-lysine, glycine-(3-(2-naphthyl)alanine)-lysine, glycine-diphenylalanine-lysine, and / or methionine-glycine-lysine and any derivatives thereof.

[0124] In some embodiments, the renal enzyme cleavable group comprises methionine-X-lysine, wherein X is a hydrophobic amino acid. In some embodiments, the renal enzyme cleavable group comprises glycine-tyrosine. In some embodiments, the renal enzyme cleavable group comprises glycine-lysine.

[0125] In some embodiments, the renalase cleavable group comprises a , and The structure of

[0126] R1 is selected from H, , , , and ,

[0127] R2 is selected from H, , , and ,and

[0128] R3 is selected from H, , , , , and .

[0129] In some embodiments, the functional linker comprises at least one spacer. In some embodiments, the spacer is linear or branched. In some embodiments, the spacer comprises a structure selected from the group consisting of a PEG group as described herein, a C 5-20 Aryl group, C 1-20 Alkyl group, C 2-20 Alkenyl groups and C 2-20Alkynyl group. In some embodiments, C 5-20 aryl group, C 1-20 alkyl group, C 2-20 alkenyl group or C 2-20 alkynyl group is unsubstituted or substituted with one or more substituents selected from N, S, O, Se, P, and halogen atoms. In some embodiments, C 5-20 aryl group, C 1-20 alkyl group, C 2-20 alkenyl group or C 2-20 alkynyl group, 0, 1, or 2 carbon atoms thereof are replaced by a group selected from C 6-10 arylene, 5- to 10-membered heteroarylene group, C 3-7 carbocyclylene, 5- to 10-membered heterocyclylene group, and wherein the arylene, heteroarylene, carbocyclylene, and heterocyclylene groups are unsubstituted or substituted with one or more substituents selected from N, S, O, Se, P, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthiol, -N(C 1-6 alkyl)(C 1-6 alkyl), nitro, and sulfonic acid groups. In some embodiments, C 1-20 alkyl group, C 2-20 alkenyl group or C 2-20 alkynyl group, 0, 1, or 2 -CH- or -CH2- groups thereof are replaced by a group selected from -O-, -S-, -S-S-, -C(O)-, and -N(C 1-6 alkyl)- groups. In some embodiments, the spacer comprises natural amino acid residues and / or unnatural amino acid residues. In some embodiments, the natural or unnatural amino acid residues are selected from lysine, aspartate, asparagine, diaminobutyric acid, phenylalanine, tyrosine, threonine, serine, proline, leucine, isoleucine, valine, arginine, histidine, glutamate, glutamine, and alanine.

[0130] In some embodiments, the functional linker comprises a structure selected from the following:

[0131] , , , , , , , , , , , , , , , , , , , , and , where n is any integer in the range of 2 to 25, such as including any one of 2 - 5, 5 - 10, 10 - 15, 15 - 20, and 20 - 25.

[0132] In some embodiments, the functional linker further comprises a chelating factor group. Exemplary chelating factor groups that can be used in the functional linker of the present application include, but are not limited to, NODASA, NODAGA, TETA, TRITA, TRAP, DTPA, CHX - DTPA, EDTA, CDTA, CPTA, DOTP, DOTPI, EGTA, HBED, TTHA, DTPA, DOTA, DOTAGA, NOTA, HP - DOA3, CBTE2a, TE2A, TMT, DPDP, HYNIC, DFO, HEDTA, NOPO, MAG3, NCS - MP - NODA, NH2 - MPAA - NODA, H2DEDPA, H4octapa, Macropa, Pypa, Py4pa, THP, and SarAr. In some embodiments, the functional linker comprises a DOTA or DOTAGA group.

[0133] In some embodiments, the functional linker comprises a conjugation group capable of linking the functional linker to the sdAb. In some embodiments, prior to conjugation, the conjugation group is selected from amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, phosphines, disulfides, alkoxyamines, azides, alkyl halides, isothiocyanates (NCS), epoxides, isocyanates, hydrazines, and acyl halides.

[0134] In some embodiments, the functional linker comprises a group resulting from any one of the structures provided in Conjugation Table 2. Thus, in addition to the conjugation group, the functional linker may further comprise the structure of any one of the groups provided below.

[0135] Table 2: Exemplary functional linker structures

[0136]

[0137] Single-domain antibody (sdAb)

[0138] In some embodiments, the conjugate of the present application comprises a single-domain antibody (sdAb) that specifically binds to a tumor antigen. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -12 and about 10 -8 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -12 and about 10 -11 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -12 and about 10 -10 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -12 and about 10 -9 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -11 and about 10 -10 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -11 and about 10 -9 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -11 and about 10 -8 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -10 and about 10 -9 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -10 and about 10 -8 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is in the range of about 10 -9 and about 10 -8 M. In some embodiments, the binding affinity of the sdAb for the tumor antigen is about 10 -12 、about 10 -11 、about 10 -10 、about 10 -9 or about 10 -8 M.

[0139] In some embodiments, the binding affinity of the conjugate for the tumor antigen is in the range of about 10 -12 and about 10 -8within the range between. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -12 and about 10 -11 M. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -12 and about 10 -10 M. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -12 and about 10 -9 M. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -11 and about 10 -10 M. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -11 and about 10 -9 M. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -11 and about 10 -8 M. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -10 and about 10 -9 M. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -10 and about 10 -8 M. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -9 and about 10 -8 M. In some embodiments, the binding affinity of the conjugate to the tumor antigen is about 10 -12 or about 10 -11 or about 10 -10 or about 10 -9 or about 10 -8 M.

[0140] In some embodiments, the conjugate of the present application comprises a functional linker, and the functional linker comprises an albumin-binding group. In some embodiments, the ratio of the binding affinity of the conjugate to the tumor antigen to the binding affinity of the conjugate to human serum albumin is from about 10 to about 10 4Between. In some embodiments, the binding affinity of the conjugate for the tumor antigen relative to the binding affinity of the conjugate for human serum albumin is between about 10 and about 100. In some embodiments, the binding affinity of the conjugate for the tumor antigen relative to the binding affinity of the conjugate for human serum albumin is between about 10 and about 1000. In some embodiments, the binding affinity of the conjugate for the tumor antigen relative to the binding affinity of the conjugate for human serum albumin is between about 100 and about 1000. In some embodiments, the binding affinity of the conjugate for the tumor antigen relative to the binding affinity of the conjugate for human serum albumin is between about 100 and about 10 4 Between. In some embodiments, the binding affinity of the conjugate for the tumor antigen relative to the binding affinity of the conjugate for human serum albumin is between about 1000 and about 10 4 Between.

[0141] In some embodiments, the sdAb in the conjugate specifically binds to a tumor antigen selected from the group consisting of prostate-specific membrane antigen (PSMA), fibroblast activation protein alpha (FAP-α, FAP), folate receptor, luteinizing hormone-releasing hormone (LHRH), norepinephrine transporter (NAT), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-2 (HER-2), vascular endothelial growth factor (VGFR), mucin-1 (MUC-1), mucin-4 (MUC-4), urokinase-type plasminogen activator receptor (uPAR), tumor-associated glycoprotein 72 (TAG-72), Claudin 18.2 (CLDN18.2), vascular endothelial growth factor receptor (VEGFR), C-X-C chemokine receptor type 4 (CXCR4), Hepsin, transmembrane serine protease 2 (TMPRSS2), and tyrosine protein kinase Met (cMET).

[0142] Claudin 18.2 (or CLDN18.2) is a protein encoded by the CLDN18 gene in humans. CLDN18.2 is highly expressed in a large proportion of gastric adenocarcinomas and pancreatic adenocarcinomas, while normal tissue expression is limited to gastric epithelium. See G. Zhu, et al. (2019) Sci. Rep. 9(1):8420.

[0143] In some embodiments, the present application provides an anti-CLDN18.2 sdAb comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 80% (such as any one of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 1.

[0144] In some embodiments, the sdAb in the conjugate as described herein specifically binds to CLDN18.2 and comprises CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO. 1. In some embodiments, CDR1, CDR2, and CDR3 follow the IMGT numbering. In some embodiments, CDR1, CDR2, and CDR3 follow the Kabat numbering. In some embodiments, CDR1, CDR2, and CDR3 follow the Chothia numbering. In some embodiments, CDR1, CDR2, and CDR3 follow the Abm numbering. In some embodiments, CDR1, CDR2, and CDR3 follow the Contact numbering.

[0145] In some embodiments, the sdAb in the conjugate specifically binds to CLDN18.2 and comprises: (1) CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 2, (2) CDR2 comprising the amino acid sequence shown in SEQ ID NO. 3 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 3, and (3) CDR3 comprising the amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 4. Amino acid alterations include, for example, substitutions, deletions, and insertions. In some embodiments, the sdAb specifically binds to CLDN18.2 and comprises: (1) CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2, (2) CDR2 comprising the amino acid sequence shown in SEQ ID NO. 3, and (3) CDR3 comprising the amino acid sequence shown in SEQ ID NO. 4.

[0146] In some embodiments, the sdAb in the conjugates described herein specifically binds to CLDN18.2 and comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 80% (such as any one of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 1. In some embodiments, the sdAb specifically binds to CLDN18.2 and comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof having up to about 10 (such as any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions compared to SEQ ID NO. 1. In some embodiments, the sdAb specifically binds to CLDN18.2 and comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof having up to about 3 (such as any one of about 1, 2 or 3) amino acid substitutions in CDR1 and / or CDR2 and / or CDR3 of SEQ ID NO. 1. In some embodiments, the sdAb specifically binds to CLDN18.2 and comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof having up to about 3 (such as any one of about 1, 2 or 3) amino acid substitutions in FR1 and / or FR2 and / or FR3 and / or FR4 of SEQ ID NO. 1. In some embodiments, the sdAb specifically binds to CLDN18.2 and comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof having amino acid substitutions in both CDR and FR. In some embodiments, the sdAb specifically binds to CLDN18.2 and comprises the amino acid sequence of SEQ ID NO: 1.

[0147] Fibroblast activation protein alpha (FAP or FAP-α) is a 170 kDa single-pass type II transmembrane glycoprotein belonging to the dipeptidyl peptidase 4 family. This protein is highly expressed in cancer-associated fibroblasts (CAFs) and plays an important role in regulating the tumor microenvironment and supporting tumor cells through the release of enzymes, cytokines, and growth factors. FAP can provide target specificity for therapeutic agents because its expression in adults is restricted to pathological sites, including cancer, fibrosis, arthritis, trauma, or inflammation. See T. Kelly, et al. (2012) Int. Rev. Cell. Mol. Biol. 297:83-116. Studies have shown that FAP expression is highly upregulated on reactive CAFs in more than 90% of all primary and metastatic epithelial tumors, but it is generally absent in normal adult tissues. FAP overexpression is associated with poor prognosis and an increased risk of metastasis.

[0148] In some embodiments, the present application provides an anti-FAP sdAb comprising the amino acid sequence shown in SEQ ID NO: 5 or a variant thereof having at least about 80% (such as any one of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 5.

[0149] In some embodiments, the sdAb in the conjugate as described herein specifically binds to FAP. In some embodiments, the sdAb in the conjugate specifically binds to FAP and comprises CDR1, CDR2 and CDR3 of the sequence shown in SEQ ID NO. 5. In some embodiments, CDR1, CDR2 and CDR3 follow the IMGT numbering. In some embodiments, CDR1, CDR2 and CDR3 follow the Kabat numbering. In some embodiments, CDR1, CDR2 and CDR3 follow the Chothia numbering. In some embodiments, CDR1, CDR2 and CDR3 follow the Abm numbering. In some embodiments, CDR1, CDR2 and CDR3 follow the Contact numbering.

[0150] In some embodiments, the sdAb in the conjugate specifically binds to FAP and comprises: (1) CDR1 comprising the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 6, (2) CDR2 comprising the amino acid sequence shown in SEQ ID NO. 7 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 7, and (3) CDR3 comprising the amino acid sequence shown in SEQ ID NO. 8 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 8. Amino acid alterations include, for example, substitutions, deletions and insertions. In some embodiments, the sdAb specifically binds to FAP and comprises: (1) CDR1 comprising the amino acid sequence shown in SEQ ID NO. 6, (2) CDR2 comprising the amino acid sequence shown in SEQ ID NO. 7, and (3) CDR3 comprising the amino acid sequence shown in SEQ ID NO. 8.

[0151] In some embodiments, the sdAb in the conjugate specifically binds to FAP and comprises: (1) a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 9, (2) a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 10, and (3) a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 11. Amino acid alterations include, for example, substitutions, deletions, and insertions. In some embodiments, the sdAb specifically binds to FAP and comprises: (1) a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 9, (2) a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 10, and (3) a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 11.

[0152] In some embodiments, the sdAb in the conjugates described herein specifically binds to FAP and comprises the amino acid sequence as set forth in SEQ ID NO: 5 or a variant thereof having at least about 80% (such as any one of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 5. In some embodiments, the sdAb specifically binds to FAP and comprises the amino acid sequence as set forth in SEQ ID NO: 5 or a variant thereof that comprises up to about 10 (such as any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions compared to SEQ ID NO. 5. In some embodiments, the sdAb specifically binds to FAP and comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof that comprises up to about 3 (such as any one of about 1, 2 or 3) amino acid substitutions in CDR1 and / or CDR2 and / or CDR3 of SEQ ID NO. 5. In some embodiments, the sdAb specifically binds to FAP and comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof that comprises up to about 3 (such as any one of about 1, 2 or 3) amino acid substitutions in FR1 and / or FR2 and / or FR3 and / or FR4 of SEQ ID NO. 5. In some embodiments, the sdAb specifically binds to FAP and comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof that comprises amino acid substitutions in both the CDR and FR. In some embodiments, the sdAb specifically binds to FAP and comprises the amino acid sequence of SEQ ID NO: 5.

[0153] Each SEQ ID NO is depicted in Table 3.

[0154] Table 3 Sequence Listing

[0155]

[0156] Drug

[0157] The drug in the conjugates as described herein can be any suitable drug known in the art. In some embodiments, the drug is selected from chemotherapeutic agents, toxins, cytokines, enzymes, immunomodulators, chelating complexes, diagnostic agents, nanoparticles and radioisotopes.

[0158] In some embodiments, the drugs in the conjugates as described herein include chemotherapeutic agents that can be used to treat cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVA®, Genentech / OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, Astrazeneca), Sutent (SUl 1248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen); alkylating agents such as Thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimine and methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; polyacetylenes; camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin);Cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I; dynemicin, including dynemicin A); bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoproteide enediyne antibiotic chromophores, aclacinomysin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino doxorubicin, cyano morpholino doxorubicin, 2-pyrrolino doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin;Sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’-trichloroethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoid, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), CREMOPHOR-free ABRAXANETM, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and a pharmaceutically acceptable salt, acid, or derivative of any of the foregoing.;

[0159] In some embodiments, the chemotherapeutic agent further includes, but is not limited to, (i) antihormonal agents for modulating or inhibiting the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON-toremifene; (ii) aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, MEGASE® medroxyprogesterone acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those that inhibit gene expression in signal pathways associated with abnormal cell proliferation, such as, for example, PKC-α, Ralf, and H-Ras; (viii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; (ix) vaccines, such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; (x) antiangiogenic agents, such as bevacizumab (AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0160] In some embodiments, the drugs in the conjugates as described herein include cytokines. The term "cytokine" is a general term for proteins released by one cell population and acting as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental prolactin; tumor necrosis factor-a and tumor necrosis factor-β; mullerian-inhibiting substance; murine gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-derived growth factor; transforming growth factors (TGF) such as TGF-a and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); osteogenic factor; interferons such as interferon-a, interferon-β, and interferon-γ; colony stimulating factors (CSF) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; tumor necrosis factors such as TNF-a or TNF-β; and other polypeptide factors including LIF and kit ligand (KL).

[0161] In some embodiments, the drug in the conjugates as described herein includes a radioisotope. In some embodiments, the radioisotope can be selected from bismuth-213, cesium-131, cesium-137, cesium-131, cobalt-60, holmium-166, iodine-125, iodine-131, iridium-192, lead-212, lutetium-177, palladium-103, phosphorus-32, potassium-42, radium-223, rhenium-186, rhenium-188, samarium-153, scandium-47, selenium-75, sodium-24, strontium-89, technetium-99m, thorium-227, xenon-133, ytterbium-169, ytterbium-177, yttrium-90, actinium-225, astatine-211, bismuth-213, carbon-11, nitrogen-13, oxygen-15, fluorine-18, cobalt-57, copper-64, copper-67, gallium-67, gallium-68, indium-111, iodine-123, iodine-124, krypton-81m, rubidium-82, strontium-82, thallium-201, or zirconium-89. In some embodiments, the radioisotope is selected from: actinium-225, fluorine-18, iodine-125, technetium-99m, lutetium-177, indium-111, gallium-68, copper-64, and zirconium-89. In some embodiments, the radioisotope is lutetium-177 or indium-111.

[0162] In some embodiments, the drug in the conjugates as described herein includes a radioisotope bound to a chelating agent (chelating complex). In some embodiments, the radioisotope is bound to a chelating agent selected from: NODASA, NODAGA, TETA, TRITA, TRAP, DTPA, CHX-DTPA, EDTA, CDTA, CPTA, DOTP, DOTPI, EGTA, HBED, TTHA, DTPA, DOTA, DOTAGA, NOTA, HP-DOA3, CBTE2a, TE2A, TMT, DPDP, HYNIC, DFO, HEDTA, NOPO, MAG3, NCS-MP-NODA, NH2-MPAA-NODA, H2DEDPA, H4octapa, Macropa, Pypa, Py4pa, THP, and SarAr.

[0163] Preparation of Conjugates

[0164] The conjugates of the present application can be prepared by any suitable method known in the art. For example, the functional linkers as described herein can be synthesized by liquid phase as well as solid phase synthesis (such as by methods employing 9-fluorenylmethoxycarbonyl (Fmoc) and / or tert-butoxycarbonyl (Boc) chemistry and / or other synthetic methods).

[0165] Solid-phase synthesis methods and techniques are well-established in the art. For example, oligopeptides can be synthesized by sequentially incorporating amino acid residues of interest one by one. In such methods, synthesis typically begins by attaching the C-terminal amino acid of the oligopeptide of interest to a suitable resin. Prior to this, the reactive side chains and α-amino groups of the amino acids are protected by suitable protecting groups to prevent reactions, thereby allowing only the α-carboxyl group to react with a functional group (such as an amine group, a hydroxyl group, or an alkyl halide group) on the solid support. After coupling the C-terminal amino acid to the support, the protecting groups on the side chains and / or α-amino groups of the amino acid are selectively removed, thus allowing the coupling of the next amino acid of interest. This process is repeated until the desired oligopeptide is fully synthesized, at which point the oligopeptide can be cleaved from the support and purified.

[0166] Coupling between each component of the functional linker may require the formation of a thioether (-S-) or ether (-O-) bond and can be achieved either on a solid phase or in a solution phase. For example, the formation of a thioether (-S-) bond can be achieved by coupling a thiol compound (such as the thiol group on the cysteine side chain) and an alkyl halide (such as 3-(Fmoc-amino)propyl bromide) in a suitable solvent (such as N,N-dimethylformamide) in the presence of a base (such as N,N-diisopropylethylamine). The formation of an ether (-O-) bond can be achieved via the Mitsunobu reaction between an alcohol (such as the hydroxyl group on the serine or threonine side chain) and a phenolic group (such as the side chain of tyrosine) in an aprotic solvent (such as 1,4-dioxane) in the presence of triphenylphosphine and diisopropyl azodicarboxylate (DIAD). If the reaction is carried out in the solution phase, the reactants used are preferably in an equimolar ratio (1 to 1), and the desired product can be purified by column chromatography or high-performance liquid chromatography (HPLC). If the reaction is carried out on a solid phase, meaning that one reactant has been attached to the solid phase, the other reactant is typically used in excess (3 equivalents of the reactant attached to the solid phase). After the reaction, the excess unreacted reactants and reagents can be removed, for example, by washing the solid phase (resin) sequentially with a solvent combination (such as N,N-dimethylformamide, methanol, and dichloromethane).

[0167] In some embodiments, each component of the functional linker of the present application further comprises a reactive linking group capable of joining one component of the functional linker to another component, e.g., via a preparation method as described herein. In some embodiments, these components include, but are not limited to, PEG groups, renin-cleavable groups, blood protein-binding groups, spacers, and chelating factor groups. In some embodiments, the reactive linking groups include, but are not limited to, amines, imines, carboxylic acid esters, alcohols, thiols, selenols, phenols, esters, acetones, aldehydes, carbenes, sulfonyl halides, imidoesters, anhydrides, disulfides, maleimides, phosphines, disulfides, alkoxyamines, azides, alkynes, strained alkynes, strained alkenes, halogens, sulfonates, haloacetyls, hydrazides, bisaziridines, phosphines, tetrazines, isothiocyanates, and oxaziridines.

[0168] In some embodiments, the PEG group used in the functional linker comprises a reactive linking group capable of joining the PEG group to other components of the functional linker. In some embodiments, the PEG group comprises two reactive linking groups capable of joining the PEG group to two other components of the functional linker. Exemplary reactive linking groups for the PEG group include, but are not limited to, amines, imines, carboxylic acid esters, alcohols, thiols, selenols, phenols, esters, acetones, aldehydes, carbenes, sulfonyl halides, imidoesters, anhydrides, disulfides, maleimides, phosphines, disulfides, alkoxyamines, azides, alkynes, strained alkynes, strained alkenes, halogens, sulfonates, haloacetyls, hydrazides, bisaziridines, phosphines, tetrazines, isothiocyanates, and oxaziridines.

[0169] In some embodiments, the renin-cleavable group used in the functional linker comprises a reactive linking group capable of joining the renin-cleavable group to other components of the functional linker. In some embodiments, the renin-cleavable group comprises two reactive linking groups capable of joining the renin-cleavable group to two other components of the functional linker. Exemplary reactive linking groups for the renin-cleavable group include, but are not limited to, amines, imines, carboxylic acid esters, alcohols, thiols, selenols, phenols, esters, acetones, aldehydes, carbenes, sulfonyl halides, imidoesters, anhydrides, disulfides, maleimides, phosphines, disulfides, alkoxyamines, azides, alkynes, strained alkynes, strained alkenes, halogens, sulfonates, haloacetyls, hydrazides, bisaziridines, phosphines, tetrazines, isothiocyanates, and oxaziridines.

[0170] In some embodiments, the blood protein binding group (such as an albumin binding group) used in the functional linker comprises a reactive linking group capable of conjugating the blood protein binding group (such as an albumin binding group) to other components of the functional linker. In some embodiments, the blood protein binding group (such as an albumin binding group) comprises two reactive linking groups capable of conjugating the blood protein binding group (such as an albumin binding group) to two other components of the functional linker. Exemplary reactive linking groups for the blood protein binding group (such as an albumin binding group) include, but are not limited to, amine, imine, carboxylate, alcohol, thiol, selenol, phenol, ester, acetone, aldehyde, carbene, sulfonyl halide, imido ester, anhydride, disulfide, maleimide, phosphine, disulfide, alkoxyamine, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, hydrazide, bisaziridine, phosphine, tetrazine, isothiocyanate, and oxaziridine.

[0171] In some embodiments, the chelating factor group used in the functional linker comprises a reactive linking group capable of conjugating the chelating factor group to other components of the functional linker. In some embodiments, the chelating factor group comprises two reactive linking groups capable of conjugating the chelating factor group to two other components of the functional linker. Exemplary reactive linking groups for the chelating factor group include, but are not limited to, amine, imine, carboxylate, alcohol, thiol, selenol, phenol, ester, acetone, aldehyde, carbene, sulfonyl halide, imido ester, anhydride, disulfide, maleimide, phosphine, disulfide, alkoxyamine, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, hydrazide, bisaziridine, phosphine, tetrazine, isothiocyanate, and oxaziridine.

[0172] In some embodiments, the spacer used in the functional linker comprises a reactive linking group capable of conjugating the spacer to other components of the functional linker. In some embodiments, the spacer comprises two reactive linking groups capable of conjugating the spacer to two other components of the functional linker. Exemplary reactive linking groups for the spacer include, but are not limited to, amine, imine, carboxylate, alcohol, thiol, selenol, phenol, ester, acetone, aldehyde, carbene, sulfonyl halide, imido ester, anhydride, disulfide, maleimide, phosphine, disulfide, alkoxyamine, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, hydrazide, bisaziridine, phosphine, tetrazine, isothiocyanate, and oxaziridine.

[0173] In some embodiments, the functional linker comprises a conjugating group capable of linking the functional linker to the sdAb. In some embodiments, the conjugating group is selected from amine, thiol, alcohol, ketone, aldehyde, nitrile, carboxylic acid, ester, alkene, alkyne, anhydride, succinimide, maleimide, phosphine, disulfide, alkoxyamine, azide, alkyl halide, isothiocyanate (NCS), epoxide, isocyanate, hydrazine, and acyl halide.

[0174] In some embodiments, the functional linker may comprise, starting from the attachment site to the sdAb: an optional spacer - PEG group - an optional spacer - renin-cleavable group. In some embodiments, the functional linker may comprise, starting from the attachment site to the sdAb: an optional spacer - renin-cleavable group - an optional spacer - PEG group. In some embodiments, the functional linker may comprise, starting from the attachment site to the sdAb: an optional spacer - PEG group - an optional spacer - blood protein-binding group. In some embodiments, the functional linker may comprise, starting from the attachment site to the sdAb: an optional spacer - renin-cleavable group - an optional spacer - blood protein-binding group. In some embodiments, the functional linker may comprise, starting from the attachment site to the sdAb: an optional spacer - PEG group - an optional spacer - renin-cleavable group - an optional spacer - blood protein-binding group. In some embodiments, the functional linker may comprise, starting from the attachment site to the sdAb: an optional spacer - renin-cleavable group - an optional spacer - PEG group - an optional spacer - blood protein-binding group.

[0175] The functional linker can be conjugated to the sdAb via any suitable method known in the art. In some embodiments, the functional linker is conjugated to the sdAb via an amino acid of the sdAb. In some embodiments, the amino acid can be cysteine, lysine, histidine, aspartate, glutamate, arginine, tyrosine, or serine. In other embodiments, the functional linker is conjugated to the sdAb through a chemical group. The chemical group can be amine, thiol, alcohol, ketone, aldehyde, nitrile, carboxylic acid, ester, alkene, alkyne, anhydride, succinimide, maleimide, phosphine, disulfide, alkoxyamine, azide, alkyl halide, isothiocyanate, epoxide, isocyanate, hydrazine, or acyl halide.

[0176] Therapeutic methods and uses

[0177] In some embodiments, a method for treating a disorder in a subject in need thereof is provided. The method comprises administering to the subject an effective amount of the conjugate of the present application. In some embodiments, the disorder is cancer.

[0178] Exemplary cancers that can be treated by the conjugates of the present application include, but are not limited to, prostate cancer, gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer, breast cancer, thyroid cancer, neuroendocrine cancer, endocrine cancer, esophageal cancer, or leukemia. In some embodiments, the cancer is selected from colorectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and lung cancer.

[0179] In some embodiments, the disease is a disease associated with tumor antigen expression. For example, in some embodiments, the disease (such as cancer) is associated with CLDN18.2. In some embodiments, the disease (such as cancer) is associated with FAP.

[0180] In some embodiments, there is provided the use of any one of the conjugates described herein for the manufacture of a medicament for treating a disease (such as cancer, for example any one of the cancers described herein).

[0181] In some embodiments, there is provided a method of imaging a tumor of a subject, which comprises administering to the subject (such as a human subject) an effective amount of any one of the conjugates described herein. In some embodiments, there is provided a conjugate described herein for imaging a tumor of a subject. In some embodiments, there is provided the use of any one of the conjugates described herein for the manufacture of a medicament for imaging a tumor of a subject.

[0182] Examples

[0183] In order that the present disclosure may be more fully understood, the following examples are presented. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the present disclosure in any way.

[0184] Example 1: Synthesis of the conjugate of the present application

[0185] 1.1 Synthesis of the functional linker

[0186] The linker of the present application is synthesized via solution-phase or solid-phase methods and further characterized by analytical HPLC and LCMS, respectively. Taking L007 as an example, the linker is synthesized via the Figure 1 scheme shown. Additional linkers are synthesized based on similar techniques. The analytical data are summarized in Table 4 below.

[0187] Table 4 Characterization of exemplary synthetic functional linkers

[0188]

[0189] 1.2 Preparation of the SdAb-linker conjugate

[0190] Claudin 18.2 sdAb (SEQ ID NO. 1, CLDN18.2) and FAP sdAb (SEQ ID NO. 5, FAP) were used as exemplary sdAbs for preparing conjugates. Briefly, CLDN18.2 sdAb and FAP sdAb were treated with 1 mM EDTA in 1xPBS at room temperature for 30 min, followed by desalting using a PD-10 (Cytiva, #17085101) column to remove trace metal ions, and then the buffer was exchanged into 50 mM carbonate-bicarbonate (pH 9.5).

[0191] A predetermined amount (3x equivalent of the sdAb sample) of DOTAGA (used as a control for chelating imaging radioisotopes) and the linker were diluted in carbonate-bicarbonate buffer (pH 9.5), and then mixed with the sdAb sample solution with an antibody concentration of 0.5 - 2 mg / mL, respectively. The mixture was incubated at 25 °C for 16 hours. Then, the unconjugated linker was removed using a PD-10 column, and subsequently, an Amicon ultrafiltration device (Merck, #UFC901024) was used to further remove the unconjugated linker and exchange the buffer into 1xPBS to purify the conjugate. Then, the purified conjugate was analyzed by SEC-HPLC, and 100 mM PB buffer (5 g NaH2PO4 and 25 g Na2HPO4 in 1 L of Milli-Q water) was used as the mobile phase. The sample was further analyzed by LC-MS. The DOL (degree of labeling) was calculated based on the LC-MS results, as shown in Table 5 below.

[0192] Table 5 DOL of sdAb-linker conjugates

[0193]

[0194] Example 2: Binding affinity of the conjugate

[0195] The binding affinity of the prepared conjugate to the tumor antigen was evaluated by ELISA. Briefly, 60 ng / 100 μL of human Claudin 18.2-VLP or 50 ng / 100 μL of human FAP-hFc in DPBS buffer was coated onto the ELISA plate and incubated overnight at 4 °C. On the next day, the plate was washed with 0.05% PBST and blocked with 10% StartingBlock buffer (Thermo, #37538) for 1 hour at room temperature. Then, 100 μL of serially diluted conjugate samples in 1% StartingBlock buffer were added to the plate. The plate was further incubated with shaking at 100 rpm for 2 hours at room temperature and washed with 0.05% PBST. Then, 100 μL of 0.4 μg / mL HRP-conjugated anti-sdAb secondary antibody (Jackson, #128-035-232) was added and incubated with shaking at 100 rpm for 1 hour at room temperature. Then, the plate was washed with 0.05% PBST, and the color was developed using TMB solution (KPL, #5120-00820). The absorbance was read at 450 nm and the EC 50 , as shown in Table 6. According to Table 6, the conjugate showed comparable binding affinity to CLDN18.2 sdAb or FAP sdAb conjugated with DOTAGA.

[0196] Table 6 Binding affinity of the conjugate

[0197]

[0198] Example 3: Pharmacokinetic study of CLDN18.2-linker conjugate

[0199] The in vivo pharmacokinetic curve of the CLDN18.2-linker conjugate was analyzed. Briefly, the conjugate was administered to BALB / c mice by intravenous injection at a dose of approximately 1 mg / kg. Then, whole blood of the animals was collected at different time points within 48 hours.

[0200] The whole blood was collected into tubes without anticoagulant and kept at room temperature for more than 30 minutes, followed by centrifugation at approximately 5000 rpm for 5 minutes at 4 °C to obtain serum. Then, the concentration of the conjugate in the serum was measured by ELISA using an HRP-labeled goat anti-lama IgG VHH domain secondary antibody. The data were further analyzed using PK Solver software, and a non-compartmental model (NCA) was used to evaluate the pharmacokinetic parameters, as shown in Table 7 and Figure 2 shown. Figure 2The conjugation with linker 007, linker 009, linker 010 and linker 011 significantly prolonged the blood circulation of the CLDN18.2 conjugate compared to the control group.

[0201] Table 7 Pharmacokinetic curves of CLDN18.2 conjugates

[0202]

[0203] Example 4: Pharmacokinetic study of FAP-linker conjugates

[0204] The in vivo pharmacokinetic curves of FAP-linker conjugates were analyzed. Briefly, the conjugate was administered to BALB / c mice by intravenous injection at a dose of approximately 1 mg / kg. Then, whole blood of the animals was collected at different time intervals within 48 hours.

[0205] The whole blood was collected into tubes without anticoagulant and kept at room temperature for more than 30 minutes, and then centrifuged at approximately 5000 rpm for 5 minutes at 4°C to obtain serum. Then, the concentration of the conjugate in the serum was measured by ELISA using an HRP-labeled goat anti-lama IgG VHH domain secondary antibody. The data was further analyzed using PK Solver software, and a non-compartmental model (NCA) was used to evaluate the pharmacokinetic parameters, as shown in Table 8 and Figure 3 as shown. As Figure 3 shown, the conjugation with linker 007 significantly prolonged the blood circulation of the FAP conjugate compared to the control group.

[0206] Table 8 Pharmacokinetic curves of FAP-linker conjugates

[0207]

[0208] Example 5: 111 In vivo SPECT imaging of In-labeled CLDN18.2 conjugates

[0209] Via 111 In labeling, the in vivo biodistribution of the CLDN18.2 conjugate was analyzed by SPECT imaging. Briefly, the CLDN18.2 conjugate was labeled with In at 30°C for 60 min and purified by a NAP-5 column, and then the radiochemical purity was analyzed by radio-TLC and radio-HPLC on the day of labeling. The release standard for the radiolabeled sample was ≥ 90% radiochemical purity. 111 For the imaging study, female athymic nude mice (approximately 6 - 8 weeks old) were each inoculated with 5×10

[0210] via subcutaneous injection into the shoulder 6CLDN18.2 HEK293T cells. When the tumor volume reached approximately 200 to 300 mm 3 at that time, the animals were injected with the labeled conjugate at a dose of 10 MBq / mouse by a single intravenous bolus injection through the tail vein. Then the animals were subjected to whole-body static SPECT at different time points under isoflurane anesthesia, followed by CT. As Figure 4 shown, compared with the control group, functional linkers 007 and 009 significantly increased the retention of the CLDN18.2 conjugate in the tumor within 48 hours. In addition, the tumor / kidney ratio increased to approximately 5 - 10 times compared with the control group.

[0211] Example 6: 177 In vivo SPECT imaging of Lu-labeled FAP conjugate

[0212] Via 177 Lu-labeling, the in vivo biodistribution of the FAP conjugate was analyzed by SPECT imaging. Briefly, the FAP conjugate was labeled with 177 Lu at a molar activity of 1 MBq / μg at 37 °C for 60 min and purified by size exclusion chromatography, and then the radiochemical purity was analyzed by radio-TLC and radio-HPLC on the day of labeling. The release criteria for the radiolabeled sample were ≥ 90% radiochemical purity.

[0213] For the imaging study, female athymic nude mice (approximately 6 - 8 weeks old) were each inoculated with 5×10 6 HEK293-hFAP cells expressing FAP via subcutaneous injection into the shoulder. When the tumor volume reached approximately 150 to 250 mm 3 at that time, the animals were injected with the labeled conjugate at a dose of 30 MBq / mouse by a single intravenous bolus injection through the tail vein. Then the animals were subjected to whole-body static SPECT at different time points under isoflurane anesthesia, followed by CT. Figure 5 It was demonstrated that functional linker 007 was able to increase the retention of the FAP conjugate in the tumor at different time points. In addition, 168 hours after administration, functional linker 007 further increased the tumor / kidney (T / K) ratio from 1:5 to 1:1.

[0214] The present disclosure should not be limited in scope by the specific embodiments described, which are intended as a single illustration of separate aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Accordingly, the present disclosure is intended to cover modifications and variations of the present disclosure provided that such modifications and variations are within the scope of the appended claims and their equivalents.

[0215] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0216] The present invention has been described in terms of specific embodiments, which have been found or proposed by the inventors of the present invention as including preferred ways of practicing the present invention. Those skilled in the art will understand that, in light of the present disclosure, many modifications and changes can be made to the specific embodiments illustrated without departing from the intended scope of the present invention. For example, due to codon redundancy, changes can be made to the underlying DNA sequence without affecting the protein sequence. Additionally, for biological functional equivalence considerations, changes can be made to the protein structure without affecting the type or amount of biological action. All such modifications are intended to be included within the scope of the appended claims.

Claims

1. A conjugate, comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the sdAb, wherein when the conjugate is administered to a subject, the functional linker increases the tumor uptake, accumulation, and / or retention of the drug compared to a conjugate without the functional moiety.

2. A conjugate, comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the sdAb, wherein when the conjugate is administered to a subject, the functional linker decreases the renal uptake, accumulation, and / or retention of the drug compared to a conjugate without the functional moiety.

3. A conjugate, comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the sdAb, wherein when the conjugate is administered to a subject, the functional linker increases the tumor / kidney ratio of the drug compared to a conjugate without the functional moiety.

4. A conjugate, comprising: (a) a single domain antibody (sdAb) that specifically binds to a tumor antigen; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the sdAb, wherein when the conjugate is administered to a subject, the functional linker prolongs the blood circulation and / or half-life of the drug compared to a conjugate without the functional moiety.

5. A conjugate, comprising: (a) an antibody mimetic that specifically binds to a tumor antigen, wherein the antibody mimetic is selected from affibody, DARPin, Anticalin, Avimer, Versabody, or Duocali; (b) a drug; and (c) a functional linker comprising a functional moiety that links the drug to the antibody mimetic, wherein when the conjugate is administered to a subject, the functional linker is capable of: (i) increasing the tumor uptake, accumulation, and / or retention of the drug, (ii) decreasing the renal uptake, accumulation, and / or retention of the drug, (iii) increasing the tumor / kidney ratio of the drug, (iv) prolonging the blood circulation of the drug, and / or (v) prolonging the half-life of the drug compared to a conjugate without the functional moiety.

6. The conjugate according to any one of claims 1-5, wherein the functional moiety comprises one or more components selected from the group consisting of polyethylene glycol (PEG) groups, renin-cleavable groups, blood protein-binding groups, spacers, and any combination thereof.

7. The conjugate according to claim 6, wherein the PEG group comprises and wherein n is any integer in the range from about 1 to about 25.

8. The conjugate according to any one of claims 1-7, wherein the functional moiety comprises the blood protein-binding group.

9. The conjugate according to claim 8, wherein the blood protein is selected from albumin, fetuin, transferrin, and IgG.

10. The conjugate according to claim 9, wherein the blood protein binding group comprises an albumin binding group.

11. The conjugate according to claim 10, wherein the albumin-binding group comprises a structure of, where n is any integer ranging from 1 to 20, and R m is H, CH3, or COOH.

12. The conjugate according to claim 10 or 11, wherein the albumin-binding group comprises a structure of, wherein R x is selected from: , , , , , , , , , , , , and wherein n is any integer ranging from 1 to 6, and R x1 , R x2 , R x3 , R x4 , R x5 , R x6 and R x7 each independently is selected from H, N, S, O, Se, P, halogen, C 5-20 aryl group, C 1-20 alkyl group, C 2-20 alkenyl group and C 2-20 alkynyl group, and wherein: (i) said C 5-20 aryl group, said C 1-20 alkyl group, said C 2-20 alkenyl group or said C 2-20 alkynyl group is unsubstituted or substituted with one or more substituents selected from N, S, O, Se, P, and halogen atoms, (ii) said C 5-20 aryl group, said C 1-20 alkyl group, said C 2-20 alkenyl group or said C 2-20 0, 1 or 2 carbon atoms in the alkynyl group are replaced by a group selected from C 6-10 arylene, 5- to 10-membered heteroarylene group, C 3-7 cycloalkylene, 5- to 10-membered heterocycloalkylene group, and wherein said arylene, heteroarylene, carbocycloalkylene and heterocycloalkylene groups are unsubstituted or are substituted by one or more substituents selected from N, S, O, Se, P, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthiol, -N(C 1-6 alkyl)(C 1-6 alkyl), nitro and sulfonic acid groups, and / or (iii) said C 5-20 aryl group, said C 1-20 alkyl group, said C 2-20 alkenyl group or said C 2-20 alkynyl group, 0, 1 or 2 -CH- or -CH2- groups in said group are replaced by a group selected from -O-, -S-, -S-S-, -C(O)- and -N(C 1-6 alkyl)- group.

13. The conjugate according to claim 10, wherein the albumin-binding group comprises a structure of, wherein R y is selected from H, F, Cl, Br, I, -CH3, -OCH3, COOH, -CF3, and n is any integer ranging from 1 to 6.

14. The conjugate according to any one of claims 1-13, wherein the functional moiety comprises the renin-cleavable group.

15. The conjugate according to claim 14, wherein the renin-cleavable group is selected from brush border enzyme-cleavable groups, lysosome-cleavable enzyme-cleavable groups, and combinations thereof.

16. The conjugate according to claim 14 or 15, wherein the renin-cleavable group comprises a dipeptide or oligopeptide.

17. The conjugate according to claim 16, wherein the dipeptide or oligopeptide comprises a structure selected from: methionine-isoleucine, glycine-lysine, glycine-phenylalanine-lysine, methionine-valine-lysine, glycine-tyrosine, glycine-lysine-lysine, glycine-arginine-lysine, aspartic acid-glycine-lysine, methionine-glycine-lysine, methionine-isoleucine-lysine, glycine-tyrosine-lysine, glycine-valine, glycine-isoleucine, methionine-phenylalanine-lysine, glycine-(3-(2-naphthyl)alanine)-lysine, glycine-diphenylalanine-lysine, methionine-glycine-lysine, and any derivatives thereof.

18. The conjugate according to any one of claims 1-17, wherein the functional linker comprises a spacer.

19. The conjugate according to claim 18, wherein the spacer comprises a structure selected from the following: natural amino acid residues, unnatural amino acid residues, C 5-20 aryl groups, C 1-20 alkyl groups, C 2-20 alkenyl groups and C 2-20 alkynyl groups, and wherein: (i) said C 5-20 aryl group, said C 1-20 alkyl group, said C 2-20 alkenyl group or said C 2-20 alkynyl group is unsubstituted or substituted by one or more substituents selected from N, S, O, Se, P and halogen atoms, (ii) said C 5-20 aryl group, said C 1-20 alkyl group, said C 2-20 alkenyl group or said C 2-20 0, 1 or 2 carbon atoms in the alkynyl group are replaced by a group selected from C 6-10 arylene, 5- to 10-membered heteroarylene group, C 3-7 cycloalkylene, 5- to 10-membered heterocycloalkylene group, and wherein said arylene, heteroarylene, carbocycloalkylene and heterocycloalkylene groups are unsubstituted or substituted by one or more substituents selected from N, S, O, Se, P, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthiol, -N(C 1-6 alkyl)(C 1-6 alkyl), nitro and sulfonic acid groups, and / or (iii) said C 1-20 alkyl group, said C 2-20 alkenyl group or said C 2-20 alkynyl group, 0, 1 or 2 -CH- or -CH2- groups in the group are replaced by a group selected from -O-, -S-, -S-S-, -C(O)- and -N(C 1-6 alkyl)- group.

20. The conjugate according to claim 19, wherein the amino acid residue is selected from lysine, aspartate, asparagine, diaminobutyric acid, phenylalanine, tyrosine, threonine, serine, proline, leucine, isoleucine, valine, arginine, histidine, glutamate, glutamine, and alanine.

21. The conjugate according to any one of claims 1-20, wherein the functional linker further comprises a chelating factor group.

22. The conjugate according to claim 21, wherein the chelating factor group comprises a structure selected from: NODASA, NODAGA, TETA, TRITA, TRAP, DTPA, CHX-DTPA, EDTA, CDTA, CPTA, DOTP, DOTPI, EGTA, HBED, TTHA, DTPA, DOTA, DOTAGA, NOTA, HP-DOA3, CBTE2a, TE2A, TMT, DPDP, HYNIC, DFO, HEDTA, NOPO, MAG3, NCS-MP-NODA, NH2-MPAA-NODA, H2DEDPA, H4octapa, Macropa, Pypa, Py4pa, THP, and SarAr.

23. The conjugate according to any one of claims 1-22, wherein the functional linker is conjugated to the sdAb or antibody mimetic via an amino acid of the sdAb or antibody mimetic.

24. The conjugate according to claim 23, wherein the amino acids of the sdAb or antibody mimetic used for conjugation are selected from cysteine, lysine, histidine, aspartate, glutamate, glutamine, arginine, tyrosine, tryptophan, threonine, and serine.

25. The conjugate according to any one of claims 1-24, wherein the functional linker comprises a conjugation group capable of conjugating with the sdAb or antibody mimetic.

26. The conjugate according to claim 25, wherein the conjugation group is selected from amine, thiol, alcohol, ketone, aldehyde, nitrile, carboxylic acid, ester, alkene, alkyne, anhydride, succinimide, maleimide, phosphine, disulfide, alkoxyamine, azide, alkyl halide, isothiocyanate (NCS), epoxide, isocyanate, hydrazine, and acyl halide.

27. The conjugate according to any one of claims 1-26, wherein the tumor antigen is selected from prostate-specific membrane antigen (PSMA), fibroblast activation protein α (FAP-α, FAP), folate receptor, luteinizing hormone-releasing hormone (LHRH), norepinephrine transporter (NAT), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-2 (HER-2), vascular endothelial growth factor (VGFR), mucin-1 (MUC-1), mucin-4 (MUC-4), urokinase-type plasminogen activator receptor (uPAR), tumor-associated glycoprotein 72 (TAG-72), Claudin 18.2 (CLDN18.2), vascular endothelial growth factor receptor (VEGFR), C-X-C chemokine receptor type 4 (CXCR4), Hepsin, TMPRSS2, and cMET.

28. The conjugate according to claim 27, wherein the tumor antigen is CLDN18.

2.

29. The conjugate according to claim 28, wherein the sdAb comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO.

1.

30. The conjugate according to claim 28, wherein the sdAb comprises: (1) CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2 and an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 2, (2) CDR2 comprising the amino acid sequence shown in SEQ ID NO. 3 and an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 3, and (3) CDR3 comprising the amino acid sequence shown in SEQ ID NO. 4 and an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO.

4.

31. The conjugate according to claim 27, wherein the tumor antigen is FAP.

32. The conjugate according to claim 31, wherein the sdAb comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO.

5.

33. The conjugate according to claim 31, wherein the sdAb comprises: (1) a CDR1 comprising the amino acid sequence shown in SEQ ID NO. 6 and an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 6, (2) a CDR2 comprising the amino acid sequence shown in SEQ ID NO. 7 and an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 7, and (3) a CDR3 comprising the amino acid sequence shown in SEQ ID NO. 8 and an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO.

8.

34. The conjugate according to any one of claims 1-33, wherein the drug is selected from chemotherapeutic agents, toxins, cytokines, enzymes, immunomodulators, chelate complexes, diagnostic agents, nanoparticles, and radioisotopes.

35. The conjugate according to claim 34, wherein the drug is a radioisotope selected from the following: bismuth-213, cesium-131, cesium-137, cesium-131, cobalt-60, holmium-166, iodine-125, iodine-131, iridium-192, lead-212, lutetium-177, palladium-103, phosphorus-32, potassium-42, radium-223, rhenium-186, rhenium-188, samarium-153, scandium-47, selenium-75, sodium-24, strontium-89, technetium-99m, thorium-227, xenon-133, ytterbium-169, ytterbium-177, yttrium-90, actinium-225, astatine-211, bismuth-213, carbon-11, nitrogen-13, oxygen-15, fluorine-18, cobalt-57, copper-64, copper-67, gallium-67, gallium-68, indium-111, iodine-123, iodine-124, krypton-81m, rubidium-82, strontium-82, thallium-201, and zirconium-89.

36. A method for treating a disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the conjugate according to any one of claims 1-35.

37. The method according to claim 36, wherein the disorder is cancer.

38. A method for preparing the conjugate according to any one of claims 1-35, the method comprising conjugating the functional linker and the drug to the sdAb.

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