WO2025080751A2 - Protéines de liaison à l'antigène anti-cdh17 et leurs utilisations - Google Patents

Protéines de liaison à l'antigène anti-cdh17 et leurs utilisations Download PDF

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WO2025080751A2
WO2025080751A2 PCT/US2024/050633 US2024050633W WO2025080751A2 WO 2025080751 A2 WO2025080751 A2 WO 2025080751A2 US 2024050633 W US2024050633 W US 2024050633W WO 2025080751 A2 WO2025080751 A2 WO 2025080751A2
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seq
amino acid
acid sequence
antigen
binding protein
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WO2025080751A3 (fr
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Luigi Franchi
Anthony W. Opipari
Elaine Maria PINHEIRO
Robert Thomas ABRAHAM
Laura PREISS
Ferdinand Huber
Paul-Albert KÖNIG
Tamara DAKE
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Odyssey Therapeutics Inc
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Odyssey Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application relates to antigen-binding proteins (e.g., antibodies such as singledomain antibodies) that specifically bind cadherin 17 (CDH17), methods for their preparation, and uses thereof.
  • antigen-binding proteins e.g., antibodies such as singledomain antibodies
  • CDH17 cadherin 17
  • CDH17 contains an RGD (Arg-Gly-Asp) motif through which it interacts with integrin a201, and this interaction has been associated with especially aggressive forms of cancer during late-stage metastasis. Accordingly, there is a need in the art to develop high-affinity molecules which can efficiently target and specifically bind cadherin 17 (CDH17) expressed in tumor cells while having minimal off-tumor toxicity.
  • RGD Arg-Gly-Asp
  • ALCLLRFETCLEYNRAQYPY (SEQ ID NO: 3); AAVRSGSDWWTTMTQRHYDF (SEQ ID NO: 8);
  • VATGNTYRGAYDRPAEYDF SEQ ID NO: 29
  • AAQFSLPVDASPLRRYYY SEQ ID NO: 34
  • the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 9, 14, 19, 23, 26, 30, 35, 40, 45, 50, 55, 60, 65, 187, and 1619-1622, or an amino acid sequence having at least 75% identity thereto.
  • the antigen-binding protein binds to cyno CDH17 with a K D of about
  • the antigen-binding protein binds to mouse CDH17.
  • the antigen-binding protein binds to the extracellular cadherin domain 6
  • the antigen-binding protein binds to an extracellular region C-terminal to the tripeptide motif, RGD, in EC6 of human CDH17. [0033] In some embodiments, the antigen-binding protein binds to the extracellular cadherin domain 1 (ECI) of human CDH17.
  • ECI extracellular cadherin domain 1
  • the antigen-binding protein comprises one or more modifications that reduce binding of said antigen-binding protein by pre-existing antibodies found in human blood or serum.
  • fusion protein that specifically binds CDH17, comprising one or more of any of various antigen-binding proteins described herein.
  • the fusion protein comprises three or more said antigen-binding proteins.
  • the one or more antigen-binding proteins bind to the same epitope on CDH17.
  • the one or more antigen-binding proteins are one or more single-domain antibodies.
  • one or more single-domain antibodies are one or more VHHs.
  • the fusion protein described herein further comprises an immunoglobulin Fc region.
  • the immunoglobulin Fc region is an Fc region of a human immunoglobulin. In some embodiments, the immunoglobulin Fc region is an Fc region of human IgGl, lgG2, lgG3 or lgG4, or a variant thereof.
  • the immunoglobulin Fc region is an Fc region of human IgGl, or a variant thereof.
  • the Fc region of human IgGl comprises one or more mutations selected from Leu234Ala (L234A), Leu234Gly (L234G), Leu234Ser (L234S), Leu234Thr (L234T), Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Leu235Ser (L235S), Leu235Thr (L235T), Leu235Val (L235V), Leu235Gln (L235Q), Gly236Arg (G236R), Met252Tyr (M252Y), Ser254Thr (S254T), Thr256Glu (T256E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329
  • the Fc region of human IgGl comprises a set of mutations selected from 1). L234A and L235A; 2). L234A, L235A, and P329A;
  • the Fc region of human lgG4 comprises a set of mutations selected from
  • a recombinant vector comprising a polynucleotide molecule described herein.
  • the cancer is a solid tumor.
  • the cancer is selected from colorectal cancer, familial GIST, familial pancreatic cancer, gastrointestinal stromal tumor (GIST), hereditary diffuse gastric cancer, hereditary pancreatitis, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the pancreas, peritoneal cancer, pancreatic cancer, small bowel cancer, and stomach cancer.
  • the cancer is gastrointestinal cancer.
  • the gastrointestinal cancer is colorectal cancer, gastric cancer, esophageal cancer, or pancreatic cancer.
  • the method further comprises administering one or more additional therapeutic agents.
  • the EGFR inhibitor is selected from cetuximab and/or panitumumab.
  • the apoptosis-inducing agent is a B-cell lymphoma 2 (BCL2) inhibitor, a BCL-extra large (BCL-XL) inhibitor, or an inhibitor of apoptosis proteins (IAP) inhibitor, or a combination thereof.
  • the immunotherapeutic agent is an anti-CTLA4 agent, anti-PDl agent, anti-PD-Ll agent, anti-LAG3 agent, or anti-TIM3 agent, or a combination thereof.
  • the subject is a mammal. In some embodiments, the mammal is human.
  • Figure 1 depicts an exemplary immune library construction and general panning strategy for discovery of CDH17 binders.
  • Figure 2 shows sample selection for next generation sequencing (NGS) throughout the phage display process.
  • NGS next generation sequencing
  • FIG. 3 shows a schematic diagram of an exemplary NGS workflow.
  • the VHH region of the phage elutions was polymerase chain reaction (PCR) amplified, unique and sample-specific barcodes were fused, and NGS was performed with the Illumina NovaSeq platform from Genewiz.
  • the raw data were demultiplexed, and then processed by the Pipebio NGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions and the VHHs, including CDRs, were annotated. Based on CDR3 identity, V-body sequences were clustered, allowing for a detailed analysis of V-body enrichment during phage display, sequence diversity, CDR3 length distribution and cluster abundance.
  • Extracellular cadherin (EC) repeats are shown positioned superior to the membrane. N-glycans and calcium-binding sites are shown as stars and black dots, respectively.
  • the structure prediction of CDH17 was downloaded from alphafold.ebi.ac.uk/. Information about the amino acid position and domain organization were extracted from Uniport.
  • Figures 5A-5E illustrate on-cell binding data.
  • Figure 5A illustrates on-cell binding of anti-CDH17 V-body monomers to GP2d cell line.
  • Figure 5B illustrates on-cell binding of anti-CDH17 V-body monomers to Colo205 cell line.
  • Figure 5C illustrates on-cell binding of anti-CDH17 V-body monomers to DLD-1 cell line.
  • Figure 5D illustrates on-cell binding of anti-CDH17 V-body monomers to T84 cell line.
  • Figure 5E illustrates on-cell binding of anti-CDH17 V-body monomers to HCT-15 cell line.
  • Figure 6 shows a schematic diagram of an exemplary experimental setup for determination of epitope binning.
  • Figure discloses "HHHHHH” as SEQ ID NO: 1838.
  • Figure 7 shows epitope binning of the V-bodies.
  • Figure 8 shows a summary of fluorescence activated cell sorting (FACS) domain mapping.
  • the term "about,” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 5%.
  • the expression “about 100” includes 95 and 105 and all values in between (e.g., 96, 97, 98, 99, etc.).
  • antigen encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor.
  • the antigen described herein is CDH17, including human, cynomolgus (cyno), and/or mouse CDH17.
  • epitope can refer to an antigenic determinant on the surface of an antigen to which an antibody molecule binds.
  • a single antigen may have more than one epitope.
  • different antibodies may bind to different areas on an antigen and may have different biological effects (e.g., agnostic or antagonistic effects).
  • Epitopes may be either conformational or linear.
  • a conformational epitope is formed by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is formed by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include non-peptidic moieties on the antigen, such as saccharides, phosphoryl groups, or sulfonyl groups.
  • an antigen-binding protein refers in its broadest sense to a protein that specifically binds an antigen (e.g., CDH17).
  • an antigen-binding protein is an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single chain antibody (e.g., single chain fragment variable (scFv)); a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab') 2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an lgG2 antibody; an lgG3 antibody; or an lgG4 antibody, and fragments thereof.
  • antigen-binding protein also encompasses, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
  • Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer.
  • peptide antibody mimetics can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components (e.g., fibronectin type III domain (FN3)) as a scaffold.
  • fibronectin components e.g., fibronectin type III domain (FN3)
  • antibody and "immunoglobulin” or “Ig” are used interchangeably herein, and is used in the broadest sense and encompasses, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies (e.g., VHH), single chain antibodies, intrabodies, anti-idiotypic (anti-ld) antibodies, and antigen-binding fragments of antibodies, as described below.
  • an antibody can be human, humanized, camelized, recombinantly produced, chimeric, synthetic, affinity de-matured and/or affinity matured as well as an antibody from other species, for example mouse, camel, llama, rabbit, etc.
  • the specific target antigen that can be bound by an antibody provided herein includes a CDH17 polypeptide, CDH17 fragment or CDH17 epitope.
  • An "antigen-binding fragment" generally refers a portion of an antibody heavy and/or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • Non-limiting examples of antigen-binding fragments include single-domain antibody (e.g., VHH), single-chain Fvs (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody, or a chemically modified derivative thereof.
  • antibodies provided herein include immunoglobulin molecules and molecules that contain immunologically active portion(s) of an immunoglobulin molecule, for example, one or more complementarity determining regions (CDRs) of an antibody that binds to CDH17.
  • CDRs complementarity determining regions
  • Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189- 224 (1993); Pliickthun and Skerra, Meth. EnzymoL, 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990).
  • the antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2), or any subclass (e.g., lgG2a and lgG2b) of immunoglobulin molecule.
  • any type e.g., IgG, IgE, IgM, IgD, IgA and IgY
  • any class e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2
  • any subclass e.g., lgG2a and lgG2b
  • single-domain antibody refers to an antibody or antibody fragment containing a single antibody variable domain that is able to bind to a specific antigen alone, without the requirement of another antibody variable domain.
  • the complementarity determining regions (CDRs) of a single-domain antibody are part of a single antibody variable domain.
  • single-domain antibodies include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered antibodies, variable domains derived from the aforementioned antibodies, and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, shark, goat, rabbit, and/or bovine.
  • a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains.
  • the variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four-chain immunoglobulins.
  • Such a VHH molecule can be derived from antibodies raised in Camelidae species, e.g., camel, llama, dromedary, alpaca and guanaco.
  • a single-domain antibody may be obtained from a Camelidae VH domain.
  • a single-domain antibody may be obtained from human VH by camelization. See Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which being incorporated by reference, for review of single-domain antibodies.
  • the term "specifically binds" as used herein means that an antigen-binding protein forms a complex with a target antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a dissociation constant (K D ) of about 1x10 6 M or less (e.g., less than 10 5 M, less than 5xl0 -7 M, less than 10' 7 M, less than 5xl0 -8 M, less than 10' 8 M, less than 5xl0' 9 M, less than 10' 9 M, or less than IO 10 M).
  • K D dissociation constant
  • an antigen-binding protein e.g., an antibody or an antibody fragment
  • a target antigen e.g., an antibody or an antibody fragment
  • surface plasmon resonance e.g., BIACORE® assays
  • bio-layer interferometry e.g., bio-layer interferometry
  • ligand binding assays e.g., enzyme-linked immunosorbent assay (ELISA)
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescent-activated cell sorting
  • flow cytometry-based binding assays and the like e.g., specific binding to a particular target antigen from a certain species does not exclude that the antigen-binding protein can also specifically bind to the analogous target from a different species.
  • specific binding to human CDH17 does not exclude that the antigen-binding protein can also specifically bind to CDH17 from cynomolgus monkeys (“cyno") or mice.
  • isolated when used in the context of antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides, and vectors, means the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides and vectors are at least partially free of other biological molecules from the cells or cell culture from which they are produced.
  • biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium.
  • An isolated antigen-binding protein may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof.
  • the term “isolated” is not intended to refer to a complete absence of such biological molecules (e.g., minor or insignificant amounts of impurity may remain) or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies).
  • the term "operably linked” as used herein can refer to a functional relationship between two or more regions of a polypeptide chain in which the two or more regions are linked so as to produce a functional polypeptide.
  • the term "variant”, “derivative” or “derived from” in the context of proteins or polypeptides refer to: (a) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the polypeptide it is a variant or derivative of; (b) a polypeptide encoded by a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleotide sequence encoding the polypeptide it is a variant or derivative of; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and/or
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FAST A, BLAST or Gap, as discussed below.
  • a nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
  • the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference.
  • Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur- containing side chains are cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamateaspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM 250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443- 1445, herein incorporated by reference.
  • a "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1.
  • FASTA e.g., FASTA2 and FASTA3
  • FASTA2 and FASTA3 provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra).
  • Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.
  • the terms “enhance” or “promote,” or “increase,” or “expand,” or “improve” refer generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition.
  • a measurable physiological response may include an increase in immune cell expansion, activation, effector function, persistence, and/or an increase in tumor cell death killing ability, among others apparent from the understanding in the art and the description herein.
  • an "increased” or “enhanced” amount can be a "statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.
  • a “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refer generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition.
  • a “decrease” or “reduced” amount can be a "statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle or a control composition.
  • the terms "treat” or "treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub- clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms.
  • the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
  • an effective amount refers to a quantity and/or concentration of a composition containing an active ingredient (e.g., anti-CDH17 antigen-binding protein) that when administered into a patient either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a significant decrease in disease progression as, for example, by ameliorating or eliminating symptoms and/or the cause of the disease.
  • An effective amount may be an amount that relieves, lessens, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents progression of the disease or disorder, or improves physical functioning of the patient.
  • a therapeutically effective amount of a composition containing an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects.
  • a therapeutically effective amount may be delivered in one or more administrations.
  • a therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic and/or prophylactic result.
  • the terms "individual”, “subject” and “patient” are used interchangeably herein to refer to an animal; for example a mammal.
  • the terms include human and veterinary subjects.
  • methods of treating mammals including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided.
  • the subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • a subject can be a subject in need of treatment for a disease or disorder.
  • the subject is a human.
  • antigen-binding proteins e.g., antibodies, such as single-domain antibodies
  • CDH17 cadherin 17
  • Cadherin 17 is a calcium-dependent cell adhesion glycoprotein belonging to the 7D cadherin subfamily within the cadherin superfamily. It consists of seven extracellular cadherin (EC) repeats with a total length of 764 amino acids, a single transmembrane helix and a very short intracellular domain of only 23 amino acids, lacking binding sites for interactions with the cytoskeleton proteins such as catenins. CDH17 is predicted to harbor eight N-glycans located throughout all ECs except for ECI, with several of them confirmed experimentally.
  • CDH17 exhibits the typical canonical calcium binding motif that coordinates three calcium ions, which were shown to contribute to the rigidity of the protein.
  • CDH17 Similar to other cadherins, CDH17 contributes to cell adhesion via trans-interaction with cadherins of neighboring cells. However, instead of dimerizing via ECI and EC2, it was shown that in CDH17 EC2 of one chain interacts with EC4 of a second chain. Further it was suggested that additional EC domains may be involved in the dimerization.
  • CDH17 comprises an RGD (Arg-Gly-Asp) tripeptide motif in EC6, through which the protein interacts with integrin a2[31. This CDH17 a201 interaction was associated with aggressive forms of cancer during late-stage metastasis. The soluble form of CDH17 was reported to contain the RGD motif suggesting proteolytic cleavage C-terminal of EC6 or even more proximal to the membrane.
  • RGD Arg-Gly-Asp
  • antigen-binding proteins that specifically bind to cadherin 17 (CDH17) described herein can act as an anchor and/or a guide to a target cancer cell, while avoiding impact on CDH17 biology. Since CDH17 is not expressed on normal adult hepatocytes, CDH17 can be used as a targeting antigen so as to avoid liver toxicity that has plagued various therapeutic molecules aimed at targeting cancer.
  • An anti-CDH17 antigen-binding protein of the present disclosure can target membrane proximal epitopes of CDH17 in order to not impair its dimerization. In some embodiments, membrane proximal targeting of can avoid binding to potentially shed CDH17 thus retaining targeting to tumor cells.
  • binding of the anti-CDH17 antigen-binding proteins of the present disclosure to the integrin binding tripeptide RGD motif in extracellular cadherin domain 6 (EC6) of human CDH17 can be avoided.
  • anti-CDH17 antigen-binding proteins described herein can specifically bind to CDH17 from human, cyno and/or mouse.
  • a human CDH17 is encoded by a CDH17 gene (NCBI Gene ID 1015; AA Sequence: >sp
  • a cyno CDH17 is encoded by a CDH17 gene (NCBI Gene ID 102129651 ;
  • a mouse CDH17 is encoded by a Cdhl7 gene (NCBI Gene ID 12557; AA Sequence: >sp
  • antigen-binding proteins of the present disclosure bind to human CDH17.
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • a K D of less than about 1 ⁇ 10 ⁇ 6 M for example, less than about 5 ⁇ 10 ⁇ 7 M, less than about 3 ⁇ 10 ⁇ 7 M, less than about 1 ⁇ 10 ⁇ 7 M, less than about 8 ⁇ 10 ⁇ 8 M, less than about 5 ⁇ 10 ⁇ 8 M, less than about 3 ⁇ 10 ⁇ 8 M, less than about 1 ⁇ 10 ⁇ 8 M, less than about 8 ⁇ 10 ⁇ 9 M, less than about 5 ⁇ 10 ⁇ 9 M, less than about 3 ⁇ 10 ⁇ 9 M, or less than about 1 ⁇ 10 ⁇ 9 M, or about 1 ⁇ 10 ⁇ 10 to 1 ⁇ 10 ⁇ 9 M, 1 ⁇ 10 ⁇ 10 to 5 ⁇ 10 ⁇ 9 M, about 1 ⁇ 10 ⁇ 10 to 1 ⁇ 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 10 to 5 ⁇ 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 9 to 1 ⁇ 10 ⁇ 10 ⁇ 9 to 1 ⁇ 10 ⁇ 10 ⁇ 9 M,
  • antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human CDH17 with a K D of less than about 3 ⁇ 10 ⁇ 7 M. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human CDH17 with a K D of less than about 2.2 ⁇ 10 ⁇ 7 M. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human CDH17 with a K D of less than about 1 ⁇ 10 ⁇ 7 M.
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins may bind to human CDH17 with a K D of about 1.54 x 10 -7 M.
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins of the present disclosure bind to cynomolgus monkey (“cyno”) CDH17.
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • a K D of less than about 1 ⁇ 10 ⁇ 6 M for example, less than about 5 ⁇ 10 ⁇ 7 M, less than about 3 ⁇ 10 ⁇ 7 M, less than about 1 ⁇ 10 ⁇ 7 M, less than about 8 ⁇ 10 ⁇ 8 M, less than about 5 ⁇ 10 ⁇ 8 M, less than about 3 ⁇ 10 ⁇ 8 M, less than about 2 ⁇ 10 ⁇ 8 M, less than about 1 ⁇ 10 ⁇ 8 M, less than about 8 ⁇ 10 ⁇ 9 M, less than about 5 ⁇ 10 ⁇ 9 M, less than about 3 ⁇ 10 ⁇ 9 M, or less than about 1 ⁇ 10 ⁇ 9 M, or about 1 ⁇ 10 ⁇ 10 to 1 ⁇ 10 ⁇ 9 M, 1 ⁇ 10 ⁇ 10 to 5 ⁇ 10 ⁇ 9 M, about 1 ⁇ 10 ⁇ 10 to 1 ⁇ 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 10 to 5 ⁇ 10 ⁇ 10 ⁇ 9 M, about 1 ⁇ 10 ⁇ 10 to 1 ⁇ 10 ⁇ 8 M
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins may bind to cyno CDH17 with a K D of about 1.1 x 10 -8 M.
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno CDH17 with a K D of about 5.8 x 10 -9 M.
  • antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno CDH17 with a K D of less than about 3 ⁇ 10 ⁇ 7 M.
  • antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno CDH17 with a K D of about 1 ⁇ 10 ⁇ 9 to 1 ⁇ 10 ⁇ 7 M.
  • antigen- binding proteins of the present disclosure may bind to mouse CDH17 with a K D of less than about 2 ⁇ 10 ⁇ 7 M (e.g., about 160 nM, 120 nM, 115 nM, 110 nM, 100 nM, 95 nM, 90 nM, 75 nM, 50 nM, 45 nM, 30 nM, 20 nM, or less).
  • antigen-binding proteins of the present disclosure may bind to mouse CDH17 with a K D of about 1x10 -6 M.
  • antigen-binding proteins of the present disclosure may bind to mouse CDH17 with a K D of about 7x10 -8 M.
  • antigen- binding proteins of the present disclosure may bind to mouse CDH17 with a K D of about 5.2x10 -8 M. [0122] In some embodiments, antigen-binding proteins of the present disclosure do not bind to mouse CDH17. [0123] In some embodiments, antigen-binding proteins of the present disclosure bind to rat CDH17.
  • antigen-binding proteins of the present disclosure may bind to rat CDH17 with a K D of less than about 1 ⁇ 10 ⁇ 6 M, for example, less than about 5 ⁇ 10 ⁇ 7 M, less than about 3 ⁇ 10 ⁇ 7 M, less than about 1 ⁇ 10 ⁇ 7 M, less than about 8 ⁇ 10 ⁇ 8 M, less than about 5 ⁇ 10 ⁇ 8 M, less than about 3 ⁇ 10 ⁇ 8 M, less than about 1 ⁇ 10 ⁇ 8 M, less than about 8 ⁇ 10 ⁇ 9 M, less than about 5 ⁇ 10 ⁇ 9 M, less than about 3 ⁇ 10 ⁇ 9 M, or less than about 1 ⁇ 10 ⁇ 9 M, or about 1 ⁇ 10 ⁇ 10 to 1 ⁇ 10 ⁇ 9 M, 1 ⁇ 10 ⁇ 10 to 5 ⁇ 10 ⁇ 9 M, about 1 ⁇ 10 ⁇ 10 to 1 ⁇ 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 10 to 5 ⁇ 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 9 to 1 ⁇ 10 ⁇ 8 M, about 1 ⁇ 10 ⁇ 9 to 5 ⁇ 10 ⁇
  • antigen- binding proteins of the present disclosure do not bind to rat CDH17.
  • anti-CDH17 antigen-binding proteins described herein do not block dimerization of CDH17.
  • anti-CDH17 antigen-binding proteins described herein do not impact CDH17 biological functions.
  • anti-CDH17 antigen-binding proteins described herein do not bind to shed CDH17 thus retaining targeting to tumor cells.
  • anti-CDH17 antigen-binding proteins described herein do not bind to the integrin binding RGD (Arg-Gly-Asp) motif in extracellular cadherin domain 6 (EC6) of human CDH17.
  • anti-CDH17 antigen-binding proteins described herein bind to the extracellular cadherin domain 6 (EC6) and/or extracellular cadherin domain 7 (EC7) of human CDH17. [0127] In some embodiments, anti-CDH17 antigen-binding proteins described herein bind to an extracellular region C-terminal to the RGD motif in EC6 of human CDH17. [0128] In some embodiments, anti-CDH17 antigen-binding proteins described herein bind to the extracellular cadherin domain 1 (EC1) of human CDH17.
  • EC6 extracellular cadherin domain 6
  • EC7 extracellular cadherin domain 7
  • Binding affinity of a molecular interaction between two molecules can be measured via various techniques, such as surface plasmon resonance (SPR), bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescent-activated cell sorting (FACS), flow cytometry binding assays, or isothermal titration calorimetry (ITC), and the like.
  • SPR surface plasmon resonance
  • BLI bio-layer interferometry
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescent-activated cell sorting
  • ITC isothermal titration calorimetry
  • Bio-layer Interferometry is a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam).
  • a change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface.
  • association and dissociation rates and affinities can be determined.
  • BLI can for example be performed using the Octet® Systems.
  • affinities can be measured in Kinetic Exclusion Assay (KinExA) (see e.g., Drake et al. 2004, Anal. Biochem., 328: 35-43), which is a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules.
  • Antigen-binding proteins of the present disclosure can include an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single chain antibody (e.g., single chain fragment variable (scFv)); a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab') 2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an lgG2 antibody; an lgG3 antibody; or an lgG4 antibody, and fragments thereof.
  • an antibody or an antigen-binding fragment of an antibody such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a
  • an antigen-binding protein that binds to CDH17 is a single-domain antibody (also termed as "sdAb").
  • the single-domain antibodies of the present disclosure can be derived from numerous sources, including but not limited to VHHs, VNARs, or VH domains (naturally occurring or engineered VH domains).
  • VHHs can be generated from camelid heavy chain only antibodies and libraries (e.g., synthetic libraries) thereof.
  • VNARs can be generated from cartilaginous fish heavy chain only antibodies and libraries (e.g., synthetic libraries) thereof.
  • Various methods have been implemented to generate monomeric sdAbs from conventionally heterodimeric VH and VL domains, including interface engineering and selection of specific germline families.
  • the sdAb of the present invention are human or humanized.
  • a single-domain antibody described herein is a VHH fragment (also known as a nanobody). VHH fragments are also referred to as "V-bodies" in the present disclosure.
  • the VHH is a camelid VHH, a humanized VHH or a camelized VH.
  • a single-domain antibody described herein is a VH domain.
  • a single-domain antibody described herein is a naturally occurring VH domain or engineered VH domain.
  • the variable domain of an antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises at least three complementarity determining regions (CDRs) which determine its binding specificity.
  • the CDRs are distributed between framework regions (FRs).
  • the variable domain typically contains 4 framework regions interspaced by 3 CDR regions, resulting in the following typical antibody variable domain structure: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDRs and/or FRs of the single domain antibody of the invention may be fragments or derivatives from a naturally occurring antibody variable domain or may be synthetic.
  • Sequence identifiers corresponding to exemplary anti-CDH17 VHH antibodies provided herein are listed in Table 1-1.
  • Table 1-1 sets forth the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DNA sequences of the full-length camelid VHH antibodies, as well as amino acid sequences of corresponding humanized VHH antibodies.
  • Amino acid sequences of additional exemplary anti-CDH17 VHH antibodies and corresponding humanized VHH antibodies are provided in Table 1-2.
  • an anti-CDH17 antigen-binding protein (e.g., antibody such as a singledomain antibody) described herein may comprise a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from a). A(A/L)CLLRFE(S/T)CLEYNRAQY(N/P)Y (SEQ ID NO: 83); b). AAVR(A/S)GSDWWTTM(R/T)QR(D/H)YD(F/Y) (SEQ ID NO: 85); c). AARDSR(K/R)GGLFADLN(E/G)YDY (SEQ ID NO: 88); d).
  • CDR3 complementarity determining region 3
  • AATG(D/N)(L/S)YRGAYDRP(A/T)EYDY (SEQ ID NO: 90); e). VATGNTYRGAYDRPAEYDF (SEQ ID NO: 29); f). AAQFSLPVDA(S/T)PLRRY(A/Y)(H/Y) (SEQ ID NO: 93); g). AATRKTRRSTVAGTEVDY (SEQ ID NO: 39); h). AARNGGYDLNDYAY (SEQ ID NO: 44); i). NAGG(G/P/A)(A/R)(L/R)GY (SEQ ID NO: 98); j). NVGGQL(K/L/R)GY (SEQ ID NO: 101); k).
  • NQGG(Q/S)KGY (SEQ ID NO: 104); l). AADG(L/P)PY(G/S)(D/S)WFGDQFDV (SEQ ID NO: 1633); m). AFNKWGRLSADL(D/N)DYFR (SEQ ID NO: 1636); n). N(M/T)HRSY(A/D)I(D/N/R/S)FYDN (SEQ ID NO: 1639); and o). RRYDDY(D/G)S (SEQ ID NO: 1642).
  • the CDR3 comprises an amino acid sequence selected from ALCLLRFETCLEYNRAQYPY (SEQ ID NO: 3); AAVRSGSDWWTTMTQRHYDF (SEQ ID NO: 8);
  • AARDSRRGGLFADLNEYDY (SEQ ID NO: 13); AATGDSYRGAYDRPAEYDY (SEQ ID NO: 18);
  • AATGNSYRGAYDRPTEYDY SEQ ID NO: 22
  • AATGDLYRGAYDRPAEYDY SEQ ID NO: 25
  • VATGNTYRGAYDRPAEYDF SEQ ID NO: 29
  • AAQFSLPVDASPLRRYYY SEQ ID NO: 34
  • AATRKTRRSTVAGTEVDY (SEQ ID NO: 39); AARNGGYDLNDYAY (SEQ ID NO: 44); NAGGGRLGY (SEQ ID NO: 49); NAGGAALGY (SEQ ID NO: 54); NVGGQLLGY (SEQ ID NO: 59); NQGGSKGY (SEQ ID NO: 64);
  • AFNKWGRLSADLDDYFR (SEQ ID NO: 1612); NMHRSYDISFYDN (SEQ ID NO: 1615); and RRYDDYGS (SEQ ID NO: 1618).
  • an anti-CDH17 antigen-binding protein (e.g., antibody such as a singledomain antibody) described herein may further comprise a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from a). GFTLSN(T/Y)N (SEQ ID NO: 81); b). GSPLDYYA (SEQ ID NO: 6); c). RL(A/N/T)(F/S)(N/S)(R/S)(S/T)T; d). GRTFS(E/T)PI (SEQ ID NO: 89); e). GRTFSSPI (SEQ ID NO: 28); f).
  • CDR1 complementarity determining region 1
  • TRTF(D/N)MYA (SEQ ID NO: 91); g). GRTF(D/S)S(L/Y)(L/V) (SEQ ID NO: 94); h). GRTDSILN (SEQ ID NO: 42); i). G(I/M)RFS(S/Q)YA (SEQ ID NO: 96); j). GIRFS(A/S)YA (SEQ ID NO: 99); k). GS(I/R)FS(R/S)(W/Y)A (SEQ ID NO: 102); l). G(G/R)TASEYG (SEQ ID NO: 1631); m). EQTMTGF(T/W) (SEQ ID NO: 1634); n). GLRFS(S/N)YA (SEQ ID NO: 1637); and o). (G/R)GT(F/V)SGYA (SEQ ID NO: 1640).
  • the CDR1 comprises an amino acid sequence selected from GFTLSNYN (SEQ ID NO: 1); GSPLDYYA (SEQ ID NO: 6); RLNFSRTT (SEQ ID NO: 11); GRTFSEPI (SEQ ID NO: 16);
  • GRTFSTPI SEQ ID NO: 21
  • GRTFSEPI SEQ ID NO: 16
  • GRTFSSPI SEQ ID NO: 28
  • TRTFNMYA SEQ ID NO: 32
  • GRTFSSYL SEQ ID NO: 37
  • GRTDSILN SEQ ID NO: 42
  • GIRFSSYA SEQ ID NO: 47
  • GMRFSQYA SEQ ID NO: 52
  • GIRFSAYA SEQ ID NO: 57
  • GSRFSSYA SEQ ID NO: 62
  • TRTFDMYA SEQ ID NO: 637
  • GGTASEYG (SEQ ID NO: 1607); EQTMTGFW (SEQ ID NO: 1610); GLRFSNYA (SEQ ID NO: 1613); and GGTVSGYA (SEQ ID NO: 1616).
  • an anti-CDH17 antigen-binding protein (e.g., antibody such as a singledomain antibody) described herein may further comprise a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from a). (F/I)SRGGRT (SEQ ID NO: 82); b). ISTSGR(C/S)T (SEQ ID NO: 84); c). SGW(A/S)R(G/T)RT (SEQ ID NO: 87); d). LISTGGST (SEQ ID NO: 17); e). I(N/S)RSG(A/T)NT (SEQ ID NO: 92); f).
  • CDR2 complementarity determining region 2
  • ISWN(D/G)RST (SEQ ID NO: 95); g). ISWFRGET (SEQ ID NO: 43); h). I(F/T)(I/K/N/S)(D/G)(G/Y)(R/S/T)T; i). MT(A/N/T)GGMT (SEQ ID NO: 100); j). IT(N/S)GG(G/R/S)T (SEQ ID NO: 103); k). ISTSGGVT (SEQ ID NO: 1632); l). ISASGSRV (SEQ ID NO: 1635); m). IT(N/K)GG(I/N/S)T (SEQ ID NO: 1638); and n). INSGGPT (SEQ ID NO: 1641).
  • the CDR2 comprises an amino acid sequence selected from ISRGGRT (SEQ ID NO: 2); ISTSGRCT (SEQ ID NO: 7); SGWARGRT (SEQ ID NO: 12); LISTGGST (SEQ ID NO: 17);
  • ISRSGTNT SEQ ID NO: 33
  • ISWNDRST SEQ ID NO: 38
  • ISWFRGET SEQ ID NO: 43
  • ITSGYRT SEQ ID NO: 48
  • IFKDGTT SEQ ID NO: 53
  • MTAGGMT SEQ ID NO: 58
  • ITSGGRT SEQ ID NO: 63
  • INRSGANT SEQ ID NO: 875
  • ISTSGGVT SEQ ID NO: 1608
  • ISASGSRV SEQ ID NO: 1611
  • ITKGGIT SEQ ID NO: 1614
  • INSGGPT SEQ ID NO: 1617
  • the antigenbinding protein comprises i). a CDR1 comprising an amino acid sequence of GFTLSN(T/Y)N (SEQ ID NO: 81), a CDR2 comprising an amino acid sequence of (F/I)SRGGRT (SEQ ID NO: 82), and a CDR3 comprising an amino acid sequence of A(A/L)CLLRFE(S/T)CLEYNRAQY(N/P)Y (SEQ ID NO: 83); ii).
  • a CDR1 comprising an amino acid sequence of GSPLDYYA (SEQ ID NO: 6), a CDR2 comprising an amino acid sequence of ISTSGR(C/S)T (SEQ ID NO: 84), and a CDR3 comprising an amino acid sequence of AAVR(A/S)GSDWWTTM(R/T)QR(D/H)YD(F/Y) (SEQ ID NO: 85); iii).
  • a CDR1 comprising an amino acid sequence of RL(A/N/T)(F/S)(N/S)(R/S)(S/T)T
  • a CDR2 comprising an amino acid sequence of SGW(A/S)R(G/T)RT (SEQ ID NO: 87)
  • a CDR3 comprising an amino acid sequence of AARDSR(K/R)GGLFADLN(E/G)YDY (SEQ ID NO: 88); iv).
  • a CDR1 comprising an amino acid sequence of GRTFS(E/T)PI (SEQ ID NO: 89), a CDR2 comprising an amino acid sequence of LISTGGST (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of AATG(D/N)(L/S)YRGAYDRP(A/T)EYDY (SEQ ID NO: 90); v).
  • a CDR1 comprising an amino acid sequence of GRTFSSPI (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LISTGGST (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of VATGNTYRGAYDRPAEYDF (SEQ ID NO: 29); vi).
  • a CDR1 comprising an amino acid sequence of TRTF(D/N)MYA (SEQ ID NO: 91), a CDR2 comprising an amino acid sequence of l(N/S)RSG(A/T)NT (SEQ ID NO: 92), and a CDR3 comprising an amino acid sequence of AAQFSLPVDA(S/T)PLRRY(A/Y)(H/Y) (SEQ ID NO: 93); vii).
  • a CDR1 comprising an amino acid sequence of GRTF(D/S)S(L/Y)(L/V) (SEQ ID NO: 94), a CDR2 comprising an amino acid sequence of ISWN(D/G)RST (SEQ ID NO: 95), and a CDR3 comprising an amino acid sequence of AATRKTRRSTVAGTEVDY (SEQ ID NO: 39); viii) a CDR1 comprising an amino acid sequence of GRTDSILN (SEQ ID NO: 42), a CDR2 comprising an amino acid sequence of ISWFRGET (SEQ ID NO: 43), and a CDR3 comprising an amino acid sequence of AARNGGYDLNDYAY (SEQ ID NO: 44); ix).
  • a CDR1 comprising an amino acid sequence of G(I/M)RFS(S/Q)YA (SEQ ID NO: 96), a CDR2 comprising an amino acid sequence of I ( F/T)(I/K/N/S)(D/G)(G/Y)(R/S/T)T, and a CDR3 comprising an amino acid sequence of NAGG(G/P/A)(A/R)(L/R)GY (SEQ ID NO: 98); x).
  • a CDR1 comprising an amino acid sequence of GIRFS(A/S)YA (SEQ ID NO: 99), a CDR2 comprising an amino acid sequence of MT(A/N/T)GGMT (SEQ ID NO: 100), and a CDR3 comprising an amino acid sequence of NVGGQL(K/L/R)GY (SEQ ID NO: 101); xi).
  • a CDR1 comprising an amino acid sequence of GS(I/R)FS(R/S)(W/Y)A (SEQ ID NO: 102), a CDR2 comprising an amino acid sequence of IT(N/S)GG(G/R/S)T (SEQ ID NO: 103), and a CDR3 comprising an amino acid sequence of NQGG(Q/S)KGY (SEQ ID NO: 104); xii).
  • a CDR1 comprising an amino acid sequence of G(G/R)TASEYG (SEQ ID NO: 1631), a CDR2 comprising an amino acid sequence of ISTSGGVT (SEQ ID NO: 1632), and a CDR3 comprising an amino acid sequence of AADG(L/P)PY(G/S)(D/S)WFGDQFDV (SEQ ID NO: 1633); xiii).
  • a CDR1 comprising an amino acid sequence of EQTMTGF(T/W) (SEQ ID NO: 1634), a CDR2 comprising an amino acid sequence of ISASGSRV (SEQ ID NO: 1635), and a CDR3 comprising an amino acid sequence of AFNKWGRLSADL(D/N)DYFR (SEQ ID NO: 1636); xiv).
  • a CDR1 comprising an amino acid sequence of GLRFS(S/N)YA (SEQ ID NO: 1637), a CDR2 comprising an amino acid sequence of IT(N/K)GG(I/N/S)T (SEQ ID NO: 1638), and a CDR3 comprising an amino acid sequence of N(M/T)HRSY(A/D)I(D/N/R/S)FYDN (SEQ ID NO: 1639); or xv).
  • a CDR1 comprising an amino acid sequence of (G/R)GT(F/V)SGYA (SEQ ID NO: 1640), a CDR2 comprising an amino acid sequence of INSGGPT (SEQ ID NO: 1641), and a CDR3 comprising an amino acid sequence of RRYDDY(D/G)S (SEQ ID NO: 1642).
  • the antigenbinding protein comprises i). a CDR1 comprising an amino acid sequence of GFTLSNYN (SEQ ID NO: 1), a CDR2 comprising an amino acid sequence of ISRGGRT (SEQ ID NO: 2), and a CDR3 comprising an amino acid sequence of ALCLLRFETCLEYNRAQYPY (SEQ ID NO: 3); ii).
  • a CDR1 comprising an amino acid sequence of GSPLDYYA (SEQ ID NO: 6), a CDR2 comprising an amino acid sequence of ISTSGRCT (SEQ ID NO: 7), and a CDR3 comprising an amino acid sequence of AAVRSGSDWWTTMTQRHYDF (SEQ ID NO: 8); iii).
  • a CDR1 comprising an amino acid sequence of RLNFSRTT (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of SGWARGRT (SEQ ID NO: 12), and a CDR3 comprising an amino acid sequence of AARDSRRGGLFADLNEYDY (SEQ ID NO: 13); iv).
  • a CDR1 comprising an amino acid sequence of GRTFSEPI (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of LISTGGST (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of AATGDSYRGAYDRPAEYDY (SEQ ID NO: 18); v).
  • a CDR1 comprising an amino acid sequence of GRTFSTPI (SEQ ID NO: 21), a CDR2 comprising an amino acid sequence of LISTGGST (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of
  • a CDR1 comprising an amino acid sequence of GRTFSEPI (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of LISTGGST (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of AATGDLYRGAYDRPAEYDY (SEQ ID NO: 25); vii). a CDR1 comprising an amino acid sequence of GRTFSSPI (SEQ ID NO: 28), a CDR2 comprising an amino acid sequence of LISTGGST (SEQ ID NO:17), and a CDR3 comprising an amino acid sequence of VATGNTYRGAYDRPAEYDF (SEQ ID NO: 29); viii).
  • a CDR1 comprising an amino acid sequence of TRTFNMYA (SEQ ID NO: 32), a CDR2 comprising an amino acid sequence of ISRSGTNT (SEQ ID NO: 33), and a CDR3 comprising an amino acid sequence of AAQFSLPVDASPLRRYYY (SEQ ID NO: 34); ix).
  • a CDR1 comprising an amino acid sequence of GRTFSSYL (SEQ ID NO: 37), a CDR2 comprising an amino acid sequence of ISWNDRST (SEQ ID NO: 38), and a CDR3 comprising an amino acid sequence of AATRKTRRSTVAGTEVDY (SEQ ID NO: 39); x).
  • a CDR1 comprising an amino acid sequence of GRTDSILN (SEQ ID NO: 42), a CDR2 comprising an amino acid sequence of ISWFRGET (SEQ ID NO: 43), and a CDR3 comprising an amino acid sequence of AARNGGYDLNDYAY (SEQ ID NO: 44); xi).
  • a CDR1 comprising an amino acid sequence of GIRFSSYA (SEQ ID NO: 47), a CDR2 comprising an amino acid sequence of ITSGYRT (SEQ ID NO: 48), and a CDR3 comprising an amino acid sequence of NAGGGRLGY (SEQ ID NO: 49); xii).
  • a CDR1 comprising an amino acid sequence of GMRFSQYA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IFKDGTT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of NAGGAALGY (SEQ ID NO: 54); xiii).
  • a CDR1 comprising an amino acid sequence of GIRFSAYA (SEQ ID NO: 57), a CDR2 comprising an amino acid sequence of MTAGGMT (SEQ ID NO: 58), and a CDR3 comprising an amino acid sequence of NVGGQLLGY (SEQ ID NO: 59); xiv).
  • a CDR1 comprising an amino acid sequence of GSRFSSYA (SEQ ID NO: 62), a CDR2 comprising an amino acid sequence of ITSGGRT (SEQ ID NO: 63), and a CDR3 comprising an amino acid sequence of NQGGSKGY (SEQ ID NO: 64); xv).
  • a CDR1 comprising an amino acid sequence of TRTFDMYA (SEQ ID NO: 637), a CDR2 comprising an amino acid sequence of INRSGANT (SEQ ID NO: 875), and a CDR3 comprising an amino acid sequence of AAQFSLPVDATPLRRYAH (SEQ ID NO: 1113); xvi).
  • a CDR1 comprising an amino acid sequence of GGTASEYG (SEQ ID NO: 1607), a CDR2 comprising an amino acid sequence of ISTSGGVT (SEQ ID NO: 1608), and a CDR3 comprising an amino acid sequence of AADGLPYGDWFGDQFDV (SEQ ID NO:1609); xvii) a CDR1 comprising an amino acid sequence of EQTMTGFW (SEQ ID NO: 1610), a CDR2 comprising an amino acid sequence of ISASGSRV (SEQ ID NO: 1611), and a CDR3 comprising an amino acid sequence of AFNKWGRLSADLDDYFR (SEQ ID NO: 1612); xviii) a CDR1 comprising an amino acid sequence of GLRFSNYA (SEQ ID NO: 1613), a CDR2 comprising an amino acid sequence of ITKGGIT (SEQ ID NO: 1614), and a CDR3 comprising an amino acid sequence of NMHRSYDISFYDN (SEQ ID
  • a CDR1 comprising an amino acid sequence of GGTVSGYA (SEQ ID NO: 1616), a CDR2 comprising an amino acid sequence of INSGGPT (SEQ ID NO: 1617), and a CDR3 comprising an amino acid sequence of RRYDDYGS (SEQ ID NO: 1618).
  • the antigen-binding protein comprises a), a CDR1 comprising an amino acid sequence of GRTFSSYL (SEQ ID NO: 37), a CDR2 comprising an amino acid sequence of ISWNDRST (SEQ ID NO: 38), and a CDR3 comprising an amino acid sequence of AATRKTRRSTVAGTEVDY (SEQ ID NO: 39); b). a CDR1 comprising an amino acid sequence of RLNFSRTT (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of SGWARGRT (SEQ ID NO: 12), and a CDR3 comprising an amino acid sequence of AARDSRRGGLFADLNEYDY (SEQ ID NO: 13); or c).
  • a CDR1 comprising an amino acid sequence of GMRFSQYA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IFKDGTT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of NAGGAALGY (SEQ ID NO: 54).
  • the antigen-binding protein comprises a), a CDR1 comprising an amino acid sequence of GGTASEYG (SEQ ID NO: 1607), a CDR2 comprising an amino acid sequence of ISTSGGVT (SEQ ID NO: 1608), and a CDR3 comprising an amino acid sequence of AADGLPYGDWFGDQFDV (SEQ ID NO: 1609); b).
  • a CDR1 comprising an amino acid sequence of EQTMTGFW (SEQ ID NO: 1610), a CDR2 comprising an amino acid sequence of ISASGSRV (SEQ ID NO: 1611), and a CDR3 comprising an amino acid sequence of AFNKWGRLSADLDDYFR (SEQ ID NO: 1612); c). a CDR1 comprising an amino acid sequence of GMRFSQYA (SEQ ID NO: 52), a CDR2 comprising an amino acid sequence of IFKDGTT (SEQ ID NO: 53), and a CDR3 comprising an amino acid sequence of NAGGAALGY (SEQ ID NO: 54); or d).
  • a CDR1 comprising an amino acid sequence of GGTVSGYA (SEQ ID NO: 1616), a CDR2 comprising an amino acid sequence of INSGGPT (SEQ ID NO: 1617), and a CDR3 comprising an amino acid sequence of RRYDDYGS (SEQ ID NO: 1618).
  • the antigenbinding protein is a single-domain antibody.
  • the single-domain antibody is a VHH, a VNAR, or a VH domain.
  • the VHH is a camelid VHH.
  • the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 9, 14, 19, 23, 26, 30, 35, 40, 45, 50, 55, 60, 65, 105-342, 1619-1622, 1643-1671, and 1778-1806, or an amino acid sequence having at least 75% identity thereto.
  • the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 9, 14, 19, 23, 26, 30, 35, 40, 45, 50, 55, 60, 65, 187, and 1619-1622, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
  • the VHH comprises an amino acid sequence selected from any one of SEQ ID Nos: 40, 19, and 55, or an amino acid sequence having at least 75% identity thereto.
  • the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 55, 1619, 1620, and 1622, or an amino acid sequence having at least 75% identity thereto. [0152] In some embodiments, the VHH is a humanized VHH.
  • the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 10, 15, 20, 24, 27, 31, 36, 41, 46, 51, 56, 61, 66 343-554, 1623-1626, and 1672- 1691, or an amino acid sequence having at least 75% identity thereto.
  • the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 10, 15, 20, 24, 27, 31, 36, 41, 46, 51, 56, 61, 66, 421, and 1623-1626, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.
  • the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 15, 41, and 56, or an amino acid sequence having at least 75% identity thereto.
  • the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 56, 1623, 1624, and 1626, or an amino acid sequence having at least 75% identity thereto.
  • anti-CDH17 antigen-binding proteins e.g., antibodies such as singledomain antibodies
  • a CDR1 comprising an amino acid sequence selected from any of the CDR1 amino acid sequences listed in Table 1-1 or Table 6, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
  • an anti-CDH17 antigen-binding protein (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 1, 6,
  • anti-CDH17 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • a CDR2 CDR2
  • an anti-CDH17 antigen-binding protein (e.g., antibody such as singledomain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 2, 7,
  • anti-CDH17 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • a CDR3 comprising an amino acid sequence selected from any of the CDR3 amino acid sequences listed in Table 1-1 or Table 6, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • an anti-CDH17 antigen-binding protein (e.g., antibody such as singledomain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 3, 8,
  • anti-CDH17 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • anti-CDH17antigen-binding proteins e.g., antibodies such as singledomain antibodies
  • anti-CDH17antigen-binding proteins comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within a VHH amino acid sequence as defined by any of the exemplary anti-CDH17 VHH antibodies listed in Table 1-1, Table 1-2, or Table 6.
  • antibodies, or antigen-binding fragments thereof comprising the set of CDR1-CDR2-CDR3 amino acid sequences contained within a VHH amino acid sequence selected from SEQ ID NOs: 4, 9, 14, 19, 23, 26, 30, 35, 40, 45, 50, 55, 60, 65, 105-342, 1619-1622, 1643-1671, and 1778-1806.
  • an anti-CDH17 antigen-binding protein e.g., antibody such as a singledomain antibody
  • an anti-CDH17 antigen-binding protein of the present disclosure can include: a) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4; b) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9; c) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 14; d) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 23 e) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 26; f)
  • variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 187;
  • variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1619;
  • variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1620;
  • variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1621;
  • variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1622.
  • the present disclosure also provides an anti-CDH17 antigen-binding protein (e.g., antibody such as a single-domain antibody) that competes for binding to CDH17 with any one of the exemplary anti-CDH17 VHH antibodies listed in Table 1-1, Table 1-2, or Table 6.
  • an anti-CDH17 antigen-binding protein e.g., antibody such as a single-domain antibody
  • the present disclosure also provides an anti-CDH17 antigen-binding protein (e.g., antibody such as a single-domain antibody) that binds to the same epitope on CDH17 as any one of the exemplary anti-CDH17 VHH antibodies listed in Table 1-1, Table 1-2, or Table 6.
  • an anti-CDH17 antigen-binding protein e.g., antibody such as a single-domain antibody
  • Single-domain antibodies e.g., antibody such as a single-domain antibody
  • a single-domain antibody (e.g., VHH) can be obtained by immunization of dromedaries, camels, llamas, alpacas, or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies.
  • Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and/or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens. By reverse transcription and polymerase chain reaction (PCR), a gene library of single-domain antibodies containing several million clones can be produced. Screening techniques such as phage display, yeast display, and ribosome display help to identify the clones binding the antigen.
  • Affinity maturation strategies can be categorized as either targeted/rational approaches or untargeted/random approaches.
  • targeted approaches information about the VHH of interest is needed, such as hot spots for affinity maturation or structural information on the VHH:antigen complex, whereas for untargeted approaches no prior information is needed.
  • Targeted approaches that may be applied for affinity maturation of VHHs include site-directed in-vitro mutagenesis and in- s/7/co/computational approaches.
  • Common untargeted approaches used for affinity maturation of VHHs include random in-vitro mutagenesis, CDR swapping and autonomous hypermutation yeast surface display, with the latter two being novel, emerging and very time efficient techniques.
  • the resulting library can be screened by employing standard display techniques such as yeast, phage or ribosome display to select for the best binders.
  • standard display techniques such as yeast, phage or ribosome display to select for the best binders.
  • the choice of the display system is often guided by the library size to be displayed, with yeast display being able to handle library sizes of ⁇ 10 7 - 10 9 , phage display ⁇ 1O 8 -1O 10 and ribosome display ⁇ 1O 12 -1O 13 (Chan and Groves, 2021).
  • yeast display being able to handle library sizes of ⁇ 10 7 - 10 9
  • phage display ⁇ 1O 8 -1O 10 and ribosome display ⁇ 1O 12 -1O 13 Choan and Groves, 2021).
  • the number of highly interactive residues such as aromatic amino acids usually increase in the CDR regions.
  • the selected affinity matured clones may be further evaluated by a developability assessment to test for undesired properties, such as un
  • a set of selected residues within the CDRs of a VHH may be mutated (Tiller et al., 2017; Yau et al., 2005). Pre-selection of these residues can be either performed using alanine scanning to identify hot spot residues for mutation or by using structural data of the antigen:VHH complex to identify positions to be mutated. These sites can then be either submitted to saturating mutagenesis to substitute a specific site with all possible amino acids or specific amino acid substitutions yielding several smaller libraries. After mutagenesis binders can be displayed to select the best matured candidate.
  • Untargeted/random affinity maturation strategies that can be applied to affinity mature VHHs include random in vitro mutagenesis, CDR shuffling/swapping and in vivo affinity maturation via yeast display.
  • random in vitro mutagenesis the sequence of either the entire VHH or only the CDRs are mutated randomly (Chen et aL, 2021; Ye et aL, 2021; Zupancic et aL, 2021).
  • the most commonly used technique is error prone PCR employing a DNA polymerase that lacks proof reading activity and PCR conditions that increase the polymerase error rate even further. This technique can be applied without further structural knowledge or information on the importance of residues that contribute to antigen:VHH interaction.
  • the resulting mutational library can then be displayed to select the best matured candidate.
  • This technique may also be combined with NGS sequencing of the display elutions to get an in-depth readout of all obtained candidates, enabling the identification of low abundant but still promising clones (Chen et aL, 2021).
  • CDR shuffling or swapping is applied for VHH affinity maturation, such as described in Zupancic et aL, 2021.
  • enriched libraries can be used as input material for a PCR reaction to individually amplify the CDR of the VHHs.
  • the PCR products can then be mixed and reassembled using overlapping PCR to generate the entire plasmid for further rounds of display to select for the best matured binder.
  • One limitation of this approach is that it can only be used for VHHs comprising the same framework as it is the case for synthetic libraries.
  • in vivo affinity maturation via yeast display is applied for VHH affinity maturation, such as described in Wellner et al., 2021.
  • the method is based on an autonomous hypermutation yeast surface display (AHEAD), which imitates somatic hypermutation during VHH selection using engineered yeast strains.
  • AHEAD autonomous hypermutation yeast surface display
  • the yeast's error prone orthogonal DNA replication system can generate new variants during plasmid replication by randomly introducing mutations.
  • the new variants can then be displayed and selected using yeast surface display to identify the best binders. This enables the production of high affinity clones in very little time (about 2 weeks), which is significantly faster than classical affinity maturation procedures.
  • the method can be applied using synthetic or immune libraries using unenriched libraries enriched libraries or a subset of preselected clones.
  • binders with medium affinity are required, as it is the case for the anti-CDH17 V-bodies and the affinity of the identified candidates need to be decreased, very similar techniques can be applied. For example, mutations that are aiming at lowering the affinity can be introduced using the same targeted or untargeted approaches as described for the affinity maturation. The selection afterwards can be adapted accordingly. If larger libraries are generated that need to be screened via a display technique, the selection strategy can be adapted to enrich medium affinity binders while excluding high affinity candidates. This could, for example be a pre-panning in phage display with low antigen concentration to remove all higher affinity candidates, followed by a selection with high antigen concentration to obtain medium affinity VHHs. For library sizes of up to 1000 candidates a kinetic off- rate characterization can be used to get immediate information about the kinetic behavior of the candidates.
  • Single-domain antibodies can also be derived from conventional antibodies.
  • single-domain antibodies can be made from conventional murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and display techniques for identification of the most specific antigens.
  • the binding region of a conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity. Monomerization can be accomplished by replacing lipophilic by hydrophilic amino acids. (See e.g., Borrebaeck, C. A. K.; Ohlin, M. (2002). "Antibody evolution beyond Nature". Nature Biotechnology 20 (12): 1189-90.) If affinity can be retained after monomerization, the single-domain antibodies can likewise be produced in E. coli, S. cerevisiae or other suitable organisms.
  • a “humanized antibody” refers to a chimeric, genetically engineered, antibody in which the amino acid sequences (typically CDRs) from an antibody (donor antibody), e.g., a camelid antibody, are grafted onto a human antibody (acceptor antibody).
  • a humanized antibody typically comprises CDRs from a donor antibody and variable region framework and constant regions, when present, from a human antibody.
  • a “humanized VHH” comprises CDRs that corresponds to the CDRs of a naturally occurring VHH domain (e.g., a camelid VHH), but that has been "humanized”.
  • Humanized VHH may be prepared by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4- chain human antibody.
  • Such humanized VHHs can be obtained in any suitable manner known to a skilled person in the art and thus not strictly limited to methods described herein.
  • a human germline reference that is most similar to the camelid germline sequence of the selected VHH may be identified.
  • Most of the isolated camelid VHHs in literature belong to the camelid IGHV3 subfamily 2 (Nguyen et aL, 2000, EMBO J) with DP-47/VH3-23 from the IGHV3 family commonly used as human reference.
  • the framework of the camelid VHH can then be compared to the human reference sequence. Surface exposed residues are substituted to their human counterpart as it is assumed that their contribution to protein stability is rather low. Buried residues however remain of camelid origin, as they likely contribute to the overall VHH stability.
  • VHHs use framework 2 residues H44, H45 and H47 for antigen binding (Zavrtanik et aL, 2018, J Mol Biol).
  • a full humanization of these residues hence frequently results in reduced solubility or aggregation of the VHHs and a reduced or complete loss of binding affinity for the target antigen (van Faassen et aL, 2020, Vincke et aL, 2009).
  • all or at least some of these hallmark residues in framework 2 remain of camelid origin when humanizing VHHs.
  • CDR grafting Another approach that may be applied to humanize VHHs is CDR grafting.
  • CDRs of the selected VHHs can be transplanted onto a universal VHH framework that has been partially or fully humanized (Saerens et al., 2009 J Biol Chem, Soler et al., 2021, Vincke et al., 2009 J Biol Chem).
  • CDR grafting has been successfully used in some cases but failed for several others, with VHHs frequently losing their potential to bind to the desired antigen and/or becoming structurally instable with a high tendency to aggregate (van Faassen et al., 2020, FASEB).
  • VHH sequences are used fully or partially humanized synthetic VHH libraries instead of camelid immune libraries for VHH discovery (Moutel et al. 2016, eLife; McMahon, 2018, NSMB; Zimmermann et al., 2018, eLife). In many of these libraries the hallmark residues are still of camelid origin for reasons discussed above.
  • humanized VHH sequences still retain the residues that are relevant for protein A binding.
  • the engineering activities during humanization may be applied to engineer protein A binding properties into a VHH that did previously not interact with protein A (Graille et al., 2000, PNAS).
  • a “camelized antibody” refers to an antibody having amino acid sequences (typically CDRs) from a donor antibody, e.g., a human antibody, and variable region framework and constant regions, when present, from a camelid antibody. Accordingly, a “camelized VH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized”. Camelized VH may be prepared by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody.
  • the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, or the VH sequence of a human antibody.
  • camelized VH can be obtained in any suitable manner known to a skilled person in the art and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.
  • the amino acid residues of a single-domain antibody can be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids described in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example FIG.
  • the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. For example, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. As a result, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • the total number of amino acid residues in a VH domain and a VHH domain is usually in the range of from 110 to 120, often between 112 and 115. However, smaller and longer sequences may also be suitable for the purposes described herein.
  • Determination of CDR regions in a single-domain antibody may be accomplished using different methods, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat” numbering scheme); Al-Lazikani et aL, (1997) JMB 273,927-948 ("Chothia” numbering scheme); MacCallum et aL, J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol.
  • the boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification.
  • the Kabat scheme is based on structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular's AbM antibody modeling software.
  • CDRs can be defined in accordance with any of the Kabat numbering scheme, the Chothia numbering scheme, a combination of Kabat and Chothia, the AbM numbering scheme, and/or the Contact numbering scheme.
  • a VHH typically comprises three CDRs, designated CDR1, CDR2, and CDR3.
  • Table 1-3 below, lists exemplary position boundaries of CDR-H1, CDR-H2, CDR- H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively.
  • residue numbering is listed using both the Kabat and Chothia numbering schemes.
  • FRs are located between CDRs, for example, with FR-H1 located before CDR-H1, FR-H2 located between CDR-H1 and CDR-H2, FR- H3 located between CDR-H2 and CDR-H3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
  • CDR complementary metal-oxide-semiconductor
  • CDR-H1, CDR-H2, CDR-H3 individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) CDR as defined by any of the above-mentioned schemes.
  • a particular CDR e.g., a CDR-H3
  • a CDR-H3 contains the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence
  • a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the VHH, as defined by any of the above-mentioned schemes.
  • specific CDR sequences are specified.
  • Exemplary CDR sequences of provided antibodies are described using various numbering schemes (see e.g., Table 1-3), although it is understood that a provided antibody can include CDRs as described according to any of the other above-mentioned numbering schemes or other numbering schemes known to a person of ordinary skill in the art.
  • the framework sequences may be any suitable framework sequences.
  • the framework sequences may be framework sequences derived from a heavy chain variable domain (e.g., a VH sequence or VHH sequence).
  • the framework sequences are either framework sequences that have been derived from a VHH sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences (in which said framework sequences may optionally have been partially or fully camelized).
  • an antigen-binding protein of the present disclosure may comprise naturally occurring sequences (from a suitable species), recombinant sequences, or synthetic or semi-synthetic sequences.
  • nucleotide sequences encoding antigen-binding proteins of the present disclosure may comprise naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (for example, sequences that are prepared by PCR or isolated from a library).
  • Anti-CDH17 antigen-binding proteins e.g., antibodies such single-domain antibodies
  • Anti-CDH17 antigen-binding proteins of the present disclosure may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy chain variable domains as compared to the exemplary antibody sequences provided herein. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • the antigen-binding molecules of the present disclosure may comprise antigen-binding domains which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations").
  • Germline mutations A person of ordinary skill in the art, starting with the heavy chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigenbinding fragments which comprise one or more individual germline mutations or combinations thereof.
  • all of the framework and/or CDR residues within the VHH domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).
  • the antigen-binding domains may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced biological properties (e.g., agonistic effect), reduced immunogenicity, etc.
  • Antigen-binding proteins comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.
  • anti-CDH17 antigen-binding proteins comprising variants of any of the VHH and/or CDR amino acid sequences disclosed herein having one or more amino acid substitutions.
  • the present disclosure includes anti-CDH17 antigen-binding proteins having VHH and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 amino acid substitutions relative to any of the VHH and/or CDR amino acid sequences set forth in Tables 1-1 and 1-2 herein.
  • Amino acid substitutions may be introduced into an antigen-binding protein of interest and the resultant variants can screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or reduced ADCC or CDC.
  • Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
  • an amino acid substitution is a conservative substitution, meaning exchanging an amino acid with another amino acid of the same class.
  • amino acid substitutions may also include a non-conservative substitution, meaning exchanging an amino acid with an amino acid of a different class. Other exemplary amino acid substitutions are shown in Table 2.
  • single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations to reduce oxidation levels of oxidation-labile residues such as Met (M). In certain embodiments, it may be desirable to address Met (M) oxidation liability by mutation of a Met (M) residue.
  • the single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations (e.g., substitution mutations) of a Met residue to reduce oxidation.
  • a Met residue may be substituted in any of the single-domain antibodies described herein with e.g., He (I), Ala (A), or Leu (L), to reduce oxidation.
  • single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications that reduce binding of the single-domain antibodies (e.g., VHH) by preexisting antibodies found in human blood or serum.
  • single-domain antibodies (e.g., VHHs) of the present disclosure are modified by mutation of amino acid position 11, for example LeullGlu (L11E), LeullLys (L11K), or LeullVal (L11V).
  • a single-domain antibody (e.g., VHH) of the present disclosure may comprise a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering).
  • a single-domain antibody (e.g., VHH) of the present disclosure may comprise an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the single-domain antibody (e.g., VHH).
  • A alanine
  • the C-terminus of a VHH is normally VTVSS (SEQ ID NO: 1582).
  • a singledomain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering).
  • a single-domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering).
  • the C-terminus of a single-domain antibody can be any one of VKVSS (SEQ ID NO: 1583), VQVSS (SEQ ID NO: 1584), VTVKS (SEQ ID NO: 1585), VTVQS (SEQ ID NO: 1586), VKVKS (SEQ ID NO: 1587), VKVQS (SEQ ID NO: 1588), VQVKS (SEQ ID NO: 1589), or VQVQS (SEQ ID NO: 1590).
  • single-domain antibodies e.g., VHH
  • single-domain antibodies are modified by changes in carboxy-terminal region, for example to a terminal sequence having the sequence GQGTLVTVKPGG (SEQ ID NO: 1594) or GQGTLVTVEPGG (SEQ ID NO: 1595) or modification thereof. Additional modification to reduce binding by pre-existing antibodies in human serum can be found in e.g., W02012/175741, WO2015/173325, WO2016/150845, W02011/003622, W02013/024059; US 11,426,468, US 10,526,397, which are incorporated herein by reference in their entireties.
  • a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 1836) or VPAG (SEQ ID NO: 1837).
  • a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 1836).
  • a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VPAG (SEQ ID NO: 1837).
  • a single-domain antibody e.g., VHH
  • VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 9, 10, 14, 15, 19, 20, 23, 24, 26, 27 , 30, 31, 35, 36, 40, 41, 45, 46, 50, 51, 55, 56, 60, 61, 65, 66, 105-554, 1619, 1620-1626, and 1778-1806, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxyterminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 1836) or VPAG (SEQ ID NO: 1837).
  • a single-domain antibody e.g., VHH
  • VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 9, 14, 19, 23, 26, 30, 35, 40, 45, 50, 55, 60, 65, 105-342, 1619-1622, and 1643-1671, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 1836) or VPAG (SEQ ID NO: 1837).
  • a single-domain antibody e.g., VHH
  • VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 9, 14, 19, 23, 26, 30, 35, 40, 45, 50, 55, 60, 65, 187, and 1619-1622, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 1836) or VPAG (SEQ ID NO: 1837).
  • a single-domain antibody e.g., VHH
  • VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 10, 15, 20, 24, 27, 31, 36, 41, 46, 51, 56, 61, 66, 343-554, 1623-1626, and 1672-1691, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 1836) or VPAG (SEQ ID NO: 1837).
  • a single-domain antibody e.g., VHH
  • VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 10, 15, 20, 24, 27, 31, 36, 41, 46, 51, 56, 61, 66, 421, 1623-1626, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 1836) or VPAG (SEQ ID NO: 1837).
  • single-domain antibodies e.g., VHH
  • VHH single-domain antibodies
  • SpA staphylococcal protein A
  • SpG streptococcal protein G
  • binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of the antibodies or antibody fragments.
  • the high-affinity interaction of the IgG Fc region with SpA and SpG has been extensively exploited and became the gold standard for monoclonal antibody purification (Bjdrck and Kronvall, 1984).
  • Other non-Fc containing antibody fragments, such as VHHs and Fabs do not have the capacity to bind to SpA or SpG via their Fc regions.
  • single-domain antibodies e.g., VHH
  • VHH single-domain antibodies
  • the VHH-SpA interface has been mapped to thirteen residues, which cluster within the framework at the back side of the V-body, distant to the CDRs (Grail le et al., 2000, Henry et al., 2016).
  • VHH-SpA co-structure superposition of a SpA-Fab crystal structure and a VHH allows for visualizing the binding mode.
  • a SpA-binding motif included in a single-domain antibody (e.g., VHH) of the present disclosure may include one or more, or all of the thirteen residues.
  • anti-CDH17 antigen-binding proteins of the present disclosure can adopt an alternative protein scaffold.
  • Such alternative protein scaffold may be a single chain polypeptidic framework, optionally with a reduced size (e.g., less than about 200 amino acids), that contains a highly structured core associated with variable domains of high conformational tolerance allowing insertions, deletions, or other substitutions.
  • Such antigen-binding proteins may be generated by grafting CDRs or variable regions described herein onto a suitable protein scaffold.
  • the structure of alternative scaffolds may vary, but preferably are of human origin for those developed as therapeutics.
  • an alternative protein scaffold of the present disclosure can be based either on a conventional immunoglobulin (Ig) backbone, or are derived from a completely unrelated protein. These variable domains can be modified to create novel binding interfaces toward any targeted antigen.
  • an alternative protein scaffold of the present disclosure can be derived from Protein A, e.g., the Z-domain thereof (affibodies), lmmE7 (immunity proteins), BPTI/APPI (Kunitz domains), Ras- binding protein AF-6 (PDZ-domains), charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neokarzinostatin, a fibronectin domain (used in "adnectin”), an ankyrin repeat (AR) domain (used in "DARPins”), avidity multimers (also known as “avimers”), or thioredoxin (Skerra, A
  • Anticalins are a suitable type of non-lg based alternative scaffolds for use in the antigen-binding molecules of the present disclosure.
  • Anticalins are a class of engineered ligand-binding proteins that are based on the lipocalin scaffold.
  • Lipocalins are a family of proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have limited sequence homology, but share a common tertiary structure architecture based on eight antiparallel 0-barrels. Lipocalins contain four exposed loops built on the rigid
  • Exemplary anticalin proteins that are commonly used are about a size of about 180 amino acids and a mass of about 20 kDa.
  • DARPins are another suitable non-lg based alternative scaffold that can be used in the antigenbinding molecules of the present disclosure.
  • DARPins are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat (AR) proteins, which usually contain a 33 amino acid protein motif consisting of two a-helices separated by loops, which repeats mediate protein— protein interactions.
  • AR ankyrin repeat
  • DARPins can be generated using combinatorial AR libraries constructed based on the 33 amino acid AR motif with seven randomized positions.
  • DARPin libraries can be screened using ribosome display, and library members typically are well produced in Escherichia coli, do not aggregate, and display high thermodynamic stability.
  • DARPins contain two to four of these motifs flanked by N- and C- terminal capping motifs to shield hydrophobic regions and allow increased solubility.
  • the avimer structure can also be used as a protein backbone to generate a suitable non-lg based alternative scaffold.
  • Avimers typically consist of two or more peptide sequences of 30 to 35 amino acids each, connected by peptide linker. The individual sequences are derived from A-domains of various membrane receptors and have a rigid structure, stabilized by disulfide bridges and calcium. Each A- domain can bind to a certain epitope of the target protein. The combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity.
  • Proteins derived from fibronectin III (FN3) domains can also be used to generate a suitable non- lg based alternative scaffold (also known as "monobody”).
  • FN10 fibronectin type III domain
  • the connecting loops of FN10 each about 15 to 21 amino acids in length, can be randomized and the domains displayed on both phage and yeast to select for a scaffold with the desirable properties.
  • AdnectinsTM is an exemplary scaffold generated using 10 th FN3 domains randomized and displayed in this way.
  • CentyrinTM Another exemplary scaffold comprising FN3 domains is a CentyrinTM.
  • CentryrinsTM contain the consensus sequence of FN3 domains of human Tenascin C (TNC), which is found in the extracellular matrix of various tissues.
  • CentyrinTM scaffolds have loops that have structural homology to antibody variable domains (i.e., CDR1, CDR2 and CDR3), and are small (about 10 kDa), simple, and highly stable single domain proteins that do not contain cysteine, disulfides or glycosylated residues.
  • CentyrinTM possess excellent biophysical properties such as stability to heat, pH, denaturant and organic solvents, reversible unfolding and monodispersity.
  • FLAPs fluctuation-regulated affinity proteins
  • fusion proteins and conjugates comprising at least one anti- CDH17 antigen-binding protein (e.g., antibody such as a single-domain antibody) linked, directly or indirectly, to one or more additional domains or moieties.
  • the at least one anti-CDH17 antigen-binding protein can specifically bind CDH17.
  • the fusion protein or conjugate of the present disclosure comprises a single polypeptide. In other embodiments, the fusion protein or conjugate of the present disclosure comprises more than one polypeptide. In some embodiments, the fusion protein or conjugate of the present disclosure comprises two polypeptides.
  • the fusion protein or conjugate of the present disclosure comprises at least one anti-CDH17 antigen-binding protein (e.g., antibody such as a single-domain antibody) described herein.
  • the fusion protein or conjugate is multivalent.
  • the fusion protein or conjugate of the present disclosure may be at least bivalent, but can also be e.g., trivalent, tetravalent, pentavalent, hexavalent, etc.
  • the terms "bivalent”, “trivalent”, “tetravalent”, “pentavalent”, or “hexavalent” all fall under the term “multivalent” and indicate the presence of two, three, four, five or six binding units (e.g., VHHs), respectively.
  • the fusion protein or conjugate is multispecific.
  • the one or more additional domain or moieties may be one or more additional binding domain that binds to one or more further antigen or protein.
  • the fusion protein or conjugate of the present disclosure may be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc.
  • a fusion protein or conjugate of the present disclosure comprises one or more of an anti-CDH17 antigen-binding protein described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises two or more of an anti-CDH17 antigen-binding protein described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises three or more of an anti-CDH17 antigen-binding protein described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises four or more of an anti- CDH17 antigen-binding protein described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises one, two, three, four, five, six, seven, eight, nine, or ten, or more of an anti-CDH17 antigen-binding protein described herein.
  • the one or more antigen-binding proteins can bind to the same epitope on CDH17. In some embodiments, the one or more antigen-binding proteins can bind to different epitopes on CDH17.
  • the one or more antigen-binding proteins can be one or more singledomain antibodies disclosed herein, for example, one or more VHHs disclosed herein.
  • the two or more anti-CDH17 antigen-binding proteins may comprise the same sequence or may comprise different sequences.
  • the two or more anti-CDH17 antigen-binding proteins may bind to the same epitope on CDH17 or different epitopes on CDH17.
  • a fusion protein or conjugate of the present disclosure may be biparatopic, e.g., if two VHHs bind two different epitopes on CDH17.
  • a fusion protein or conjugate of the present disclosure comprises at least one anti-CDH17 antigen-binding protein (e.g., antibody such as a single-domain antibody) provided herein operably linked to an immunoglobulin Fc region.
  • An immunoglobulin Fc region may be linked indirectly or directly to the at least one anti-CDH17 antigen-binding protein (e.g., antibody such as a single-domain antibody).
  • a fusion protein or conjugate of the present disclosure comprises one, two, three, four, five, six or more anti-CDH17 antigen-binding proteins provided herein operably linked to an Fc region.
  • an Fc region refers to a portion of a heavy chain constant region comprising CH2 and CH3.
  • an Fc region comprises a hinge, CH2, and CH3.
  • the hinge can mediate dimerization between two Fc-containing polypeptides.
  • An Fc region included in a fusion protein or conjugate may be an Fc region from any species, or derived from any species, including, but not limited to, human, mouse, rat, monkey (e.g., cyno), camel, llama, shark, goat, rabbit, and/or bovine.
  • an Fc region included in a fusion protein or conjugate of the present disclosure is a human immunoglobulin Fc region, or is derived from a human immunoglobulin Fc region.
  • the immunoglobulin Fc region is of IgG, IgE, IgM, IgD, IgA or IgY isotype.
  • the immunoglobulin Fc region is an IgG isotype, such as IgGl, lgG2, lgG3, or lgG4 subclass, or a variant thereof.
  • the immunoglobulin Fc region may comprise a variant or fragment of a native IgG Fc region.
  • an Fc region included in a fusion protein or conjugate described herein may be a murine (e.g., a mouse or a rat) immunoglobulin Fc region, or derived from a murine immunoglobulin Fc region.
  • an Fc region included in a fusion protein or conjugate described herein may be a cyno immunoglobulin Fc region, or derived from a cyno immunoglobulin Fc region.
  • a native Fc region typically possesses an effector function, including but not limited to, Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (for example B-cell receptor); and B-cell activation, etc.
  • effector functions generally require the Fc region to be combined with a binding domain (for example, an antibody variable domain) and can be assessed using various assays.
  • a fusion protein or conjugate of the present disclosure can comprise a dimer of Fc regions.
  • an Fc region mediates dimerization of the CDH17-binding units at physiological conditions, such as when expressed from a cell, such that a dimer is formed that doubles the number of CDH17 binding units.
  • a fusion polypeptide comprising one VHH domain that binds CDH17 and an Fc region is monovalent as a monomer, but the Fc region can mediate dimerization; as a result, the fusion protein is bivalent (i.e., having two anti-CDH17 VHH domains per molecule).
  • two anti-CDH17 VHH domains (2x) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is tetravalent (i.e., having four anti-CDH17 VHH domains per molecule).
  • three anti-CDH17 VHH domain (3x) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is hexavalent (i.e., having six anti-CDH17 VHH domains per molecule).
  • a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (anti-CDH17 VHH)n- Linker-Fc, wherein n can be any integral number (e.g., 1, 2, 3, 4, 5, etc). When n>2, each anti-CDH17 VHH may be optionally operably linked to another anti-CDH17 VHH via a linker.
  • a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (anti-CDH17 VHH)n- Linker-Fc-(anti-CDH17 VHH)m, wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc).
  • n>2 or m>2 each anti-CDH17 VHH may be optionally operably linked to another anti- CDH17 VHH via a linker.
  • a fusion protein or conjugate of the present disclosure is bivalent.
  • the bivalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CDH17 VHH)-Linker-Fc.
  • a fusion protein or conjugate of the present disclosure is tetravalent.
  • the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CDH17 VHH)-Linker- (anti-CDH17 VHH)-Linker-Fc.
  • the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CDH17 VHH)-Linker-Fc-Linker-(anti-CDH17 VHH).
  • the multiple linkers used in the fusion protein are not necessarily the same.
  • a fusion protein or conjugate of the disclosure is hexavalent.
  • the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CDH17 VHH)-Linker-(anti-CDH17 VHH)-Linker-(anti-CDH17 VHH)-Linker-Fc.
  • the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CDH17 VHH)-Linker-(anti-CDH17 VHH)-Linker-Fc-linker-(anti-CDH17 VHH).
  • the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-CDH17 VHH)-Linker-Fc- Linker-(anti-CDH17 VHH)-Linker-(anti-CDH17 VHH).
  • the multiple linkers used in the fusion protein are not necessarily the same.
  • the CH3 domain of the Fc region can be used as homodimerization domain, such that the resulting fusion protein may be formed from two identical polypeptides.
  • the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization.
  • a heterodimerization domain can be incorporated into the fusion protein such that the construct is a heterodimeric fusion protein.
  • the first and second Fc regions may be of the same IgG isotype such as, e.g., IgGl/IgGl, lgG2/lgG2, lgG4/lgG4.
  • the first and second Fc regions may be of different IgG isotypes such as, e.g., lgGl/lgG2, lgGl/lgG4, lgG2/lgG4, etc.
  • the Fc region included in a fusion protein or conjugate of the present disclosure can be mutated or modified.
  • the mutations include one or more amino acid substitutions to reduce an effector function of the Fc region.
  • mutations to Fc regions to alter, such as reduce, effector function are known, including any as described below.
  • the numbering of the residues in an immunoglobulin heavy chain or portion thereof, such as an Fc region is according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).
  • the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • amino acid modifications that can alter ADCC and/or CDC are described in Alegre et al, 1992 J Immunol, 148: 3461-3468; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Shields et al., 2001 JBC, 276(9): 6591-6604; Lazar et al., 2006 PNAS, 103(11): 4005-4010; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Stavenhagen et aL, 2008 Advan.
  • an Fc region included in a fusion protein or conjugate of the present disclosure exhibits reduced effector functions (such as CDC and ADCC).
  • Various in vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the fusion protein construct and/or cleaved components thereof lack FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • the primary cells for mediating ADCC are NK cells which express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII.
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in e.g., US 5,500,362; US 5,821,337; Hellstrom. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986); and Hellstrom et al., Proc. Nat'l Acad. Sci.
  • nonradioactive assay methods may be employed, such as ACTITM non-radioactive cytotoxicity assay for flow cytometry or CytoTox96TM non-radioactive cytotoxicity assay.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).
  • Clq binding assays may also be carried out to confirm that the fusion protein construct or cleaved components thereof is unable to bind Clq and hence lacks CDC activity (see, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402).
  • a CDC assay may be performed (see, e.g., Gazzano- Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).
  • mutations that enhance ADCC include modification at Ser239 and Ile332, for example Ser239Asp and He332Glu (S239D, 1332E).
  • mutations that enhance CDC include modifications at Lys326 and Glu333.
  • the Fc region is modified at one or both of these positions, for example Lys326Ala and/or Glu333Ala (K326A and E333A) using the Kabat numbering system.
  • the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu234Ala (L234A), Leu234Gly (L234G), Leu234Ser (L234S), Leu234Thr (L234T), Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Leu235Ser (L235S), Leu235Thr (L235T), Leu235Val (L235V), Leu235Gln (L235Q), Gly236Arg (G236R), Met252Tyr (M252Y), Ser254Thr (S254T), Thr256Glu (T256E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro
  • the human IgGl Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D).
  • the Fc region of the fusion protein is modified at amino acid Leu235 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A).
  • the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A/L235A) or Leu234Val and Leu235Ala (L234V/L235A).
  • the Fc region of the fusion protein is altered at amino acids at 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A/L235A/N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A/L235A/P329A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Asp265 (Kabat Numbering) to alter Fc receptor interactions, e.g Asp265Ala (D265A).
  • Asp265 Kabat Numbering
  • the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat Numbering) to alter Fc receptor interactions, e.g., Pro329Ala (P329A) or Pro329Gly (P329G).
  • Pro329A Pro329Ala
  • Pro329Gly P329G
  • the Fc region of the fusion protein is altered at both amino acids 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A/P329A) or Asp265Ala and Pro329Gly (D265A/P329G).
  • the Fc region of the fusion protein is altered at amino acids at 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A/L235A/D265A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Gly (L234A/L235A/P329G).
  • the Fc region of the fusion protein is altered at amino acids at 234, 235, 265 and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A/L235A/D265A/P329G).
  • the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding.
  • Gly235 is deleted from the fusion protein.
  • the human IgGl Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A).
  • the human IgGl Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).
  • the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Gly, Leu235Ser, Gly236Arg (L234G/L235S/G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Thr, Gly236Arg (L234S/L235T/G236R).
  • the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Val, Gly236Arg (L234S/L235V/G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Gln, Gly236Arg (L234T/L235Q/G236R).
  • the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Thr, Gly236Arg (L234T/L235T/G236R). In some embodiments, the Fc region of the fusion protein fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Thr, Leu235Thr, Pro329Gly (L234A/L235A/P329G).
  • the Fc region of the fusion protein is altered at amino acids at 252, 254, and 256, e.g., Met252Tyr, Ser254Thr, Thr256Glu (M252Y/S254T/T256E).
  • the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235).
  • the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions Glu233 (E233), Leu234 (L234), or Leu235 (L235) and is modified at one or more of the Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding.
  • an Fc region included in a CDH17 binding polypeptide is derived from a human Fc domain, and comprises a three amino acid deletion in the lower hinge corresponding to IgGl E233, L234, and L235.
  • such Fc polypeptides do not engage FcyRs and thus are referred to as "effector silent" or "effector null.”
  • Fc deletion of these three amino acids reduces the complement protein Clq binding.
  • a polypeptide with an Fc region with Fc deletion of these three amino acids retains binding to FcRn and therefore has extended half-life and transcytosis associated with FcRn mediated recycling.
  • the immunoglobulin Fc region of the fusion protein is a variant of human IgGl Fc region, having an amino acid sequence:
  • IgGl L234A, L235A also known as "LALA” variant
  • L235A also known as "LALA” variant
  • the immunoglobulin Fc region of the fusion protein is a variant of human IgGl Fc region, having an amino acid sequence:
  • IgGl L234A, L235A, and P329A also known as "LALAPA” variant
  • the immunoglobulin Fc region of the fusion protein is a variant of human IgGl Fc region, having an amino acid sequence:
  • IgGl D265A, N297A and P329A also known as "DANAPA” variant
  • the immunoglobulin Fc region of the fusion protein is a variant of human
  • IgGl Fc region having an amino acid sequence: IgGl L234A, L235A, and G237A (also known as "LALAGA” variant) (mutations bolded in the sequence below)
  • the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
  • IgGl L234G/L235S/G236R (mutations bolded in the sequence below)
  • the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
  • IgGl L234S/L235T/G236R (mutations bolded in the sequence below)
  • the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
  • IgGl L234S/L235V/G236R (mutations bolded in the sequence below)
  • the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
  • IgGl L234T/L235Q/G236R (mutations bolded in the sequence below)
  • the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
  • the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence: IgGl L234A/L235A/P329G (mutations bolded in the sequence below) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
  • the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence: IgGl M252Y/S254T/T256E (mutations bolded in the sequence below) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPG (SEQ ID NO:
  • the human IgG Fc region is modified to enhance FcRn binding.
  • Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol.
  • the Fc region lacks or has reduced fucose attached to the N-linked glycan-chain at N297.
  • fucosylation There are numerous ways to prevent fucosylation, including but not limited to production in a FUT8 deficient cell line; addition inhibitors to the mammalian cell culture media, for example Castanospermine; and metabolic engineering of the production cell line.
  • the Fc domain included in a fusion protein or conjugate of the present disclosure is derived from a human Fc domain and comprises mutations M252Y and M428V.
  • the mutated or modified Fc polypeptide includes the following mutations: M252Y and M428L using the Kabat numbering system. In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5), while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn mediated recycling and extended half-life.
  • the Fc domain included in a fusion protein or conjugate is derived from a human Fc domain and comprises mutations to induce heterodimerization.
  • mutations include those referred to as "knob" and "hole” mutations.
  • having an amino acid modification within the CH3 domain at Thr366, which when replaced with a bulkier amino acid, e.g., Try (T366W) is able to preferentially pair with a second CH3 domain having amino acid modifications to less bulky amino acids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala and Vai, respectively (T366S/L368A/Y407V).
  • the "knob” Fc domain comprises the mutation T366W.
  • the "hole” Fc domain comprises mutations T366S, L368A, and Y407V.
  • Fc domains used for heterodimerization comprise additional mutations, such as the mutation S354C on a first member of a heterodimeric Fc pair that forms an asymmetric disulfide with a corresponding mutation Y349C on the second member of a heterodimeric Fc pair.
  • one member of a heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding.
  • one member of a heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435.
  • the hole Fc domain comprises the modification H435R or H435K (referred to as "hole-R" in some instances when the modification is H435R), while the knob Fc domain does not.
  • the hole-R mutation improves purification of the heterodimer over homodimeric hole Fc domains that may be present.
  • the human IgG Fc region is modified to prevent dimerization.
  • the fusion proteins of the present disclosure are monomeric. For example, modification at residue Thr366 to a charged residue, e.g. Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.
  • the immunoglobulin Fc region of the fusion protein is of human lgG3 isotype, or a variant thereof.
  • the lgG3 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D).
  • the human lgG3 Fc region is modified at amino acid 435 to extend the half-life, e.g., Arg435His (R435H).
  • the human lgG3 Fc region lacks Lys447 (EU index of Kabat et al 1991).
  • the immunoglobulin Fc region of the fusion protein is of human lgG4 isotype, or a variant thereof.
  • an immunoglobulin Fc region of human lgG4 isotype may have an amino acid sequence:
  • the immunoglobulin Fc region of human lgG4 isotype may comprise the amino acid sequence set forth in SEQ ID NO: 1603, or a similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • the human lgG4 Fc region comprises one or more mutations selected from, e.g., Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and/or Pro329Gly (P329G) according to EU numbering.
  • Ser228Pro S228P
  • Leu235Glu L235E
  • Leu235Ala L235A
  • Phe234Ala F234A
  • P329G Pro329Gly
  • the lgG4 Fc region of the fusion protein is altered at amino acids at 228 and 235, e.g., Ser228Pro, Leu235Glu or Leu235Ala (S228P/L235E or S228P/L235A). In some embodiments, the lgG4 Fc region of the fusion protein is altered at amino acids at 228, 234 and 235, e.g., Ser228Pro, Phe234Ala, Leu235Glu or Leu235Ala (S228P/F234A/L235E or S228P/F234A/L235A).
  • the lgG4 Fc region of the fusion protein is altered at amino acids at 228, 235, and 329, e.g., Ser228Pro, Leu235Glu and P329G (S228P/L235E/P329G).
  • the human lgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E).
  • the human lgG4 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D).
  • the human lgG4 Fc region lacks Lys447 (EU index of Kabat et al 1991).
  • the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1572) [0266] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG
  • the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, F234A, L235E (mutations bolded in the sequence below)
  • the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, F234A, L235A (mutations bolded in the sequence below)
  • the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 P329G, S228P, L235E (mutations bolded in the sequence below)
  • Additional lgG4 heavy chain modifications suitable for use in the fusion proteins or conjugates of the present disclosure include those described in Tables 1 and 2 of Dumet et aL, mAbs, 11:8, 1341-1350, which is incorporated herein by reference in its entirety.
  • the fusion protein or conjugate contains an immunoglobulin hinge region.
  • the hinge region serves as a linker to connect one or more CDH17 binding units (e.g., VHHs) to the Fc region.
  • the fusion protein can comprise a linker in addition to the hinge region to connect the one or more CDH17 binding units (e.g., VHHs) to the Fc region.
  • the hinge region can be selected from any of the human IgG subclasses.
  • the fusion protein may contain a modified IgGl hinge having the sequence of EPKSSDKTHTCPPC (SEQ ID NO: 1577), wherein the Cys220 that typically forms a disulfide bond with the C-terminal cysteine of the light chain is mutated to serine, e.g., Cys220Ser (C220S).
  • the fusion protein contains a truncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 1578).
  • the fusion protein or conjugate has a modified hinge from lgG4, which is modified to prevent or reduce strand exchange, e.g., Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 1579).
  • S228P Ser228Pro
  • ESKYGPPCPPC SEQ ID NO: 1579
  • a fusion protein or conjugate of the present disclosure may comprise sequences other than an Fc region to achieve multimerization (e.g., dimerization).
  • an amino acid sequence containing at least one cysteine residue may be included to facilitate dimerization of two polypeptides by formation of a disulfide bond between the two polypeptides.
  • such multimerizing domain may comprise one or more cysteine residues, or a short cysteine-containing peptide.
  • Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.
  • Fc mutations suitable for use in the fusion proteins disclosed herein are also discussed in, e.g., Wilkinson et al., Fc-engineered antibodies with immune effector functions completely abolished.
  • a fusion protein or conjugate of the present disclosure may comprise one or more other moieties which provide the fusion protein or conjugate with increased (in vivo) halflife.
  • In vivo half-life extension means, that the fusion protein or conjugate has an increased half-life in a mammal, such as a human subject, after administration.
  • Non-limiting examples of half-life extension moieties suitable for use in the present disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.
  • PEG polyethylene glycol
  • a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG.
  • a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to human serum albumin.
  • the binding moiety is a single-domain antibody (e.g., VHH).
  • albumin binders that are described in, e.g., WO 04/041865, WO 06/122787, W02012/175400, WO 2012/175741, WO2015/173325, W02017/080850, WO2017/085172, WO2018/104444, W02018/134235, WO2018/134234, each of which is incorporated herein by reference is its entirety, can be used in the fusion protein or conjugate of the present disclosure.
  • Anti-CDH17 antigen-binding proteins may be operably linked, directly or indirectly, to a second moiety, such as but not limited to, a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a small molecule drug, a chemotherapeutic agent, a therapeutic agent, a diagnostic agent, or a combination thereof.
  • a conjugate of the present disclosure comprises a label, which can generate a detectable signal.
  • a label can be used for research or diagnostic purposes, such as for the in vivo detection of cancer.
  • the label is capable of producing, either directly or indirectly, a detectable signal.
  • the label may be radio-opaque or a radioisotope (such as 3H, 14C, 32P, 35S, 1231, 1251, 1311); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound (such as fluorescein isothiocyanate, rhodamine or luciferin); an enzyme (such as fJ- galactosidase, alkaline phosphatase, or horseradish peroxidase); an imaging agent; or a metal ion.
  • a radioisotope such as 3H, 14C, 32P, 35S, 1231, 1251, 1311
  • a fluorescent (fluorophore) or chemiluminescent (chromophore) compound such as fluorescein isothiocyanate, rhodamine or luciferin
  • an enzyme such as fJ- galactosidase, alkaline phosphatase, or horseradish
  • the label is a radioactive atom for scintigraphic studies, for example 99Tc or 1231, or a spin label for nuclear magnetic resonance (NMR) imaging, such as zirconium-89, iodine-123, iodine- 131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • Zrconium-89 may also be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011/056983).
  • Anti-CDH17 antigen-binding proteins e.g., antibodies such as single-domain antibodies
  • Anti-CDH17 antigen-binding proteins of the present disclosure may be conjugated to another moiety, such as an epitope tag, e.g., for the purpose of purification or detection.
  • an epitope tag e.g., for the purpose of purification or detection.
  • Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is commonly employed in protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound.
  • Non-limiting examples of epitope tag molecules that can be conjugated to anti-CDH17 antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure, e.g., for the purposes of molecular recognition include a poly-histidine tag (His-tag), a myc-tag, human influenza hemagglutinin (HA) tag, a FLAG-tag, maltose-binding protein, glutathione-S-transferase, biotin, and streptavidin.
  • Conjugates containing the epitopes presented by these molecules are capable of being recognized by complementary molecules such as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively.
  • complementary molecules such as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively.
  • solid phase resins include agarose beads, which are compatible with purifications in
  • a conjugate of the present disclosure may comprise one or more anti- CDH17 VHH domains described herein conjugated to a therapeutic agent, which can be cytotoxic, cytostatic or otherwise provides some therapeutic benefit.
  • the cytotoxic agent is a drug, a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate).
  • a drug conjugates described herein may allow targeted delivery of a drug moiety to a target tissue (e.g., tumors).
  • a conjugate of the present disclosure comprises a toxin.
  • the toxin includes, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et aL, Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. NatL Acad. Sci.
  • the toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Examples of other therapeutic agents that can be conjugated to an anti-CDH17 antigen-binding protein of the present disclosure are described herein (see “Treatment Methods and Other Uses" section).
  • anti-CDH17 antigen-binding proteins e.g., antibodies such as singledomain antibodies
  • the moiety that can facilitate delivery of an anti-CDH17 antigen-binding protein to the central nervous system (CNS)/brain can be for example, a peptide, a polypeptide, small molecule, a lipid, or a synthetic polymer.
  • Various approaches to deliver single-domain antibodies into the brain are described in Pothin et aL, Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.
  • an anti-CDH17 antigen-binding protein e.g., antibody such as singledomain antibody
  • a moiety e.g., an antibody
  • TfR transferrin receptor
  • the transferrin receptor (TfR) is highly expressed by brain capillary endothelial cells (BCECs) forming the blood-brain barrier (BBB) and has been utilized as a target for brain drug delivery.
  • an anti-CDH17 antigen-binding protein e.g., antibody such as single-domain antibody
  • hydrophobic fatty acid moieties such as C18 fatty acid (stearic acid), C16 fatty acid (palmitic acid) or C8 fatty acid (octanoic acid) moieties; or amphiphilic block copolymer moieties, such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (pluronics or poloxamers) or poly(2-oxasolines).
  • fatty acid moieties and block copolymer moieties that can be utilized for brain delivery of proteins are described in, e.g., Yi and Kabanov, J Drug Target. 2013; 21(10): 940-955, which is incorporated herein by reference in its entirety.
  • Example methods for attaching a moiety, such as a label, to a binding protein include those described in Hunter, et al., Nature 144:945 (1962); David, et aL, Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Elsevier, N.Y. (1983); and Colcher et al., Meth. Enzymol., 121 :802-16 (1986).
  • the attachment between an anti-CDH17 antigen-binding protein and a second moiety can be covalent or non-covalent, e.g., via a biotin-streptavidin non-covalent interaction.
  • a second moiety can be attached to an anti-CDH17 antigen-binding protein using any of various molecular biological or chemical conjugation and linkage methods known in the art and described below.
  • linkers such as peptide linkers, cleavable linkers, non-cleavable linkers or linkers that aid in the conjugation reaction, can be used to link or conjugate a second moiety to an anti-CDH17 antigen-binding protein described herein.
  • an anti-CDH17 antigen-binding protein e.g., antibody such as singledomain antibody
  • one or more second moieties e.g., about 1 to about 20 moieties per molecule, optionally via a linker.
  • the one or more second moieties can be the same or different.
  • the linker may be composed of one or more linker components. For covalent attachment of an antibody and the second moiety, the linker typically has two reactive functional groups, i.e., bivalency in a reactive sense.
  • Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups have been described in, e.g., Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242.
  • a linker used in a conjugate of the present disclosure may include 6- maleimidocaproyl ("MC”), maleimidopropanoyl ("MP”), valine-citrulline (“val-cit”), a alaninephenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2- pyridylthio)pentanoate (“SPP”), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate (“SMCC”), or N-Succinimidyl(4-iodo-acetyl)aminobenzoate (“STAB”), or a combination thereof.
  • MC 6- maleimidocaproyl
  • MP maleimidopropanoyl
  • val-cit valine-citrulline
  • ala-phe alaninephenylalanine
  • a linker used in a conjugate of the present disclosure may comprise amino acid residues.
  • Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • Exemplary dipeptides include valine-citrulline (vc or val-cit), alaninephenylalanine (af or ala-phe).
  • Exemplary tripeptides include glyci ne-valine-citrul line (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).
  • Amino acid residues used in an amino acid linker component may include naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline.
  • Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
  • Conjugates of an anti-CDH17 antigen-binding protein e.g., antibody such as single-domain antibody
  • second moiety e.g., cytotoxic agent
  • bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyl dithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as N-succinimidyl
  • Conjugates of the present disclosure can be prepared by a variety of methods.
  • the conjugation method may include: (1) reaction of a nucleophilic group of a VHH domain with a bivalent linker reagent, to form VHH-Linker, via a covalent bond, followed by reaction with a drug moiety; or (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form drug-linker, via a covalent bond, followed by reaction with the nucleophilic group of a VHH domain.
  • Nucleophilic groups on proteins including antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups (e.g., lysine), (iii) side chain thiol groups (e.g., cysteine), and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
  • Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
  • active esters such as NHS esters, HOBt esters, haloformates, and acid halides
  • alkyl and benzyl halides such as haloacetamides
  • aldehydes, ketones, carboxyl, and maleimide groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, hal
  • Additional nucleophilic groups can be introduced into proteins (e.g., antibodies such as VHH domains) through the reaction of lysines with 2- iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.
  • Reactive thiol groups may be introduced into a protein (e.g., antibody such as a VHH domain) by introducing one, two, three, four, or more cysteine residues.
  • Conjugates such as antibody drug conjugates, may also be produced by modification of an antibody, such as a VHH domain, to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug.
  • the sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may lead with the amine group of linker reagents or drug moieties.
  • the resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages.
  • reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques).
  • proteins containing N-terminal serine or threonine residues can react with sodium metaperiodate, resulting in production of an aldehyde in place of the first amino acid.
  • Such aldehyde can be reacted with a drug moiety or linker nucleophile.
  • nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBi esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
  • a fusion protein containing a VHH domain and cytotoxic agent may be made, e.g., by recombinant DNA techniques or peptide synthesis.
  • a DNA sequence may be engineered to comprise respective regions encoding the two portions of the fusion protein either adjacent to one another or separated by a region encoding a linker peptide which does not impair the desired properties of the fusion protein.
  • the DNA sequence can be then transfected into a host cell that expresses the fusion protein.
  • the fusion protein can be recovered from the cell culture and purified using techniques known in the art.
  • the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via peptide linkers.
  • a peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects a peptide linker ranges from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length.
  • a peptide linker e.g., a peptide linker separating two VHH domains or an VHH domain and a heavy chain constant region, is at least 5 amino acids, at least 6 amino acids or at least 7 amino acids in length and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids or up to 60 amino acids in length.
  • the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length.
  • the linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length.
  • the linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids in length.
  • charged (e.g., charged hydrophilic linkers) and/or flexible linkers are used.
  • flexible linkers that can be used in the fusion proteins of the disclosure include those disclosed by Chen et ai, 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et a/., 2014, Protein Engineering, Design & Selection 27(10): 325-330.
  • GS-linker glycines and serines
  • G n S SEQ ID NO: 1553
  • SG n SEQ ID NO: 1554
  • n is an integer from 1 to 10, e.g., 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10.
  • the linker is or comprises a monomer or multimer of repeat of G 4 S (SEQ ID NO: 1507), e.g., (GGGGS)n (SEQ ID NO: 1555).
  • Polyglycine linkers can suitably be used in the fusion proteins of the disclosure.
  • a peptide linker used herein comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 1556), five consecutive glycines (5Gly) (SEQ ID NO: 1557), six consecutive glycines (6Gly) (SEQ ID NO: 1558), seven consecutive glycines (7Gly) (SEQ ID NO: 1559), eight consecutive glycines (8Gly) (SEQ ID NO: 1560), or nine consecutive glycines (9Gly) (SEQ ID NO: 1561).
  • a GS-linker used herein comprises an amino acid sequence selected from GGSGGS (SEQ ID NO: 1562), i.e., (GGS) 2 (SEQ ID NO: 1562); GGSGGSGGS (SEQ ID NO: 1563), i.e., (GGS) 3 (SEQ ID NO: 1563); GGSGGSGGSGGS (SEQ ID NO: 1564), i.e., (GGS) 4 (SEQ ID NO: 1564); and GGSGGSGGSGGSGGS (SEQ ID NO: 1565), i.e., (GGS) 5 (SEQ ID NO: 1565).
  • the fusion proteins can include a combination of a GS-linker and a glycine linker.
  • two or more VHHs are linked via a GGGGSGGGGSGGGGS (SEQ ID NO: 1508) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGS (SEQ ID NO: 1566) linker.
  • a VHH and an Fc region are linked via a GGGGSESKYGPPCPSCP (SEQ ID NO: 1547) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGS (SEQ ID NO: 1507) linker.
  • the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via a "rigid" peptide linker. Such peptidic linker may comprise a prolinerich peptide. In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO:
  • a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPAP (SEQ ID NO:
  • a rigid peptide linker comprises PAPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO:
  • a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 1551). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPGGGS (SEQ ID NO: 1552).
  • a rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 1567), where n is any integer, e.g., 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10.
  • n is any integer, e.g., 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10.
  • the fusion protein described herein may further comprise a signal sequence at its N-terminus.
  • Signal sequences may be present in the precursor molecule of the fusion protein and may be removed after the protein is secreted from the host cell during production.
  • the signal sequence is MAVMAPRTLVLLLSGALALTQTWA (SEQ ID NO: 1580) or a fragment or variant thereof.
  • the signal sequence is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 1581), or a fragment or variant thereof.
  • polynucleotide molecules encoding the anti-CDH17 antigen-binding proteins e.g., antibodies including single-domain antibodies
  • polypeptide portion(s) of a conjugate of the present disclosure are also encompassed within the present disclosure.
  • a polynucleotide molecule of the present disclosure comprises the nucleotide sequence selected from SEQ ID NOs: 67-80, 1269-1506, 1627-1630, and 1807-1835, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • a polynucleotide molecule of the present disclosure the nucleotide sequence of any one of SEQ ID NOs: 67-80, 1351, and 1627-1630, or a similar sequence thereof having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence selected from SEQ ID NOs: 5, 10, 15, 20, 24, T1 , 31, 36, 41, 46, 51, 56, 61, 66, 343-554, 1623-1626, and 1672-1691, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence selected from SEQ ID NOs: 5, 10, 15, 20, 24, T1 , 31, 36, 41, 46, 51, 56, 61, 66, 421, and 1623-1626, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • a polynucleotide molecule may be used to transform/transfect a host cell or host organism, e.g., for expression and/or production of a polypeptide.
  • Suitable hosts or host cells for production of an anti- CDH17 polypeptides described herein include any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
  • a host or host cell comprising a polynucleotide molecule encoding an anti-CDH17 antigen-binding protein polypeptide or fusion protein described herein is also encompassed by the present disclosure.
  • a polynucleotide molecule may be for example DNA, RNA, or a hybrid thereof, and may also comprise (e.g., chemically) modified nucleotides, like locked nucleic acids (LNA) or peptide nucleic acids (PNA).
  • LNA locked nucleic acids
  • PNA peptide nucleic acids
  • the polynucleotide is single-stranded.
  • the polynucleotide is double-stranded.
  • the polynucleotide is in the form of doublestranded DNA (e.g., plasmid).
  • the polynucleotide is in the form of a singlestranded RNA (e.g., mRNA).
  • Techniques for generating polynucleotides may include, for example but not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers.
  • polynucleotides of the present disclosure may be isolated from a suitable natural source. Polynucleotide sequences encoding naturally occurring (poly)peptides can for example be subjected to site-directed mutagenesis, to generate a polynucleotide molecule encoding polypeptide with sequence variation.
  • vectors comprising the polynucleotide molecules encoding the antiCD H17 antigen-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or other relevant polypeptides of the present disclosure.
  • a "vector” as used herein is a vehicle suitable for carrying genetic material into a host cell.
  • a vector can include a nucleic acid vector, such as a plasmid or mRNA, or nucleic acids embedded into a bigger structure, such as a liposome or viral vector.
  • a vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters, enhancers, terminators) that regulate the expression of a polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example,
  • promoters, enhancers, terminators e.g., promoters, enhancers, terminators
  • selectable marker genes such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example,
  • the vector is an expression vector, i.e. a vector suitable for expressing an encoded polypeptide or construct under suitable conditions in a host cell.
  • polynucleotides encoding partial or full-length polypeptide chains e.g., obtained as described above (e.g., VHH, VHH-Fc) can be inserted into expression vectors such that the genes are operatively linked to one or more transcriptional and translational control sequences.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • Polynucleotides encoding the two or more polypeptide chains (when present and differ from one another) of an anti-CDH17 antigen-binding protein or fusion protein of the present disclosure can be inserted into separate vectors, or, optionally, incorporated into the same expression vector.
  • the recombinant expression vectors of the invention may include regulatory sequences that control the expression of genes encoding the polypeptide chain(s) in a host cell.
  • the design of the expression vector, including the selection of regulatory sequences, may depend on the choice of the host cell to be transformed and/or the desired level of protein expression.
  • suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • viral elements include those described in, e.g., U.S. Pat. Nos. 5, 168,062; 4,510,245; and 4,968,615; the disclosures of each of which are incorporated herein by reference.
  • Recombinant expression vectors of the present disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • a selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., US4,399,216; US 4,634,665; and US 5,179,017; the disclosure of each of which is incorporated herein by reference in its entirety).
  • the selectable marker gene confers resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin, or cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced.
  • antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin
  • cytotoxic drugs such as G418, puromycin, blasticidin, hygromycin or methotrexate
  • Vectors of the present disclosure may further include sequence elements that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription.
  • sequence elements include, e.g., 5' and 3' untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • IRS internal ribosomal entry site
  • polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • Viral vectors can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral vectors are particularly useful for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents to induce gene integration.
  • Suitable viral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses (AAV) such as AAV2, AAV8, AAV9), negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
  • a retrovirus e.g., Ad5, Ad26, Ad34, Ad35, and Ad48
  • parvovirus e.g., adeno-associated viruses (AAV) such as AAV2, AAV8, AAV9
  • negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
  • RNA viruses such as picornavirus and alphavirus
  • double-stranded DNA viruses including adenovirus, herpes virus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), baculovirus, coronavirus, and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
  • herpes virus e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus
  • baculovirus e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox.
  • VVA modified vaccinia Ankara
  • viruses useful for delivering polynucleotides encoding polypeptides of the present disclosure include, for example Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus.
  • retroviruses include, but are not limited to, avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M.1996. Fundamental Virology, DMKDN Fields, PM Howley, ed.
  • viral genomes useful in the compositions and methods of the present disclosure include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline sarcoma virus, feline leukemia virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.
  • the present disclosure also provides host cells or host organisms that comprise the polynucleotides or vectors encoding the anti-CDH17 antigen-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or other relevant polypeptides described herein.
  • Suitable host cells or host organisms can be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • Host cells can also include cells transfected in vivo with a polynucleotide(s) or vector provided herein.
  • Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris); plant cells; and insect cells.
  • mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), COS cells, SP2/0 cells, and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells.
  • Exemplary prokaryotic cells include bacterial cells such as Escherichia coli.
  • the present disclosure also provides methods of producing the anti-CDH17 antigen-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or conjugates described herein.
  • anti-CDH17 antigen-binding proteins e.g., antibodies including single-domain antibodies
  • fusion proteins e.g., fusion proteins, or conjugates described herein.
  • a method may comprise transforming/transfecting a host cell or host organism with a polynucleotide encoding an anti-CDH17 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or other relevant polypeptide(s) described herein, expressing the anti-CDH17 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or other relevant polypeptide(s) in the host, optionally followed by one or more isolation and/or purification steps.
  • an anti-CDH17 antigen-binding protein e.g., antibody such as single-domain antibody
  • fusion protein e.g., antibody such as single-domain antibody
  • recombinant expression vectors encoding one or more polypeptide(s) of an anti-CDH17 antigen-binding protein e.g., antibody such as single-domain antibody
  • fusion protein e.g., fusion protein, or conjugate of the present disclosure
  • the host cells are cultured for a period of time sufficient to allow for expression of the protein(s) or polypeptide(s) in the host cells or secretion of the protein(s) or polypeptide(s) into the culture medium in which the host cells are grown.
  • Protein(s) or polypeptide(s) can be recovered from the culture medium using standard protein purification methods.
  • Host cells can also be used to produce portions of intact antibodies, such as VHH domains.
  • a protein or polypeptide of the present disclosure can be purified by any method known in the art for purification of a protein or polypeptide, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for CDH17 after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the proteins or polypeptides of the present disclosure can be fused to heterologous polypeptide sequences described herein (e.g., His-tag) or otherwise known in the art to facilitate purification or to produce therapeutic conjugates below). Once isolated, a protein or polypeptide of the present disclosure can, if desired, be further purified, e.g., by high performance liquid chromatography, or by gel filtration chromatography, such as on a SuperdexTM column.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for CDH17 after Protein A or Protein G selection, and sizing
  • the present disclosure also provides a composition comprising anti-CDH17 antigen-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or conjugate of the present technology, at least one polynucleotide molecule encoding the same, at least one vector comprising such a polynucleotide molecule, or at least one host cell comprising the polynucleotide molecule or vector.
  • the composition may be a pharmaceutical composition.
  • the composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions.
  • suitable formulations include, but are not limited to, solutions, suspensions, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Phdomain Sci Technol 52:238- 311.
  • a pharmaceutical composition of the present disclosure may be formulated according to its intended route of administration.
  • suitable routes of administration include, e.g., intravenous, subcutaneous, intratumoral, oral (e.g., buccal, sublingual), intranasal, inhalation, intraocular, intramuscular, intradermal, transdermal (i.e., topical), intraperitoneal, transmucosal, vaginal, and rectal administration, or injection to the CNS/brain (e.g., intraspinal, intracerebral, or intrathecal administration).
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; fixed oils; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as phosphates, acetates, or citrates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of plastic or glass.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include, for example, physiological saline, bacteriostatic water, Cremophor EL®, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition is preferably sterile and has a proper fluidity. In most embodiments, the composition is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the contamination by microorganisms can be achieved by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation include vacuum drying and/or freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously steri I e-f i Itered solution thereof.
  • Oral compositions may include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, capsules, or liquid forms. Formulation in tablet and liquid forms may be used for protease insensitive VHHs. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • compounds of the present disclosure may be formulated to facilitate crossing of the blood-brain barrier.
  • anti-CDH17 antigen-binding proteins e.g., antibody such as single-domain antibody
  • fusion proteins e.g., fusion proteins, or conjugates of the present disclosure may be encapsulated into brain targeted liposomes, lipid nanoparticles, lipid microparticles, or lipid microcapsules for brain delivery.
  • Example liposomes delivery systems are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.
  • the active compounds are prepared with carriers that can protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,811, which is incorporated herein by reference in its entirety.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure is dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the pharmaceutical compositions (or components thereof) can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in diagnostic kits with instructions for use.
  • compositions are administered in an amount effective for treatment or prophylaxis of the specific indication.
  • the therapeutically effective amount is typically dependent on the weight of the subject being treated, the physical or health condition of the subject, the extensiveness of the condition to be treated, or the age of the subject being treated.
  • the pharmaceutical composition may be administered in an amount in the range of about 50 pg/kg body weight to about 50 mg/kg body weight per dose.
  • the pharmaceutical composition may be administered in an amount in the range of about 100 pg/kg body weight to about 50 mg/kg body weight per dose.
  • the pharmaceutical composition may be administered in an amount in the range of about 100 pg/kg body weight to about 20 mg/kg body weight per dose.
  • the pharmaceutical composition may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.
  • the frequency and the duration of the treatment can be adjusted.
  • Effective dosages and schedules for administering a pharmaceutical composition of the present disclosure may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • interspecies scaling of dosages can be performed using well- known methods in the art (e.g., Mordenti et al., 1991, Phdomainaceut. Res. 8:1351).
  • the pharmaceutical composition may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 200 mg per dose.
  • the antigen-binding proteins of the present disclosure are administered as a viral vector (e.g., an AAV)
  • dose ranges and frequency of administration of the viral vector described herein can vary depending on the nature of the viral vector, and the medical condition, as well as parameters of a specific patient and the route of administration used.
  • viral vector compositions can be administered to a subject at a dose ranging from about Ixio 5 plaque forming units (pfu) to about lxlO 15 pfu, depending on mode of administration, the route of administration, the nature of the disease and condition of the subject.
  • the viral vector compositions can be administered at a dose ranging from about lxlO 8 pfu to about lxlO 15 pfu, or from about lxlO 10 pfu to about lxlO 15 pfu, or from about lxlO 8 pfu to about lxlO 12 pfu.
  • a more accurate dose can also depend on the subject in which it is being administered. For example, a lower dose may be required if the subject is juvenile, and a higher dose may be required if the subject is an adult human subject. In certain embodiments, a more accurate dose can depend on the weight of the subject.
  • a juvenile human subject can receive from about lxlO 8 pfu to about lxlO 10 pfu, while an adult human subject can receive a dose from about lxlO 10 pfu to about lxlO 12 pfu.
  • Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, intraspinal, intracerebral, intrathecal and oral routes.
  • the composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • a pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Grit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida.
  • a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, intramuscular, intratumoral, intraperitoneal, intraspinal, intracerebral, and intrathecal injections, drip infusions, etc.
  • the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • the amount of the antigen-binding proteins described herein may be about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, the antigen-binding proteins described herein may be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.
  • the pharmaceutical composition may be administered as needed to a subject.
  • an effective dose of the pharmaceutical composition is administered to a subject one or more times.
  • an effective dose of the pharmaceutical composition is administered to the subject once a month, less than once a month, such as, for example, every two months, every three months, or every six months.
  • an effective dose of the pharmaceutical composition is administered more than once a month, such as, for example, every two weeks, every week, twice per week, three times per week, daily, or multiple times per day.
  • An effective dose of the pharmaceutical composition is administered to the subject at least once.
  • the effective dose of the pharmaceutical composition may be administered multiple times, including for periods of at least a month, at least six months, or at least a year.
  • the pharmaceutical composition is administered to a subject as needed to alleviate one or more symptoms of a condition.
  • a pharmaceutical composition of the present disclosure may be administered to a subject at levels lower than that required to achieve the desired therapeutic effect and the dosage may be gradually increased until the desired effect is achieved.
  • a pharmaceutical composition of the present disclosure may be administered at a high dose and subsequently administered progressively lower doses until a therapeutic effect is achieved.
  • a suitable daily dose of an antigen-binding protein of the invention is an amount of the antibody which is the lowest dose effective to produce a therapeutic effect.
  • compositions of the present disclosure may optionally include more than one active agent.
  • compositions of the present disclosure may contain an anti-CDH17 antigenbinding protein conjugated to, admixed with, or administered separately from another pharmaceutically active molecule, e.g., a chemotherapeutic agent, a vascular endothelial growth factor (VEGF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, or an apoptosis-inducing agent, an immunotherapeutic agent, or a combination thereof.
  • a chemotherapeutic agent e.g., a chemotherapeutic agent, a vascular endothelial growth factor (VEGF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, or an apoptosis-inducing agent, an immunotherapeutic agent, or a combination thereof.
  • VEGF vascular endothelial growth factor
  • EGFR epidermal growth factor receptor
  • an apoptosis-inducing agent e.g
  • an anti-CDH17 antigen-binding protein may be admixed with one or more additional active agents, such as a chemotherapeutic agent, a vascular endothelial growth factor (VEGF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, or an apoptosis-inducing agent, an immunotherapeutic agent, or a combination thereof, to treat a cancer described herein.
  • additional active agents such as a chemotherapeutic agent, a vascular endothelial growth factor (VEGF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, or an apoptosis-inducing agent, an immunotherapeutic agent, or a combination thereof.
  • additional active agents such as a chemotherapeutic agent, a vascular endothelial growth factor (VEGF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, or an apoptosis-inducing agent, an immunotherapeutic agent, or a combination thereof.
  • VEGF vascular
  • Examples of additional active agents that can be used to induce cell death and that can be conjugated to, admixed with, or administered separately from an anti-CDH17 antigen-binding protein of the present disclosure include cytotoxic agents, e.g., those described herein.
  • provided herein is a method of using anti-CDH17 antigen-binding proteins, fusion proteins, and/or conjugates of the present disclosure to induce cell death in a cell (e.g., a cancer cell) and/or a population of cells expressing CDH17.
  • the methods may comprise contacting the cells and/or population of cells with an anti-CDH17 antigen-binding protein, fusion protein and/or conjugate described herein.
  • the cells and/or population of cells may comprise cancer cells.
  • the cancer cells may comprise a solid tumor.
  • the methods may be carried out in vitro or in vivo. When such methods are carried out in vivo, the methods can further comprise administering the anti-CDH17 antigen-binding protein, fusion protein or conjugate described herein into a subject in need thereof.
  • the subject may be a mammal (e.g., a human).
  • anti-CDH17 antigen-binding proteins, fusion proteins or conjugates of the present disclosure may be capable of inducing cell death in between 1% and 100% cells of a population treated cells (e.g., cancer cells) (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), as measured, e.g., by fluorescence activated cell sorting (FACS) analysis.
  • FACS fluorescence activated cell sorting
  • anti-CDH17 antigen-binding proteins of the present disclosure are capable of interacting with and promoting signal transduction events mediated by CDH17.
  • Anti-CDH17 antigen-binding proteins of the present disclosure may be able to induce conformational changes within CDH17 that lead, e.g., to receptor trimerization. This spatial configuration has been shown to render CDH17 active for apoptosis signal transduction.
  • anti-CDH17 antigen-binding proteins e.g., antibody such as single-domain antibody
  • fusion proteins, conjugates, polynucleotide molecules, vectors, and/or host cells described herein, or pharmaceutical compositions thereof are useful for the (prophylactic or therapeutic) treatment of a wide array of diseases or disorders.
  • the present technology provides an anti- CDH17 antigen-binding protein (e.g., antibody such as single-domain antibody), a fusion protein, a conjugate, a polynucleotide molecule, a vector, or a host cell for use as a medicament.
  • an anti- CDH17 antigen-binding protein e.g., antibody such as single-domain antibody
  • a fusion protein e.g., a fusion protein, a conjugate, a polynucleotide molecule, a vector, or a host cell described herein.
  • compositions and methods described herein are used to treat and/or prevent a cancer.
  • the cancer can be a solid cancer.
  • Non-limiting examples of cancers which may be treated and/or prevented using the compositions and methods described herein include colorectal cancer, familial GIST, familial pancreatic cancer, gastrointestinal stromal tumor (GIST), hereditary diffuse gastric cancer, hereditary pancreatitis, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the pancreas, and peritoneal cancer, pancreatic cancer, small bowel cancer, stomach cancer.
  • the cancer may be gastrointestinal cancer.
  • the gastrointestinal cancer is colorectal cancer, gastric cancer, esophageal cancer, or pancreatic cancer.
  • patients receiving an anti-CDH17 treatment of the present disclosure can be monitored for their responsiveness to the treatment.
  • a physician may monitor the response of a mammalian subject (e.g., a human) to treatment with anti-CDH17 antigen-binding proteins of the present disclosure by analyzing cancer cell death within a particular patient.
  • a composition of the present disclosure may be capable of inducing cancer cell death by e.g., about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.
  • Anti-CDH17 antigen-binding proteins described herein may be administered as a monotherapy or in combination with one or more additional therapeutic agents.
  • anti-CDH17 antigen-binding proteins of the present disclosure may also be admixed, conjugated, or administered with, or administered separately from, another therapeutic agent.
  • Non-limiting examples of additional therapeutic agents which can be administered in combination with the anti-CDH17 antigen-binding proteins described herein include a chemotherapeutic agent, a vascular endothelial growth factor (VEGF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an apoptosis-inducing agent, an immunotherapeutic agent, or a combination thereof.
  • VEGF vascular endothelial growth factor
  • EGFR epidermal growth factor receptor
  • an apoptosis-inducing agent an immunotherapeutic agent, or a combination thereof.
  • anti-CDH17 antigen-binding proteins of the present disclosure can be admixed with, co-administered with, or administered separately from a chemotherapeutic agent.
  • the chemotherapeutic agent may be administered prior to administration of the anti-CDH17 antigen-binding protein.
  • the chemotherapeutic agent may be used in a process of chemosensitization and the chemotherapeutic agent, when administered in combination with the anti-CDH17 antigen-binding protein (e.g., co-administered, or separately administered such as prior to the anti-CDH17 antigen-binding protein), can sensitize a cancer cell, or a population thereof (e.g., in a subject in need thereof such as a human), for the administration of the anti-CDH17 antigenbinding protein.
  • the sensitization of the cancer cell, or population thereof can render the cancer cell, or population thereof, more sensitive to the anti-CDH17 antigen-binding protein.
  • the administration of the chemotherapeutic agent in combination with the anti- CDH17 antigen-binding protein does not affect the efficacy of the anti-CDH17 antigen-binding protein. In some embodiments, the administration of the chemotherapeutic agent in combination with the anti- CDH17 antigen-binding protein enhances the efficacy of the anti-CDH17 antigen-binding protein.
  • chemotherapeutic agents include bleomycin, carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide, daunorubicin, doxorubicin or liposomal doxorubicin, mitomycin C, actinomycin, diethylstilbestrol, daunorubicin, etoposide, 5-fluorouracil, floxuridine, melphalan, methotrexate, mitomycin, 6-mercaptopurine, teniposide, 6-thioguanine, vincristine and vinblastine, leflunomide, actinomycin, tamoxifen, interferon a-2b, glutamic acid, plicamycin, 6-mercaptopurine, 6- thioguanine, carmustine, BCNU, limousine, CCNU, cytosine arabinoside, estramustine, hydroxyurea, procarbazine, busulfan, medr
  • anti-CDH17 antigen-binding proteins of the present disclosure can be admixed with, co-administered with, or administered separately from a VEGF inhibitor.
  • the VEGF inhibitor can be, e.g., bevacizumab, ramucirumab, regorafenib, ziv-aflibercept.
  • anti-CDH17 antigen-binding protein of the present disclosure can be admixed with, co-administered with, or administered separately from an EGFR inhibitor.
  • Non-limiting examples of EGFR inhibitors are cetuximab and/or panitumumab.
  • anti-CDH17 antigen-binding proteins of the present disclosure can be admixed with, co-administered with, or administered separately from an apoptosis-inducing agent such as, but not limited to, a B-cell lymphoma 2 (BCL2) inhibitor, a BCL-extra large (BCL-XL) inhibitor, or an inhibitor of apoptosis proteins (IAP) inhibitor, or a combination thereof.
  • BCL2 B-cell lymphoma 2
  • BCL-XL BCL-extra large
  • IAP apoptosis proteins
  • pharmaceutical compositions of the invention may be formulated for co-administration or sequential administration with one or more additional active agents that can be used to induce cell death or inhibit proliferation of a cell.
  • additional active agents that can be used to inhibit cell proliferation and that can be conjugated to, admixed with, or administered separately from an anti-CDH17 antigenbinding protein of the present disclosure include cytotoxic agents, e.g., those described herein.
  • Exemplary cytotoxic agents that can be conjugated to, admixed with, or administered separately from anti-CDH17 antigen-binding protein of the present disclosure include, but not limited to, 13-cis retinoic acid, 14-hydroxy-retro-retinol, 2-chloro-2'-deoxyadenosine, 2-Chloro-2'-arabino-fluoro-2'- deoxyadenosine, 2-chlorodeoxyadenosine, 2-chlorodeoxyadenosine (2-Cda), 2'-deoxycoformycin, 3- methyl TTNEB, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, 9-cis retinoic acid, aclarubicin, acodazole hydrochloride, acronine, adozelesin, adozelesin, adriamycin, aldesleukin, al l-trans retinoic acid
  • Other therapeutic agents that can be conjugated to, admixed with, or administered separately from anti-CDH17 antigen-binding protein of the present disclosure include, but are not limited to, 2' deoxycoformycin (DCF), 1,25 dihydroxyvitamin D3, 5-ethynyluracil, 9-dioxamycin, abiraterone, acylfulvene, adecypenol, ALL-TK antagonists, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, anagrelide, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, antiandrogen, prostatic carcinoma, anti-dorsalizing morphogenetic protein-1, antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid,
  • DCF
  • anti-CDH17 antigen-binding proteins of the present disclosure may be admixed, conjugated, or administered with, or administered separately from, an anti-inflammatory agent.
  • anti-inflammatory agents useful in conjunction with the compositions and methods of the invention include steroids, colchicine, hydroxychloroquine, sulfasalazine, dapsone, methotrexate, mycophenolate mofetil, azathioprine, cyclosporine, sirolimus, everolimus, azathioprine, leflunomide, mycophenolate, inhibitors of IL-l/l L-2/1 L-4/IL5/I L-6/1 L-13/1 L-17/1 L- 23/TNF/complement/BAFF/interferon/JAK/CD28/lgE/lntegrins/T cell costimulation pathway or B-cell depleting agents.
  • anti-CDH17 antigen-binding proteins of the present disclosure may be admixed, conjugated, or administered with, or administered separately from, an immunotherapy agent.
  • immunotherapy agents useful in conjunction with the compositions and methods of the invention include an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-Ll agent, an anti-PD-L2 agent, a TN Fa cross-linking agent, a TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti- CD40 agent, an anti-4-lBB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILRl agent, an anti-CDH17 agent, an anti-TWEAKR agent, an anti-TLIA agent, an anti-LIGHT agent, an anti-BTLA agent, an anti-LAG3 agent, an anti-TIM3 agent, an anti-Siglecs agent, an anti-ICOS ligand agent, an anti-B7-H
  • anti-CDH17 antigen-binding proteins of the present disclosure may be admixed, conjugated, or administered with, or administered separately from, for example, Alymsys (Bevacizumab), Avastin (Bevacizumab), Bevacizumab, Camptosar (Irinotecan Hydrochloride), Capecitabine, Cetuximab, Cyramza (Ramucirumab), Eloxatin (Oxaliplatin), Erbitux (Cetuximab), 5-FU (Fluorouracil Injection), Fluorouracil Injection, Ipilimumab, Irinotecan Hydrochloride, Keytruda (Pembrolizumab), Leucovorin Calcium, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Mvasi (Bevacizumab), Nivolumab, Opdivo (Nivolumab), Oxaliplatin, Panitumumab
  • Three juvenile alpacas were immunized by six subcutaneous injections with recombinant human CDH17 using two different adjuvants (complete/incomplete Freund's adjuvant (CFA/IFA); or Gerbu FAMA) using standard protocols to elicit a humoral immune response that includes the generation of antigen-specific conventional and heavy-chain only antibodies.
  • the immune response was determined by ELISA using sera from day 0 (before immunization), day 28 and day 43.
  • PBMCs Peripheral blood mononuclear cells
  • VHH immune libraries To generate VHH immune libraries, total RNA was reversely transcribed to cDNA using random hexamer primers. Conventional and heavy chain IGH cDNA fragments were amplified by PCR using primers annealing to the IGH leader sequence region and the CH2 region. The resulting amplicons represent the VHH and VH cDNAs, respectively.
  • the VHH fragment was isolated and used as template for a nested PCR to introduce appropriate endonuclease recognition sites for cloning into the pQ81 phagemid in frame with genelll. Libraries were transformed into electrocompetent E. coli TGI cells.
  • Phages were produced according to QVQ's standard operating procedures and phage titers were determined to ensure at least 10-fold excess over the maximum diversity of the libraries.
  • Panning substrates were commercially purchased. The panning substrates were immobilized either by direct coating on ELISA plates or by binding of biotinylated antigen on neutravidin-coated ELISA plate. Glycerol stocks were prepared from all outputs and are stored at -80°C.
  • Panning outputs were analyzed by random clone picking/PE-ELISA/Sanger sequencing (QVQ) and NGS (Genewiz/PipeBio).
  • rescued outputs of the 1st and 2nd panning rounds were plated out and 460 random single anti-CDH17 clones (equal numbers of colonies from each condition) are picked to create masterplates (96-well format). From the masterplates, expression cultures in deep-well plates were inoculated to produce periplasmic extracts containing monoclonal VHH. Periplasmic extracts were used to determine binding of individual VHHs to human and cynomolgus antigen by ELISA. For conditions where the panning substrate was biotinylated and captured by Neutravidin, background binders were identified by ELISA with Neutravidin. All masterplates had been sequenced by the Sanger method.
  • phages were eluted and corresponding phagemid DNA is extracted.
  • Identification of initial V-body candidates was performed in a parallelized fashion, employing a random colony picking as well as a next generation sequencing (NGS) approach as orthogonal techniques to yield a particularly diverse set of initial candidates.
  • NGS next generation sequencing
  • random colony picking was the prevalent method for initial hit identification, which involves transformation of a phagemid pool (from a panning elution) and picking of individual bacterial colonies to isolate single clones. Following this approach, 920 and 460 single colonies were randomly picked from the 18 samples of panning round 2 for CDH17 ( Figure 2).
  • the entire VHH region first needs to be PCR-amplified from isolated phagemid pools by primers annealing to universal phagemid sequences 5' and 3' of the VHH-encoding region.
  • the generated amplicons are fused to sequencing-compatible and sample-specific barcodes. By fusing unique barcodes, it is possible to multiplex hundreds of different samples.
  • An Illumina NovaSeq 6000 with an SP flowcell was employed for sequencing, yielding 250 bp reads from each direction and a total of ⁇ 600 million reads.
  • each library was sequenced with a total of 20 million reads, compared to the first and second round of panning with 2 million reads each.
  • This strategy allows for covering sufficient sequence space in the libraries as well as in the panning elutions, which are expected to have drastically fewer unique sequences compared to the initial libraries.
  • spike-in of 30% of a standard PhiX reference genome control into the sequencing reaction helped to provide a technical quality control for assessing sequencing accuracy.
  • the NGS raw data contains multiplexed sequencing reads, which are de-multiplexed based on the sample-specific barcodes.
  • the de-multiplexed data, containing unmerged sequencing reads are then processed by the following strategy, employing an NGS analysis platform of an external service provider.
  • a schematic domain representation and structure prediction of CDH17 is provided in Figure 4.
  • V-body sequences were clustered, allowing for a detailed analysis of V- body enrichment during phage display, sequence diversity, CDR3 length distribution and cluster abundance. 284 V-bodies were identified which can be classified into fifteen distinct clusters, as follows: N1061 (group A), N1070 (group B), N1081 (group C), 45F5-N1083-N1084 (group D), N1090 (group E), N1091-N1092 (group F), N1095 (group G), 43B5 (group H), 43E7-44A4 (group I), N1242 (group J), and 44A1 (group K), N1117 (group M), N1129 (group N), N1186 (group O), and N1264 (group P).
  • Table 5-1 to Table 5-33 display the amino acid frequency distribution at each amino acid (AA) position (IMGT) for CDR1, CDR2 and CDR3 for the fifteen clusters.
  • Table 6 provides the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DNA sequences of the full-length VHH domain for the identified V-bodies.
  • endogenous expressing cells lines GP2d (DMEM, 10% FBS), Colo205 (RPMI, 10% FBS), DLD-1 (RPMI, 10% FBS), and T84 (DMEM:F12, 5% FBS), and negative cell line HCT-15 (RPMI, 10% FBS) were grown to 80-90% confluency in growth media. Cells were harvested by washing with PBS and dissociated using Accutase then pelleted, resuspended with growth media and counted. 150,000 cells/well were added to a 96-well conical bottom non-TC treated plate.
  • Binding affinities of the V-bodies for their respective target was determined by surface plasmon resonance (SPR) using a Carterra LSA instrument. Affinity purified V-bodies were covalently crosslinked onto an LSA HC30M chip using EDC/Sulfo NHS. The interaction with human CHD17 (extracellular domain - human and mouse: His-tagged CDH17; Cyno: Fc-tagged CDH17) was measured under physiological conditions (50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 %(w/v) BSA, 0.05% (v/v) Tween20, 25°C) using 10 different antigen concentrations (2-fold serial dilution, start at 500 nM). Resulting sensorgrams were analyzed and KDs were calculated using Carterra's data analysis software Kinetics. Binding affinities are summarized in Table 7.
  • V-bodies were tested for competition to the same binding region (epitope) in a pairwise combinatorial manner via a one-on-many format. Affinity purified V-bodies were covalently crosslinked onto an LSA HC30M chip using EDC/Sulfo NHS.
  • V-bodies were tested for binding to individual domains or domain formats of human CDH17 (monomers: ECI, EC2, EC3, EC5, EC6; dimer: EC6-7; trimer: EC5-7).
  • Affinity purified V-bodies were covalently crosslinked onto an LSA HC30M chip using EDC/Sulfo NHS.
  • Human CDH17 domain formats were injected (2-fold serial dilution, start at around 4 pM) under physiological conditions (50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 %(w/v) BSA, 0.05% (v/v) Tween20, 25°C). Resulting sensograms were analyzed using Carterra's data analysis software Kinetics.
  • HEK-293 cells were grown to 80-90% confluency in growth media; EMEM supplemented with 10% FBS. Cells were harvested by washing with PBS and dissociated using TrypLE then pelleted, resuspended with growth media and counted. 4 x 10 6 cells each were seeded into 9 T75 flasks in 15 mL of growth media. Cells were grown overnight at 37°C and 5% CO 2 .
  • Cells were transfected with CDH17 full length construct hCDH17-FL, and domain swapped constructs hCDH17_deltaECl, hCDH17_deltaEC2, hCDH17_deltaEC3, hCDH17_deltaEC4, hCDH17_deltaEC5, hCDH17_deltaEC6, hCDH17_deltaEC7, and no DNA control using X-tremeGENE 9 DNA Transfection Reagent (Sigma cat# 6365787001) at a 3:1 (reagent:DNA) dilution in OptiMEM following manufacturer's protocol. Cells were incubated for 48 hours at 37°C and 5% CO 2 .
  • Cells were harvested by washing with PBS and dissociated using Accutase then pelleted, resuspended with growth media and counted. 150,000 cells/well of each transfected cell line was added to a 96-well conical bottom non-TC treated plate. Cells were washed with ice-cold FACS buffer and media aspirated. Purified V-bodies and tool compounds were diluted to 100 nM with FACS buffer. Cells were stained with 50 pL of diluted V-body or tool compounds for 30 minutes on ice then washed with 150 pL of FACS buffer three times.
  • Table 7 shows a summary of binding affinities, binning and domain mapping data.
  • SEQ ID NO: 2 ODY-N1061, Group A, CDR2, amino acid sequence
  • SEQ ID NO: 4 ODY-N1061, Group A, non-humanized, amino acid sequence
  • SEQ ID NO: 12 ODY-N1081, Group C, CDR2, amino acid sequence
  • SEQ ID NO: 14 ODY-N1081, Group C, non-humanized, amino acid sequence
  • SEQ ID NO: 40 ODY-N1095, Group G, non-humanized, amino acid sequence
  • SEQ ID NO: 41 ODY-N1095, Group G, humanized, amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYLMGWFRQAPGKEREFVAAISWNDRSTYYADSVKGRFTISRDNAKNT
  • SEQ ID NO: 65 ODY-44A1, Group K, non-humanized, amino acid sequence
  • SEQ ID NO: 67 ODY-N1061, Group A, non-humanized DNA, nucleotide sequence
  • SEQ ID NO: 69 ODY-N1081, Group C, non-humanized DNA, nucleotide sequence
  • SEQ ID NO: 70 ODY-45F5_ODY-N1083_ODY-N1084, Group D, non-humanized DNA, nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTC TGGACGCACCTTCAGTGAACCTATCGTGGGCTGGTTCCGCCAGACTCCAGGGAAGGAGCGTGACTTCGTGGCCGC TCTTATTTCAACTGGTGGTAGTACGAGGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGCTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTATTGTGCGGCAACGGGGGG GACTCGTACCGTGGCGCCTATGATCGGCCCGCTGAATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC TCA
  • SEQ ID NO: 72 ODY-45F5_ODY-N1083_ODY-N1084, Group D, non-humanized DNA, nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTC CGGACGCACCTTCAGTGAACCTATCGTGGGCTGGTTCCGCCAGACTCCAGGGAAGGAGCGTGACTTCGTGGCCGC TCTTATTTCAACTGGTGGTAGTACGAGGTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGCTGTATTTGCAAATGAACAGCCTGAAACCTGGGGACACGGCCGTGTATTATTGTGCGGCAACGGGGGG GACTTGTACCGTGGCGCCTATGATCGGCCCGCTGAATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC TCA
  • SEQ ID NO: 75 ODY-N1095, Group G, non-humanized DNA, nucleotide sequence
  • SEQ ID NO: 80 ODY-44A1, Group K, non-humanized DNA, nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTACATCCTCT
  • SEQ ID NO: 108 ODY-N1061, Group A, non-humanized, amino acid sequence
  • SEQ ID NO: 110 ODY-N1061, Group A, non-humanized, amino acid sequence
  • SEQ ID NO: 112 ODY-N1061, Group A, non-humanized, amino acid sequence
  • SEQ ID NO: 120 ODY-N1070, Group B, non-humanized, amino acid sequence
  • SEQ ID NO: 130 ODY-N1081, Group C, non-humanized, amino acid sequence
  • SEQ ID NO: 140 ODY-45F5_ODY-N1083_ODY-N1084, Group D, non-humanized, amino acid sequence
  • SEQ ID NO: 147 ODY-45F5_ODY-N1083_ODY-N1084, Group D, non-humanized, amino acid sequence EVQLVESGGGLVQAGGSLRLSCAASGRTFSEPIVGWFRQTPGKERDFVAALISTGGSTRYADSVKGRFTISRDNAKNTLY
  • SEQ ID NO: 150 ODY-45F5_ODY-N1083_ODY-N1084, Group D, non-humanized, amino acid sequence

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Abstract

La présente invention concerne des protéines de liaison à l'antigène (par exemple, des anticorps tels que des anticorps à domaine unique) qui se lient spécifiquement à la cadhérine 17 (CDH17). L'invention concerne également des protéines de fusion et des conjugués comprenant les protéines de liaison à l'antigène, des polynucléotides et des vecteurs recombinants codant pour les protéines de liaison à l'antigène, ainsi que des cellules hôtes et des procédés de préparation des protéines de liaison à l'antigène. L'invention concerne en outre des compositions pharmaceutiques comprenant les protéines de liaison à l'antigène et des méthodes de traitement d'une maladie ou d'un trouble à l'aide des protéines de liaison à l'antigène.
PCT/US2024/050633 2023-10-13 2024-10-10 Protéines de liaison à l'antigène anti-cdh17 et leurs utilisations Ceased WO2025080751A2 (fr)

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