Mutant CCL27 binders against CCR10

Description

PATENT Attorney Docket No.081906-1526373-256210PC Client Ref. No. SF2025-048-2PC Mutant CCL27 binders against CCR10 CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No.63 / 709,227, filed October 18, 2024, which is incorporated by reference for all purposes. BACKGROUND OF THE INVENTION

[0002] One of the notable clinical features of myeloma is that despite the existence of many effective therapies, there is still no known cure for this disease. Resistance to current small molecule therapeutics, particularly proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz), and immunomodulatory drugs such as lenalidomide (Len), is a widespread conundrum. Immunotherapies have been a revolution for the treatment of myeloma, with many relapse and refractory patients having positive initial responses.

[0003] Despite the rise of immunotherapies for multiple myeloma, most notably CAR T-cells, there is still a strong need for new therapeutic targets and products targeting them. The clinically approved CAR T-cells for myeloma target BCMA; however, there are several pitfalls of the current products. The majority of patients who receive BCMA CAR T-cells will relapse, opening a strong need for therapies for these patients that are refractory to the current BCMA products. Additionally, there is an emerging number of side effects of BCMA CAR T-cells that may be avoided by new therapeutic targets and products. BRIEF SUMMARY OF THE INVENTION

[0004] CCR10 was identified as a new immunotherapy target for multiple myeloma. Mutant CCL27 binders have been developed for targeted therapies against CCR10. In some embodiments, a protein is provided comprising or consisting of a CCR10-binding polypeptide comprising SEQ ID NO: 4.

[0005] In some embodiments, the CCR10-binding polypeptide comprises or consists of SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the amino terminus of the protein is the amino terminus of SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO:9 (e.g., there are no amino acid fusions at the N-terminus).

[0006] In some embodiments, the CCR10-binding polypeptide is linked to a transmembrane domain. In some embodiments, a hinge domain links the CCR10-binding polypeptide to the transmembrane domain. In some embodiments, the transmembrane domain is linked to an intracellular signaling domain. In some embodiments, the transmembrane domain is linked to two or more intracellular signaling domains. In some embodiments, the intracellular signaling domains are a CD3 zeta domain and a 4-1BB domain.

[0007] In some embodiments, the CCR10-binding polypeptide is linked an antibody or fragment thereof. In some embodiments, the antibody specifically binds to CD3 or CD16a.

[0008] In some embodiments, the CCR10-binding polypeptide is linked to a detectable label, toxin, therapeutic molecule or a radioactive molecule.

[0009] Also provided is a cell comprising and / or expressing the protein comprising the CCR10-binding polypeptide as described above or elsewhere herein. In some embodiments, the cell is a human or mammalian or bacterial cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T-cell, natural killer cell, or a macrophage.

[0010] Also provided is nucleic acid encoding the protein as described above or elsewhere herein.

[0011] Also provided is a method of killing a cancer cell expressing CCR10. In some embodiments, the method comprises contacting the cell as described above or a CAR as described herein to the cancer cell such that the cell kills the cancer cell. In some embodiments, the cancer cell is a multiple myeloma cancer cell. In some embodiments, the method is performed in vivo or in vitro or ex vivo.

[0012] Also provided is a method of making a cell expressing the protein (e.g., a modified CCL27 protein) of any as described above or elsewhere herein. In some embodiments, the method comprises, introducing into the cell a nucleic acid encoding protein into the cell andallowing the cell to express the CCR10-binding polypeptide. In some embodiments, the cell is a human or mammalian or bacterial (e.g., E. coli) cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T-cell, natural killer cell, or a macrophage. In some embodiments, the method comprises obtaining the cell from a human; introducing the nucleic acid into the cell; and administering the cell to the same of a different human. DEFINITIONS

[0013] As used in herein, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules, and the like.

[0014] As used herein, the term "antibody" means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and includes, but is not limited to, a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, single domain antibodies, such as nanobodies, diabodies, camelid-derived antibodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including, but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.

[0015] "Antibody fragments" comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific or multivalent antibodies formed from antibody fragments. A "Fab" fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. A F(ab')2fragment has a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known. An "Fv" is aminimal antibody fragment that contains a complete antigen-recognition and binding site and is a dimer of one heavy- and one light-chain variable region domain.

[0016] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4. The antibodies described herein can be of any of these classes or subclasses.

[0017] As used herein, “V-region” refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, CDR3 and Framework 4.

[0018] As used herein, "complementarity-determining region (CDR)" refers to the three hypervariable regions that interrupt the four "framework" regions of a variable domain. The CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of each heavy or light chain are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus.

[0019] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., North, Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., North method. (see, e.g., North et al., J. Mol. Biol.406(2):228-256, 2011; Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol.196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol.227, 799- 817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). Definitions of CDRs are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219–221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268–9272 (1989); Martin, et al, Methods Enzymol., 203, 121–153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141–1721996).

[0020] “Epitope" or "antigenic determinant" as used in the present disclosure in the context of antibody or protein binding refers to a site on an antigen to which an antibody or protein binds. Epitopes can be formed from contiguous amino acids and / or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2- dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, Glenn E. Morris, Ed (1996). Binding of an antibody or protein to an epitope can be influenced by other environmental factors, such as s the presence of calcium ions.

[0021] As used herein, the term “specifically binds” to a target, e.g., a CCL27 protein as described herein or a fusion thereof, refers to a binding reaction whereby the CCL27 protein binds to CCR10 (or an antibody binds to its target) with greater affinity, greater avidity, and / or greater duration than it binds to a different non-target protein. In some embodiments, a target- binding protein has at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for the target compared to an unrelated target when assayed under the same binding affinity assay conditions. The term “specific binding,” “specifically binds to," or “is specific for" a particular target, as used herein, can be exhibited, for example, by a molecule (e.g., an antibody) having an equilibrium dissociation constant KD for the target of, e.g., 10-2M or smaller, e.g., 10-3M, 10-4M, 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, or 10-12M. In some embodiments, an antibody has a KD of less than 100 nM or less than 10 nM.

[0022] The term “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down an undesired physiological change or disorder. For purpose of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0023] As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present invention, the pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to the active ingredient. The nature of the carrier differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.

[0024] The term "effective amount," as used herein, refers to that amount of CCL27 protein, or protein fusion thereof, or cell that expresses a CAR targeted by a CCL27 protein as described herein, that is sufficient to effect treatment of a disease associated with the growth and / or proliferation of cancer cells, as described herein, when administered to a subject. A therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The dosages of a CAR-expressing cell as described herein for administration can range from, for example, 5x105CAR-positive T-cells per kg to 2x107CAR-positive T-cells per kg. Dosage regiments may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (i.e., side effects) of an antibody or antigen binding portion thereof are minimized and / or outweighed by the beneficial effects.

[0025] The term “bispecific” as used herein, refers to an antibody or antibody / CCL27 protein fusion that binds to two or more different epitopes. In some embodiments, a bispecific antibody or protein binds to epitopes for two different target antigens. A modified CCL27 protein as described herein can be covalently or non-covalently-linked to a second target binding protein (e.g., an scFv or other target binding protein). Linkage can be direct or via a linker, e.g., anamino acid linker. In some embodiments, a modified CCL27 protein as described herein is fused to a first immunoglobin Fc domain and a second target binding protein is fused to a second Fc domain and the first and second Fc domains bind to each other covalently or non-covalently. In some embodiments, the Fc domains bind via the presence of “knob-in-hole” mutations. See, e.g., Xu, et al., MAbs 7(1):231-42 (2015).

[0026] An "effector" refers to any molecule or combination of molecules whose activity it is desired to deliver / into and / or localize at target cell. Effectors include, but are not limited to labels, cytotoxins, enzymes, growth factors, transcription factors, antibodies, drugs, etc.

[0027] Individual substitutions, deletions or additions to a nucleic acid, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of (e.g., 1, 2, 3, 4, or 5 or more) amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of proteins described herein. The following amino acids are typically conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0028] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, polypeptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also encompasses “conservatively modified variants” thereof (e.g., degenerate codon substitutions) and complementary sequences,as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid can include for example, a gene, cDNA, mRNA, oligonucleotide, or polynucleotide.

[0029] The terms "identical" or “percent identity,” in the context two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides, or amino acids, that are the same (i.e., about 60% identity, preferably at least 65%, 70%, 75%, 80%, or 85% identity; and often at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithm (e.g., a BLASTP algorithm with default parameters for comparison of two polypeptide sequences. See, e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. The algorithms can account for gaps and the like. Percent identity is over the entire length of the reference sequence unless indicated otherwise. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG.1: In two T-cell donors (“D24” and “D25”), the efficacy of two mutant CCL27 CAR T-cells was tested against the MM.1S and AMO1 multiple myeloma cell lines (1A). To confirm the specificity of these mutants for CCR10, CCR10 was knocked out in the AMO1 cell line and no cytotoxicity was observed (1B).

[0031] FIG.2: Increased binding affinity of the CCL27 mutants was observed for CCR10 in the AMO1 (2A) and MM.1S (2B) myeloma cell lines by looking for an increase in the formation of doublets between the CAR-Ts and tumor cells.

[0032] FIG.3: In two T-cell donors ("Donor 1" and "Donor 2"), the specificity of the mutant CCL27 CAR T-cells was tested against donor-matched peripheral blood mononuclear cells (PBMCs) with the MM.1S myeloma cell line spiked in. Results showed the CCL27 mutant CAR-Ts have no major toxicity against PBMCs while effectively depleting myeloma cells.DETAILED DESCRIPTION OF THE INVENTION

[0033] Mutant CCL27 proteins have been identified that bind with greater affinity to CCR10 compared to wildtype CCL27 (e.g., SEQ ID NO:1). The new modified CCL27 proteins can be used to target any number of linked molecules or cells to CCR10 or cells expressing CCR10. For example, the new mutant CCL27 proteins described herein can be linked to effector molecules, for example, therapeutic molecules, toxic molecules, radioligands or in some embodiments, antibodies to form molecules with bi-specificity (binding CCR10 and the target of the antibody). Moreover, the new mutant CCL27 proteins can be used as an extracellular domain for a chimeric antigen receptor (CAR), which for example can be expressed in immune cells to target the immune cells to cancer cells or other cells expressing CCR10. As described further, a variety of the above-described molecules and cells can be used to kill CCR10- expressing cells, for example cancer cells expressing CCR10. Exemplary CCL27 proteins

[0034] The present disclosure provides for modified CCL27 proteins with enhanced affinity for CCR10 (e.g., SEQ ID NO:7) compared to wildtype CCL27 (SEQ ID NO:1). As shown in the examples, introduction of hydrophobic amino acids at the amino (N-) terminus of CCL27 will enhance its ability to bind to CCR10. Specifically, as shown in the examples, addition of phenylalanine (F) or tryptophan (W) to the N-terminus of a CCL27 protein enhances affinity for CCR10. It is believed that addition of one or two phenylalanine or tryptophan residues (e.g., F, W, FW, WF, WW, or FF) will increase affinity for CCR10 compared to wildtype CCL27 (e.g., SEQ ID NO:1). While data is not provided for two hydrophobic amino acids added to the N- terminus, based on computer modeling or two amino acids added, and the results described herein for one amino acid additions, it is believed that improved affinity will also occur, albeit at a lesser extent, than addition of one hydrophobic amino acid.

[0035] The above information describing modified CCL27 proteins is summarized as SEQ ID NO:4 as shown below:(W / F)nFLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQRS IC(I / V)HPQNPSLSQWFEHQERK(L / F)HGTLPKLNFGMLRKMG (SEQ ID NO: 4), where n is1 or 2 and each position is independently selected from W and F, e.g., W, F, FF, WW, WF or FW. The parentheticals in SEQ ID NO:4 indicate either amino acid can be at the indicated positions and represent amino acids that occur in described natural human variants of CCL27. In some embodiments, a single W or F is added to the N-terminal of a CCL27 protein, for example as depicted in (e.g., having no further N-terminal amino acids aside from the single W or F) SEQ ID NO: 2 or 3, respectively. In some embodiments, the modified CCL27 protein consists of or comprises SEQ ID NO: 5 or 6 (e.g., having no further N-terminal amino acids aside from the single W or F). In some embodiments, the modified CCL27 protein comprises an amino acid sequence at least 95, 96, 97, 98, or 99% identical to SEQ ID NO:1 and further includes at the N- terminus F, FF, WW, WF or FW, e.g., without any further amino acids at the N-terminus. In some embodiments, the modified CCL27 protein consists of or comprises SEQ ID NO: 8 or 9, optionally having no further N-terminal amino acids aside from the single W or F at the amino terminus of SEQ ID NO: 8 or 9.

[0036] Relative affinity for CCR10 can be measured by standard protein binding assays or competition assays, or can be measured by determining CCR10-expressing cell killing by T-cells expressing a CAR targeted by a modified CCL27 protein, e.g., as described in the examples.

[0037] The modified CCL27 proteins described herein and having enhanced CCR10-affinity can be fused, e.g., at the carboxyl terminus, or otherwise linked, to a heterologous amino acid sequence. Exemplary heterologous sequences can include, but are not limited to, tag sequences that can be detected for example by an antibody, fluorescent or other detectable protein sequences, antibody variable regions or other protein sequences that have affinity for a second target protein, effector or toxic or, otherwise therapeutic, proteins that can be localized to CCR10-expressing cells by the modified CCL27 protein sequences, and / or antibody Fc or human albumin or other sequences which when fused to the modified CCL27 proteins, increase half-life of the protein in an individual. In some embodiments, the linkage of the heterologous amino acid sequence to the modified CCL7 protein can be a covalent linkage (e.g., via a peptide bond). In other embodiments the linkage can be non-covalent. In an example of a non-covalent linkage, the heterologous amino acid sequence can be fused to a first affinity agent (e.g., biotin) and the modified CCL27 protein can be fused to a second affinity agent (e.g., streptavidin) such that the first and second affinity agents bind non-covalently, allowing for linkage of the homologousamino acid sequence to the modified CCL27 protein. Fc domains linked to the modified CCL27 proteins can be a human Fc domain or can include one or more mutations that for example increases half-life or other desired functions. See, e.g., Ko et al., Experimental & Molecular Medicine volume 54, pages 1850–1861 (2022); Lee et al., Nature Communications volume 10, Article number: 5031 (2019); Booth et al., MAbs.2018 Oct; 10(7): 1098–1110; U.S. Patent Publication No. US20190048078. In some embodiments, the Fc domains comprise a knob-in- hole mutation allowing the Fc domain to form a non-covalent association with a second Fc (optionally linked to a fusion partner such as an antibody variable domain or scFv) having a corresponding knob-in-hole mutation such that the two Fc domains associate.

[0038] Any of the modified CCL27 proteins with enhanced affinity for CCR10 as described herein can be linked to an effector molecule. Modified CCL27 proteins can be formed by conjugating the modified CCL27 proteins described herein to an effector (e.g., a detectable label, another therapeutic agent, etc.). Illustrative therapeutic agents include, but are not limited to, for example, a cytotoxic or cytostatic agent (e.g., a chemotherapeutic agent), a toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), a radioactive isotope (e.g., a radioconjugate), or an antibody.

[0039] In certain embodiments, the modified CCL27 proteins can be used to direct detectable labels to a tumor site or otherwise detect the presence of a CCR10-expressing cell, e.g., a cancer cell. This can facilitate tumor detection and / or localization. It can be effective for detecting primary tumors, or, in certain embodiments, secondary tumors produced by cancers that express CCR10.

[0040] Thus, in certain embodiments, the effector comprises a detectable label. Suitable detectable labels include, but are not limited to radio-opaque labels, nanoparticles, PET labels, MRI labels, radioactive labels, and the like. Among the radionuclides and useful in various embodiments, gamma-emitters, positron-emitters, x-ray emitters and fluorescence-emitters are suitable for localization, diagnosis and / or staging, and / or therapy, while beta and alpha-emitters and electron and neutron-capturing agents, such as boron and uranium, also can be used for therapy.

[0041] In various embodiments the detectable labels can be used in conjunction with an external detector and / or an internal detector and provide a means of effectively localizing and / orvisualizing cancer cells expressing CCR10. Such detection / visualization can be useful in various contexts including, but not limited to pre-operative and intraoperative settings.

[0042] The modified CCL27 proteins described herein can be coupled directly to a molecule (e.g., as described herein), e.g., at an available cysteine, or they can be attached to a "package" (e.g., a chelate, a liposome, a polymer microbead, a nanoparticle, etc.) carrying, containing, or comprising the radio-opaque material.

[0043] Detectable labels suitable for use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., H, I, S, C, or P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads, nanoparticles, quantum dots, and the like.

[0044] In certain embodiments, suitable radiolabels include, but are not limited to Tc, Tc , Ru, Ru, Tc, Y, Y, Zr, Y, Br, As, Br, Se, As, Ga, Ga, Ga, Ga, Cu, Cu, Cu, Cu, Cu, F e, Co, Co, Mn, Fe, Cr, Sc, H, S, P, P, Ac, Ac, Ra, Bi, Pb, Bi, At, Pb, Hg, T1, Auexample, certain radiolabels may be detected using photographic film, scintillation detectors, PET imaging, MRI, and the like. Fluorescent markers can be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0046] In another embodiment, the effector can comprise a radiosensitizer that enhances the cytotoxic effect of ionizing radiation (e.g., such as might be produced by60Co or an x-ray source) on a cell. Numerous radiosensitizing agents are known and include, but are not limited to benzoporphyrin derivative compounds (see, e.g., U.S. Patent 5,945,439), 1,2,4-benzotriazineoxides (see, e.g., U.S. Patent 5,849,738), compounds containing certain diamines (see, e.g., U.S. Patent 5,700,825), BCNT (see, e.g., U.S. Patent 5,872, 107), radiosensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S. Patent 4,474,814 ), various heterocyclic derivatives (see, e.g., U.S. Patent 5,064,849), platinum complexes (see, e.g., U.S. Patent 4,921,963), and the like.

[0047] In certain embodiments, the effector can include an alpha emitter, i.e. a radioactive isotope that emits alpha particles. Alpha-emitters have recently been shown to be effective in the treatment of cancer (see, e.g., McDevitt et al. (2001) Science 294: 1537-1540; Ballangrud et al. (2001) Cancer Res.61: 2008-2014; Borchardt et al. (2003) Cancer Res.63 : 5084-50). Suitable alpha emitters include, but are not limited to212Pb,225Ac,227Th, Bi,213Bi,211At, and the like.

[0048] Many of the molecules described herein can be provided as a chelate. The chelating molecule is typically coupled to a molecule (e.g. biotin, avidin, streptavidin, etc.) that specifically binds an epitope tag attached to a modified CCL27 proteins described herein.

[0049] Chelating groups are well known to those of skill in the art. In certain embodiments, chelating groups are derived from ethylene diamine tetra-acetic acid (EDTA), di ethylene triamine penta-acetic acid (DTP A), cyclohexyl 1,2-diamine tetra-acetic acid (CDTA), ethyleneglycol-0,0'-bis(2-aminoethyl)-N,N,N',N' -tetra-acetic acid (EGTA), N,N- bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexa- acetic acid (TTHA), l,4,7,10-tetraazacyclododecane-N,N'-,N",N"'-tetra-acetic acid (DOTA), hydroxy ethyldiamine triacetic acid (HEDTA), 1,4,8, 11-tetra-azacycl otetradecane-N,N',N",N"'- tetra-acetic acid (TETA), substituted DTP A, substituted EDTA, and the like.

[0050] Examples of certain chelators include unsubstituted or, substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4-mercaptomethylthiolane. One chelating agent, 1,4,7,10-tetraazacyclododecane-N, N, N", N'"-tetraacetic acid (DOTA), is of particular interest because of its ability to chelate a number of diagnostically and therapeutically important metals, such as radionuclides and radiolabels. Conjugates of DOTA and proteins such as antibodies have been described previously. For example, U.S. Pat. No.5,428, 156 teaches a method for conjugating DOTA to antibodies and antibody fragments. To make these conjugates, one carboxylic acid group of DOTA is converted to an active ester which can react with an amine or sulfhydryl group on the antibody or antibody fragment. Lewis et al. (1994) Bioconjugate Chem.5: 565-576, describes a similar method wherein one carboxyl group ofDOTA is converted to an active ester, and the activated DOTA is mixed with an antibody, linking the antibody to DOTA via the epsilon-amino group of a lysine residue of the antibody, thereby converting one carboxyl group of DOTA to an amide moiety.

[0051] The modified CCL27 proteins described herein can be used to deliver a variety of cytotoxic and / or cytostatic drugs including therapeutic drugs, a compound emitting radiation, cytotoxic molecules of plant, fungal, or bacterial origin, biological proteins, and mixtures thereof. In certain embodiments the cytotoxic drugs can comprise intracellularly acting cytotoxic drugs that are, e.g., small organic molecules, cytotoxic proteins or peptides, radiation emitters, including, for example, short-range, high-energy ^-emitters as described above, and the like. Additional representative therapeutic agents include radioisotopes, chemotherapeutic agents, immunomodulatory agents, anti -angiogenic agents, anti-proliferative agents, pro-apoptotic agents, and cytolytic enzymes (e.g., RNases). An agent may also include a therapeutic nucleic acid, such as a gene encoding an immunomodulatory agent, an anti-angiogenic agent, an anti- proliferative agent, or a pro-apoptotic agent. These drug descriptors are not mutually exclusive, and thus a therapeutic agent may be described using one or more of the above-noted terms. For example, selected radioisotopes are also cytotoxins. In various embodiments therapeutic agents may be prepared as pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0052] In certain embodiments, the modified CCL27 protein is attached to a therapeutic cytotoxic / cytostatic drug. In various embodiments the drugs can include, but are not limited to microtubule inhibitors and DNA-damaging agents, polymerase inhibitors (e.g. , the polymerase II inhibitor, a-amanitin), and the like. In certain embodiments the modified CCL27 protein is conjugated to the drug directly or through a linker, while in other embodiments, the modified CCL27 protein is conjugated to a drug carrier (e.g., a liposome containing the drug, a polymeric drug carrier, a nanoparticle drug carrier, a lipid drug carrier, a dendrimeric drug carrier, and the like).

[0053] In certain embodiments the drug comprises a tubulin inhibitor, including, but not limited to auristatin, Dolastatin-10, synthetic derivatives of the natural product Dolastatin-10, and maytansine or a maytansine derivative. In certain embodiments the drug comprises an auristatin. In certain embodiments the auristatin is selected from the group consisting of auristatin E (AE), auristatin EB (AEB), auristatin EFP (AEFP), Monomethyl Auristatin D(MMAD) or monomethyl dolastatin 10, Monomethyl Auristatin F (MMAF) or N-methylvaline- valine-dolaisoleuine-dolaproine-phenylalanine), Monomethyl Auristatin E (MMAE) or N- methylvaline-valine-dolaisoleuine-dolaproine-norephedrine, 5 -benzoyl valeric acid-AE ester (AEVB), vcMMAE, and vcMMAF .

[0054] In certain embodiments the drug comprises an enediyne. Enediynes are a class of anti- tumor bacterial products characterized by either nine- and ten-membered rings or the presence of a cyclic system of conjugated triple-double-triple bonds. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin.

[0055] Calicheamicin is an enediyne antibiotic that was originally isolated as a natural product from the soil organism Micromonospora echinospora ssp. calichensis (Zein et al. Science 27; 240(4856): 1198-1201, 1988). It generates double-strand DNA breaks and subsequently induces apoptosis in target cells (Zein et al. Science 27; 240(4856): 1198-1201, 1988; Nicolaou et al. Chem. Biol. September; l(l):57-66, 1994; Prokop et al. Oncogene 22:9107-9120, 2003). In certain embodiments the drug comprises calicheamicin or a calicheamicin analog. Examples of calicheamicins and analogs thereof suitable for use are disclosed, for example, in U.S. Pat. Nos. 4,671,9584,970, 198, 5,053,394, 5,037,651, 5,079,233, 5,264,586, and 5,108,912, which are incorporated herein by reference in their entirety. In certain embodiments these compounds contain a methyltrisulfide that can be reacted with appropriate thiols to form disulfides, at the same time introducing a functional group such as a hydrazide or other functional group that is useful for conjugating calicheamicin to a modified CCL27 protein. Disulfide analogs of calicheamicin can also be used, for example, analogs described in U.S. Pat. Nos.5,606,040 and 5,770,710, which are incorporated herein by reference in its entirety. In certain embodiments the disulfide analog is N-acetyl-gamma-calicheamicin dimethyl hydrazide.

[0056] In certain embodiments the drug comprises a geldanamycin. Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90 (Heat Shock Protein 90) and have been used antitumor drugs. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N- Allylamino-17-Demethoxygeldanamycin), and 17-DMAG (17-Dimethylaminoethylamino-17- demethoxygeldanamycin). In certain embodiments the drug comprises a maytansine. Maytansines or their derivatives maytansinoids inhibit cell proliferation by inhibiting the microtubules formation during mitosis through inhibition of polymerization of tubulin (see, e.g.,Remillard et al.91975) Science 189: 1002-1005). Illustrative maytansines include, but are not limited to, Mertansine (DM1); and an analogue of maytansine such as DM3 or DM4, as well as ansamitocin.

[0057] In certain embodiments the drug comprises a taxane. Taxanes are diterpenes that act as anti -tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel and docetaxel.

[0058] In certain embodiments the drug comprises a DNA interacting agent. In certain embodiments the DNA interacting agent includes, but is not limited to calicheamicins, duocarmycins, pyrrolobenzodiazepines (PBDs), and the like.

[0059] In another illustrative, but non-limiting embodiment, the drug comprises a duocarmycin. Duocarmycins are DNA damaging agents able to exert their mode of action at any phase in the cellular cycle. Agents that are part of this class of duocarmycins typically have potency in the low picomolar range. Illustrative duocarmyhcins {e.g., duocarmycin analogues) that can be used as effectors in the chimeric constructs contemplated herein include, but are not limited to duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, duocarmycin SA, Cyclopropylbenzoindole duocarmycin (CC-1065), Centanamycin, Rachelmycin,

[0060] In another illustrative, but non-limiting embodiment, the drug comprises a pyrrolobenzodiazepine. In certain embodiments the drug comprises a synthetic derivative of two pyrrolobenzodiazepines linked by a flexible polymethylene tether. Pyrrolobenzodiazepines (PBDs) and PBD dimers are described in U.S. Patent No: 7,528,126 B2, which is incorporated herein by reference for the Pyrrolobenzodiazepines and PBD dimers described therein. In certain embodiments the pyrrolobenzodiazepine is selected from the group consisting of: Anthramycin (and dimers thereof), Mazethramycin (and dimers thereof), Tomaymycin (and dimers thereof), Prothracarcin (and dimers thereof), Chicamycin (and dimers thereof), Neothramycin A (and dimers thereof), Neothramycin B (and dimers thereof), DC-81 (and dimers thereof), Sibiromycin (and dimers thereof), Porothramycin A (and dimers thereof), Porothramycin B (and dimers thereof), Sibanomycin (and dimers thereof), Abbeymycin (and dimers thereof), SG2000, and SG2285.

[0061] In certain embodiments the drug comprise a polymerase inhibitor, including, but not limited to polymerase II inhibitors such as ^-amanitin, and poly(ADP-ribose) polymerase (PARP) inhibitors. Illustrative PARP inhibitors include, but are not limited to, Iniparib (BSI 201), Talazoparib (BMN-673), Olaparib (AZD-2281), Olaparib, Rucaparib (AG014699, PF- 01367338), Veliparib (ABT-888), CEP 9722, MK 4827, BGB-290, 3-aminobenzamide, and the like.

[0062] In certain embodiments the drug comprises a vinca alkyloid. Vinca alkyloids are also anti -tubulin agents. Exemplary vinca alkyloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0063] The foregoing drugs are illustrative and not limiting. In various embodiments other anti-cancer drugs can be linked to, or co-administered with, a modified CCL27 protein as described herein, including but not limited to anti-cancer antibodies (e.g., HERCEPTIN®), antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule targeting agents, kinase inhibitors, protein synthesis inhibitors, somatostatin analogs, glucocorticoids, aromatose inhibitors, mTOR inhibitors, protein Kinase B (PKB) inhibitors, phosphatidylinositol, 3 -Kinase (PI3K) Inhibitors, cyclin dependent kinase inhibitors, anti-TRAIL molecules, MEK inhibitors, and the like. In certain embodiments the anti-cancer compounds include, but are not limited to flourouracil (5-FU), capecitabine / XELODA, 5-Trifluoromethyl-2'-deoxyuridine, methotrexate sodium, raltitrexed / Tomudex, pemetrexed / Alimta ®, cytosine Arabinoside (Cytarabine, Ara- C) / Thioguanine, 6-mercaptopurine (Mercaptopurine, 6-MP), azathioprine / Azasan, 6-thioguanine (6-TG) / Purinethol (TEVA), pentostatin / Nipent, fludarabine phosphate / Fludara ®, cladribine (2- CdA, 2-chlorodeoxyadenosine) / Leustatin, floxuridine (5-fluoro-2) / FUDR (Hospira, Inc.), ribonucleotide Reductase Inhibitor (RNR), cyclophosphamide / Cytoxan (BMS), neosar, ifosfamide / Mitoxana, thiotepa, BCNU— l,3-bis(2-chloroethyl)-l-nitosourea, l,-(2-chloroethyl)-3- cyclohexyl-lnitrosourea, methyl CCNU, hexamethylmelamine, busulfan / Myleran, procarbazine HCL / Matulane, dacarbazine (DTIC), chlorambucil / Leukaran ®, melphalan / Alkeran, cisplatin (Cisplatinum, CDDP) / Platinol, carboplatin / Paraplatin, oxaliplatin / Eloxitan, bendamustine, carmustine, chloromethine, dacarbazine (DTIC), fotemustine, lomustine, mannosulfan, nedaplatin, nimustine, prednimustine, ranimustine, satraplatin, semustine, streptozocin, temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA, triplatin tetranitrate,trofosfamide, uramustine, doxorubicin HCL / Doxil, daunorubicin citrate / Daunoxome ®, mitoxantrone HCL / Novantrone, actinomycin D, etoposide / Vepesid, topotecan HCL / Hycamtin, teniposide (VM-26), irinotecan HCL(CPT-l l) / , camptosar ®, camptothecin, Belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbine tartrate, vindesine sulphate, paclitaxel / Taxol, docetaxel / Taxotere, nanoparticle paclitaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel, vinflunine, and the like. In certain embodiments the anti-cancer drug(s) comprise one or more drugs selected from the group consisting of carboplatin(e.g., PARAPLATIN®), Cisplatin (e.g., PLATINOL®, PLATINOL-AQ®), Cyclophosphamide (e.g., CYTOXAN®, NEOSAR®), Docetaxel (e.g., TAXOTERE®), Doxorubicin (e.g., ADRIAMYCIN®), Erlotinib (e.g. , TARCEVA®), Etoposide (e.g. , VEPESID®), Fluorouracil (e.g. , 5-FU®), Gemcitabine (e.g., GEMZAR®), imatinib mesylate (e.g., GLEEVEC®), Irinotecan (e.g., CAMPTOSAR®), Methotrexate (e.g., FOLEX®, MEXATE®, AMETHOPTERIN®), Paclitaxel (e.g., TAXOL®, ABRAXANE®), Sorafmib (e.g., NEXAVAR®), Sunitinib (e.g., SUTENT®), Topotecan (e.g., HYCAMTIN®), Vinblastine (e.g., VELBAN®), Vincristine (e.g. , ONCOVIN®, VINCASAR PFS®). In certain embodiments the anti-cancer drug comprises one or more drugs selected from the group consisting of retinoic acid, a retinoic acid derivative, doxirubicin, vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin, 5 -fluorouracil, a camptothecin derivative, interferon, tamoxifen, and taxol. In certain embodiments the anti-cancer compound is selected from the group consisting of abraxane, doxorubicin, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestroltamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, zoledronic acid, vinblastine, etc.), an antisense molecule, an siRNA, and the like.

[0064] In certain embodiments the cytotoxic / cytostatic agent comprises a protein or peptide toxin or fragment thereof. Enzymatically active toxins and fragments thereof are exemplified by diphtheria toxin A fragment, nonbinding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, a-sacrin, certain Aleurites for dii proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Morodica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin, enomycin, and the tricothecenes, for example.

[0065] In certain embodiments the cytotoxins can include, but are not limited to Pseudomonas exotoxins, Diphtheria toxins, ricin, abrin and derivatives thereof. Pseudomonas exotoxin A (PE) is an extremely active monomelic protein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa, which inhibits protein synthesis in eukaryotic cells through the inactivation of elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD onto EF-2).

[0066] In certain embodiments the modified CCL27 proteins are attached to, or co- administered with, an immunomodulator and (when attached) localizes the immunomodulator at the cancer cell / tumor site. Numerous immunomodulators that can activate an immune response are known to those of skill in the art. In one illustrative, but non-limiting embodiment the immunomodulator comprise an anti-CD3 antibody or ligand, or an anti-CD-16a antibody or ligand. Anti-CD3 monoclonal antibodies can bind T-cells, and optionally induce the proliferation of human T-cells cells in vitro and activate specific and nonspecific cytolysis by human T-cell clones and human peripheral blood lymphocytes. In vivo administration of anti-CD3 prevents tumor growth of a UV-induced mouse fibro sarcoma. Anti-CD16a antibodies bind natural killer (NK) cells.

[0067] In certain embodiments the immunomodulators comprise agents that blockade immune checkpoints. Immune checkpoints refer to a plethora of inhibitory pathways hardwired into the immune system that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. It is now clear that tumors co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens. Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors.

[0068] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibodies were the first of this class of immunotherapeutics to achieve US Food and Drug Administration (FDA) approval. The first such drug to receive approval, ipilimumab (Yervoy®), for the treatment of advanced melanoma, blocks the activity of a checkpoint protein known as CTLA4, which is expressed on the surface of activated immune cells called cytotoxic T lymphocytes. CTLA4 acts as a "switch"to inactivate these T cells, thereby reducing the strength of immune responses; ipilimumab binds to CTLA4 and prevents it from sending its inhibitory signal. Two other FDA-approved checkpoint inhibitors, nivolumab (Opdivo®) and pembrolizumab (Keytruda®), work in a similar way, but they target a different checkpoint protein on activated T cells known as PD-1. Nivolumab is approved to treat some patients with advanced melanoma or advanced lung cancer, and pembrolizumab is approved to treat some patients with advanced melanoma. Accordingly in certain embodiments the immunomodulators comprise antibodies directed against CTLA4 (e.g., ipilimumab), and / or antibodies directed against PD-L1 (e.g., nivolumab, pembrolizumab), and / or antibodies directed against PD-L2.

[0069] Other examples of immune modulators that can be attached to, or co-administered with, modified CCL27 proteins include, but are not limited to, gancyclovier, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolgate mofetil, methotrextrate, glucocorticoid and its analogs, cytokines, xanthines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferons-alpha, interferon-beta, interferon-gamma), the stem cell growth factor designated "S1 factor," erythropoietin and thrombopoietin, or a combination thereof.

[0070] Useful immunomodulatory agents also include anti-hormones that block hormone action on tumors and immunosuppressive agents that suppress cytokine production, down- regulate self-antigen expression, or mask MHC antigens. Representative anti-hormones include anti-estrogens including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)- imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapnstone, and toremifene; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and anti-adrenal agents. Illustrative immunosuppressive agents include, but are not limited to 2-amino-6-aryl-5-substituted pyrimidines, azathioprine, cyclophosphamide, bromocryptine, danazol, dapsone, glutaraldehyde, anti -idiotypic antibodies for MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticosteroids, cytokine or cytokine receptor antagonists (e.g., anti-interferon antibodies, anti-ILlO antibodies, anti-TNFa antibodies,anti-IL2 antibodies), streptokinase, TGFP, rapamycin, T-cell receptor, T-cell receptor fragments, and T cell receptor antibodies.

[0071] In certain embodiments, the effector comprises a viral particle (e.g., a filamentous phage, an adeno-associated virus (AAV), a lentivirus, and the like). The modified CCL27 protein can be conjugated to the viral particle and / or can be expressed on the surface of the viral particle (e.g. a filamentous phage). The viral particle can additionally include a nucleic acid that is to be delivered to the target (e.g., a cancer cell that expresses CCR10) cell. The use of viral particles to deliver nucleic acids to cells is described in detail in WO 99 / 55720, US 6,670,188, US 6,642,051, and US Patent No: 6,669,936.

[0072] In certain embodiments, the modified CCL27 proteins described herein can be chemically conjugated to the effector molecule (e.g., a cytotoxin, a label, a ligand, a drug, a liposome, etc.). Means of chemically conjugating molecules are well known to those of skill. The procedure for attaching an effector to an antibody will vary according to the chemical structure of the effector and / or antibody. Polypeptides typically contain variety of functional groups; e.g., carboxylic acid (COOH) or free amine (- H2) groups, that are available for reaction with a suitable functional group on an effector molecule to bind the effector thereto. Alternatively, the modified CCL27 protein and / or the effector can be derivatized to expose or attach additional reactive functional groups. The derivatization can involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Illinois.

[0073] A "linker", as used herein, is a molecule that is used to join the targeting molecule to the effector molecule. The linker is capable of forming covalent bonds to both the targeting molecule and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the targeting molecule and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine). However, in some embodiments, the linkers will be joined to the alpha carbon amino or carboxyl groups of the terminal amino acids.

[0074] The linked modified CCL27 protein / effectors can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. (1987) Science 238: 1098. Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an illustrative, but non-limiting, chelating agent for conjugation of, e.g., a radionucleotide to the antibody (see, e.g., WO 1994 / 011026 (PCT / US 1993 / 010953)).

[0075] In certain embodiments conjugation of effectors (e.g., drugs, liposomes, etc.) or linkers attached to effectors, to a modified CCL27 protein takes place at solvent accessible reactive amino acids such as lysines or cysteines that can be derived from the reduction of inter-chain disulfide bonds in the modified CCL27 protein. In certain embodiments cysteine conjugation can occur after reduction of four inter-chain disulfide bonds.

[0076] In some embodiments, a modification can optionally be introduced into the CCL27 proteins or fusions thereof (e.g., within the polypeptide chain or at either the N- or C-terminal), e.g., to extend in vivo half-life, such as PEGylation or incorporation of long-chain polyethylene glycol polymers (PEG), or by fusion to a protein that extends the in vivo half-life, such as but not limited to human albumin or an Fc domain of an antibody. Introduction of PEG or long chain polymers of PEG increases the effective molecular weight of the polypeptides, for example, to prevent rapid filtration into the urine. In some embodiments, a Lysine residue in the sequence is conjugated to PEG directly or through a linker. Such linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group for linkage to the appropriately modified PEG chain. An alternative method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or at solvent exposed residues such as replacements for Arg or Lys residues. This Cys residue is then site-specifically attached to a PEG chain containing, for example, a maleimide function. Methods for incorporating PEG or long chain polymers of PEG are known in the art (described, for example, in Veronese, F. M., et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, R. B., et al., Adv. Drug Deliv. Rev.55: 217-50 (2003); Roberts, M. J., et al., Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents of which are incorporated herein by reference.

[0077] In certain embodiments, specific mutations of the modified CCL27 proteins can be made to alter the glycosylation of the polypeptide. Such mutations may be selected to introduce or eliminate one or more glycosylation sites, including but not limited to, O-linked or N-linked glycosylation sites. In certain embodiments, the proteins have glycosylation sites and patterns unaltered relative to the naturally-occurring proteins. In certain embodiments, a variant of proteins includes a glycosylation variant wherein the number and / or type of glycosylation sites have been altered relative to the naturally-occurring proteins. In certain embodiments, a variant of a polypeptide comprises a greater or a lesser number of N-linked glycosylation sites relative to a native polypeptide. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. In certain embodiments, a rearrangement of N-linked carbohydrate chains is provided, wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created.

[0078] In some embodiments, the modified CCL27 proteins described herein (e.g., SEQ ID NO:4) can be fused to a transmembrane domain, optionally via a hinge domain. In some embodiments, the transmembrane domain is linked to one or more intracellular signaling domain. The fusion proteins can be optionally expressed in a cell and optionally anchored via the transmembrane domain in a cell membrane of a cell. Exemplary cells can include but are not limited to human cells, e.g., human immune cells, e.g., T-cells, NK cells or macrophages. As one example, in some embodiments, a modified CCL27 protein described herein acts as an extracellular targeting domain of a chimeric antigen receptor (CAR).

[0079] In some embodiments, the CAR may contain one or more hinge domains that link a CCR10-binding domain comprising a modified CCL27 protein (e.g., SEQ ID NO:4 or as described herein) and the transmembrane domain, for positioning the CCR10-binding domain. Such a hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region, e.g., a naturally occurring human immunoglobulinhinge region, or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1 , CD 152, and CD7, which may be wild-type hinge regions from these molecules or may be altered.

[0080] Any transmembrane suitable for use in a CAR construct may be employed. Such transmembrane domains, include, but are not limited to, all or part of the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGAl, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C.

[0081] A transmembrane domain incorporated into a CAR construct may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.

[0082] A CAR construct of the present disclosure can include one or more intracellular signaling domains, also referred to herein as co-stimulatory domains, or cytoplasmic domains that activate or otherwise modulate an immune cell, (e.g., a T lymphocyte). The intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. In one embodiment, a co- stimulatory domain is used that increases CAR immune T cell cytokine production. In another embodiment, a co-stimulatory domain is used that facilitates immune cell (e.g., T cell) replication. In still another embodiment, a co-stimulatory domain is used that prevents CAR immune cell (e.g., T cell) exhaustion. In another embodiment, a co-stimulatory domain is used that increases immune cell (e.g., T cell) antitumor activity. In still a further embodiment, a co-stimulatory domain is used that enhances survival of CAR immune cells (e.g., T cells) (e.g., post-infusion into patients).

[0083] In some embodiments, the CAR comprises a modified CCL27 protein as described herein (e.g., SEQ ID NO:4, 8 or 9), a CD8 hinge domain, a CD8 transmembrane domain, a 4- 1BB domain and a CD3-zeta domain.

[0084] Examples of intracellular signaling domains for use in a CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0085] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine- based activation motifs or ITAMs.

[0086] Examples of ITAM containing primary intracellular signaling domains include those of CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta.

[0087] An intracellular signaling domain of a CAR can comprise a primary intracellular signaling domain only, or may comprise additional desired intracellular signaling domain(s) useful in the context of a CAR of the invention. For example, the intracellular signaling domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds to CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity invivo (Song et al. Blood.2012; 119(3):696-706). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAMl, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, and CD 19a.

[0088] In some embodiments, a CAR may be designed as an inducible CAR, or may otherwise comprise a mechanism for reversibly expressing the CAR, or controlling CAR activity to largely restrict it to a desired environment. Thus, for example, in some embodiments, the CAR- expressing cell uses a split CAR. The split CAR approach is described in more detail in publications WO2014 / 055442 and WO2014 / 055657.

[0089] In some embodiments, a cell expressing a CAR comprising a modified CCL27 protein as described herein also expresses a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., that binds to the same target or a different target.

[0090] In some embodiments, a host cell, e.g., a host immune cell (e.g., a T-cell or other immune cell) is modified to express a modified CCL27 protein as described herein, or a CAR comprising the modified CCL27 protein , using a gene editing system, such as a Cas / CRISPR system, a Transcription activator-like effector nuclease (TALEN) system, a homing endonuclease (HE) system, or a zinc-finger nuclease (ZFN) system.

[0091] The mammalian (e.g., human) cell expressing the CARs described herein are not limited by the type of cells (which can be immune cells). Illustrative immune cells include, but are not limited to, T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, macrophages, and myeloid-derived phagocytes. In some embodiments, the T cells are CD8+ T cells Treg cells. In some embodiments, the immune cells, e.g., T cells, are autologous cells from the patient to undergoimmunotherapy. In some embodiments, the immune cells are allogeneic. Methods of making CAR-expressing cells are described, e.g., in US2016 / 0185861 and US2019 / 0000880.

[0092] Many methods for introducing nucleic acids and viral vectors (e.g., viral particles) into a target cell (e.g., a CD8+T cell) are available. Non-limiting examples of suitable methods include electroporation (e.g., nucleofection), viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, microparticle- or nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, a viral vector may be used, such as an adenovirus, adeno-associated virus (AAV), lentivirus vector, a vaccinia virus vector, or any of a number of different vectors.

[0093] In certain embodiments, the CARs comprising a modified CCL27 protein described herein, are recombinantly expressed using methods well known to those of skill in the art. For example, using the sequence information provided herein, nucleic acids encoding the desired CAR can be prepared according to a number of standard methods known to those of skill in the art. The nucleic acids are transfected into host cells that then express the desired CAR.

[0094] Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning – A Laboratory Manual (2nd ed.) Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, F.M. Ausubel et al. , eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods of producing recombinant immunoglobulins are also known in the art. See, Cabilly, U.S. Patent No.4,816,567; and Queen et al. (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033.

[0095] In some embodiments, instead of a CAR, the modified CCL27 protein is part of a bi- specific or multi-valent protein, e.g., as a function of linkage to one domain of an antigen-binding antibody. As an example, in some embodiments, the bi-specific protein comprises a modified CCL27 protein and heavy and light chain variable regions that bind to CD3, CD16a, or another target protein. In some embodiments, the modified CCL27 protein is fused to a first Fc domain and an scFv (e.g., that binds CD3, CD16a or another antigen) is linked to a second Fc, and the first and second Fc are linked covalently (e.g., via disulfide bonding between the Fc domains) or non-covalently (e.g., via knob-in-hole binding of modified Fc domains).

[0096] The cells (e.g., immune cells) expressing a CAR as described herein, or a bi-specific protein as described herein, can be used to treat cancer, i.e., cancer cells that express CCR10. Exemplary cancer cells that express CCR10 can include multiple myeloma cancer cells. Sources of immune cells for transplantation can include but are not limited to bone marrow, recruited peripheral blood and umbilical cord blood and can be autologous or allogeneic (optionally HLA- matched between the donor and recipient).

[0097] Also provided are nucleic acids encoding the modified CCL27 protein (e.g., SEQ ID NO:4, 5, or 6), or fusions thereof (e.g., as described herein). Nucleic acids can be in the form or RNA or DNA or can comprise non-natural nucleotides. In some embodiments, a polynucleotide comprising a promoter operably linked to a nucleic acid encoding the modified CCL27 protein (e.g., SEQ ID NO:4, 5, or 6), or fusions thereof (e.g., as described herein) is provided. These nucleic acids can be inserted into any of a number of well-known vectors, including viral expression vectors or plasmid-based vectors for the transfection of target cells and organisms. The nucleic acids may be transfected into cells, e.g., immune cells, ex vivo or in vivo or administered in vivo.

[0098] In some embodiments, the nucleic acid constructs encoding the modified CCL27 protein or a fusion thereof is provided as a purified nucleic acid molecule, for example, as a DNA plasmid-based vectors (“naked” DNA). Alternatively, in some embodiments, a nucleic acid construct encoding the modified CCL27 protein or a fusion thereof may be contained within a viral vector and optionally administered to a human as a virus or introduced into a cell to expressing the protein. Viral delivery systems include adenovirus vectors (e.g., Ad2, Ad5, Ad7), adeno-associated viral vectors, herpes simplex viral vectors, retroviral vectors, pox viral vectors (such as vaccinia and avian poxvirus vectors, such as the fowlpox and canarypox vectors), lentiviral vectors, alphavirus vectors, poliovirus vectors, and other positive and negative strandedRNA viruses, viroids, and virusoids, or portions thereof. Methods of constructing and using such vectors are well known in the art.

[0099] Nucleic acids for administration to a subject are formulated for pharmaceutical administration. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, including intranasal, intradermal, subcutaneous or intramuscular injection or electroporation, the carrier may comprise water, saline, and optionally an alcohol, a fat, a polymer, a wax, one or more stabilizing amino acids or a buffer. General formulation technologies are known to those of skill in the art.

[0100] In some embodiments, a nucleic acid encoding the modified CCL27 protein or a fusion thereof is introduced into a cell such that the modified CCL27 protein or a fusion thereof is expressed in the cell. In some embodiments, cells are obtained from a mammal (e.g., a human), optionally enriched for one or more characteristic (e.g., expression of a surface protein such as CD4 or CD8), and then a nucleic acid encoding the modified CCL27 protein or a fusion thereof is introduced. The cells can be introduced back to the mammal (e.g., human) such that the cells are modified autologous cells. In other embodiments, the recipient is a different individual than the source of the cells (allogeneic). Methods of making cells can occur in vitro, ex vivo or in vivo. EXAMPLES

[0101] Ferguson et al., NATURE COMMUNICATIONS (2022) 13:4121 identified CCR10 as a therapeutic target for multiple myeloma (MM) and showed it is correlated with significantly worse outcomes. Ferguson et al. developed a proof-of-concept therapeutic that utilized its natural ligand CCL27 as the binder because it is thought to bind specifically to CCR10. However, Ferguson et al. saw minimal efficacy of the wild-type natural ligand (WT-CCL27) as a binder to direct cytotoxic capabilities against CCR10.

[0102] To improve this performance, we identified the relevance of the N-terminus of CCL27 in its interaction with CCR10 and it was determined that there was scope to improve the binding of CCL27 by modifying N-terminal residues. From this, we created a small mutational library of 10 unique CCL27 binder constructs with modified N-terminal amino acids.

[0103] The library of CCL27 mutant CAR T-cells was screened for in-vitro cytotoxicity against multiple myeloma cell lines (AMO1 and MM.1S) that were transduced with firefly luciferase. This enabled the measurement of tumor cells by the intensity of luminescence when exposed to the substrate luciferin. Through this in-vitro screen, two mutants with a single additional aromatic amino acid (tryptophan and phenylalanine) performed near the level of the positive control BCMA CAR T-cells in one or both myeloma cell lines.

[0104] We further validated the performance of the W-CCL27 and F-CCL27 mutant CAR T- cells with additional T-cell donors, new myeloma cell lines, and CCR10 knockout cell lines. These experiments showed that the efficacy of these mutant CCL27 CAR T-cells was replicable across T-cell donors and myeloma cell lines with reasonably high levels of CCR10 expression (FIG.1). The CCR10 knockout cell line showed that the CCL27 mutant CAR T-cells were specifically targeting CCR10 as we saw no cytotoxicity (FIG.1). We validated the efficacy of these two CCL27 mutants in vivo with 1e6 MM.1S cells implanted I.V. in NSG mice followed by administration of CAR-Ts 8 days after tumor implantation, with n=3 mice per arm. This study showed that the W-CCL27 and F-CCL27 mutants outperformed the WT CCL27 CAR-T, which performed similarly to the negative control empty CAR. We also saw that our CCL27 mutants performed similarly to BCMA CAR, which aligned well with our in-vitro results.

[0105] Lastly, we checked the binding of these CCL27 mutants compared to the WT CCL27 binder and positive control BCMA CAR-T binder using an on-cell assay looking for the formation of doublets between CAR T-cells and tumor cells. This assay in both the MM.1S and AMO1 myeloma cell lines showed the CCL27 mutants improved the binding substantially from the WT protein, towards the level of the BCMA CAR T-cells (FIG.2).

[0106] The specificity of the CCL27 mutant CAR T-cells for myeloma was further validated through a co-culture of the CAR-Ts with donor-matched peripheral blood mononuclear cells (PBMCs) and the MM.1S myeloma cell line in two T-cell donors. This assay showed that the CCL27 mutants have no major toxicity to important PBMC populations, while robustly depleting myeloma cells as measured and quantified by flow cytometry. See, FIG.3. In contrast, the CD33 CAR T-cells showed noticeable depletion of monocyte sub-populations.

[0107] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. SEQUENCES SEQ ID NO:1 Human CCL27: FLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQRSICIHPQNPS LSQWFEHQERKLHGTLPKLNFGMLRKMG SEQ ID NO:2 W-CCL27: WFLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQRSIC(I / V)HP QNPSLSQWFEHQERK(L / F)HGTLPKLNFGMLRKMG SEQ ID NO:3 F-CCL27: FFLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQRSIC(I / V)HPQ NPSLSQWFEHQERK(L / F)HGTLPKLNFGMLRKMG SEQ ID NO:4 (W / F)nFLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQRSIC(I / V) HPQNPSLSQWFEHQERK(L / F)HGTLPKLNFGMLRKMG (SEQ ID NO: 4), where n is 1 or 2 and each position is independently selected from W and F, e.g., W, F, FF, WW, WF or FW. SEQ ID NO:5 W-CCL27: WFLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQRSICIHPQNP SLSQWFEHQERKLHGTLPKLNFGMLRKMG SEQ ID NO:6 F-CCL27: FFLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQRSICIHPQNP SLSQWFEHQERKLHGTLPKLNFGMLRKMG SEQ ID NO:7 CCR10: MGTEATEQVSWGHYSGDEEDAYSAEPLPELCYKADVQAFSRAFQPSVSLTVAALGLAGNGLVLATHLAARRAARSPTSAHLLQLALADLLLALTLPFAAAGALQGWSLGSATCRTIS GLYSASFHAGFLFLACISADRYVAIARALPAGPRPSTPGRAHLVSVIVWLLSLLLALPALL FSQDGQREGQRRCRLIFPEGLTQTVKGASAVAQVALGFALPLGVMVACYALLGRTLLA ARGPERRRALRVVVALVAAFVVLQLPYSLALLLDTADLLAARERSCPASKRKDVALLV TSGLALARCGLNPVLYAFLGLRFRQDLRRLLRGGSCPSGPQPRRGCPRRPRLSSCSAPTE THSLSWDN SEQ ID NO:8 W-CCL27: WFLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQRSICIHPQNP SLSQWFEHQERKLHGTLPKLNFGMLRKMGEQKLISEEDLEEDLTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:9 F-CCL27: FFLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQRSICIHPQNPS LSQWFEHQERKLHGTLPKLNFGMLRKMGEQKLISEEDLEEDLTTTPAPRPPTPAPTIASQ PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

WHAT IS CLAIMED IS:

1. A protein comprising a CCR10-binding polypeptide comprising SEQ ID NO:

4.

2. The protein of claim 1, wherein the CCR10-binding polypeptide comprises SEQ ID NO:8 or SEQ ID NO:

9.

3. The protein of claim 1 or 2, wherein the amino terminus of the protein is the amino terminus of SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO:

9.

4. The protein of any one of claims 1-3, wherein the CCR10-binding polypeptide is linked to a transmembrane domain.

5. The protein of claim 1, wherein a hinge domain links the CCR10-binding polypeptide to the transmembrane domain.

6. The protein of claim 4 or 6, wherein the transmembrane domain is linked to an intracellular signaling domain.

7. The protein of claim 4 or 6, wherein the transmembrane domain is linked to two or more intracellular signaling domains.

8. The protein of claim 5, wherein the intracellular signaling domains are a CD3 zeta domain and a 4-1BB domain.

9. The protein any one of claims 1-3, wherein the CCR10-binding polypeptide is linked an antibody or fragment thereof.

10. The protein of claim 9, wherein the antibody specifically binds to CD3 or CD16a.

11. The protein any one of claims 1-3, wherein the CCR10-binding polypeptide is linked to a detectable label, toxin, therapeutic molecule or a radioactive molecule.

12. A cell comprising and / or expressing the protein of any one of claims 1-10.

13. The cell of claim 13, wherein the cell is a human cell.

14. The cell of claim 12 or 13, wherein the cell is an immune cell.

15. The cell of claim 14, wherein the immune cell is a T-cell, natural killer cell, or a macrophage.

16. A nucleic acid encoding the protein of any one of claims 1-10.

17. A method of killing a cancer cell expressing CCR10, the method comprising contacting the cell of any one of claims 12-15 to the cancer cell such that the cell kills the cancer cell.

18. The method of claim 17, wherein the cancer cell is a multiple myeloma cancer cell.

19. The method of any one of claims 17 or 18, wherein the method is performed in vivo.

20. A method of making a cell expressing a the protein of any one of claims 1- 10, the method comprising, introducing into the cell a nucleic acid encoding protein into the cell and allowing the cell to express the CCR10-binding polypeptide.

21. The method of claim 20, wherein the cell is a human cell.

22. The method of claim 20 or 21, wherein the cell is an immune cell.

23. The method of claim 22, wherein the immune cell is a T-cell, natural killer cell, or a macrophage.

24. The method of any one of claims 20-23 , comprising obtaining the cell from a human; introducing the nucleic acid into the cell; and administering the cell to the same of a different human.

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