Polypeptides that bind to KV1.3, zip6, NKP46 and EGFR

Abstract

The present disclosure provides polypeptides that bind Kv1.3, Zip6, NKp46, EGFR, and GIPR. The polypeptides described herein variously comprise knob domains, VHH domains, and antibody binding domains with affinity for Kv1.3, Zip6, NKp46, or EGFR. The present disclosure also provides multispecific binding molecules comprising a subject knob domain, VHH domain, and antibody binding domain and at least one other binding domain. Also provided are various methods of use for the polypeptides described herein.

Description

[0001] Atty. Dkt: CRYS-029WO

[0002] POLYPEPTIDES THAT BIND TO Kvl.3, ZIP6, NKP46 AND EGFR

[0003] CROSS-REFERENCING

[0004] This application claims the benefit of provisional application serial no. 63 / 738,359, filed on December 23, 2024, provisional application serial no. 63 / 747,749, filed on January 21, 2025, provisional application serial no. 63 / 768,078, filed on March 6, 2025, provisional application serial no. 63 / 785,572, filed on April 8, 2025, provisional application serial no. 63 / 785,575, filed on April 8, 2025, and provisional application serial no.63 / 883,608, filed on September 17, 2025, which applications are incorporated by reference herein in their entireties.

[0005] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED As A SEQUENCE LISTING XML FILE A Sequence Listing is provided herewith as a Sequence Listing XML,

[0006] “CRYS-029WO_SEQLIST.xml” created on December 20, 2025 and having a size of 2,733,529 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0007] BACKGROUND

[0008] Kyl.3

[0009] Ion channels are pore forming integral membrane proteins that enable and modulate the flux of specific ions across the cellular membranes of cells across all domains of life. Ion channels are necessary for regulating ion concentrations across cell and organelle membranes, maintaining resting membrane potentials, and generating action potentials. Ion channels can exhibit highly selective permeability for specific ions such as sodium, potassium, or calcium ions. While some ion channels are passive, gated ion channels can actively regulate the flow of ions across their pore. Gated ion channels regulate the opening or closing of the pore in response to a specific stimulus such as pH, voltage across a membrane, mechanical stress, or the binding of a ligand.

[0010] Kvl.3 is a voltage-gated, potassium (K+) selective ion channel and a member of the mammalian Kvl family of voltage-gated K+ channels. Kvl family channels are tetramers, each subunit characterized by a large cytoplasmic domain and six transmembrane helices (S1-S6). The transmembrane helices S1-S4 form a voltage sensing domain whereas the S5-S6 helices form the ion pore. In addition, the S5-S6 helices contain the P-loop which includes a conserved selectivity filter motif that is responsible for maintaining the K+ selective permeability of the Kvl family ion channels. The S6 helix of each subunit in the tetramer assemble on the intracellular side of cell membrane to form a gate. When the cell membraneAtty. Dkt: CRYS-029WO

[0011] is at its resting potential the gate is closed. Upon depolarization of the cell membrane, the voltage gated sensing domains undergo a conformational change which opens the gate and enables K+ ion efflux.

[0012] Kvl.3 is the only one of the eight Kvl family ion channels expressed in T cells, where it plays an essential role in T cell activation. T cell activation begins when the T cell receptor (TCR) engages with an antigen-MHC complex on the surface of an antigen-presenting cell. This engagement triggers a rapid influx of Ca2+ into the cell which depolarizes the T cell membrane. This depolarization stimulates Kvl.3 in the T cell membrane to open which enables the efflux of K+ to re-polarize the cell membrane and restore the membrane potential that drives the Ca2+ influx needed for sustaining T cell activation.

[0013] The key role Kvl.3 plays in T cell activation has made it a promising therapeutic target for a diverse range of diseases caused by, characterized by, or associated with inflammation (e.g., psoriasis, inflammatory bowel disease, hepatic fibrosis, Alzheimer’s disease, Parkinson’s disease, etc.). For example, the role of Kvl.3 in the activation of auto-reactive effector memory T cells (TEMs) has been implicated in the pathogenesis of chronic autoimmune diseases such as rheumatoid arthritis (RA), Type 1 diabetes (T1D), systemic lupus erythematosus (SLE), multiple sclerosis (MS), etc.

[0014] Existing Kvl.3 modulators are typically small molecules or peptide toxins derived from plants and animal venoms. These types of modulators function by physically occluding the ion channel pore of Kvl.3 to block the permeation of K+. However, the ion channel pore region is relatively conserved across Kvl ion channel family members and thus small molecule and peptide toxins exhibit poor selectivity for Kvl.3 with the accompanying risk of toxic side effects. Engineering efforts for improving the specificity of such small molecules and peptide toxins must be done on a case-by-case basis which creates time delays and increases cost. While antibody -based approaches for modulating Kvl.3 offer the promise high specificity in targeting Kvl.3, development of such antibodies has been limited and there are currenlly no FDA approved antibody-derived therapeutics against Kvl.3.

[0015] Zip6

[0016] Zinc is an essential trace element necessary for cell growth and proliferation and is involved in variety of specific cellular processes including gene regulation, cell signaling, protein folding, and functioning as a cofactor for many enzymes. Accordingly, the regulation of zinc homeostasis is key for maintaining normal cellular function. Zinc homeostasis is maintained, in large part, through the action of zinc transporters which regulate the flux of zinc into and out of cells and cell organelles. In mammals, zinc transporters are grouped into two families: the ZIP (Zrt-, Irt-like protein) family and the Znt (zinc transporter) family. The ZIP family, otherwise known as the SLC39A (solute carrier family 39 A) family, is made up of 14 members (Zipl-14) that regulate the flux of extracellular zinc into cytoplasm across the plasma membrane and the flux of intracellular zinc into and out of organelles. Zip6 is localized to the plasma membrane where it facilitates transport of extracellular zinc into the cytoplasm. Like other ZIPAtty. Dkt: CRYS-029WO

[0017] family members, Zip6 contains eight trans-membrane domains (TMDs) with a histidine -rich cytoplasmic loop between transmembrane domains 3 and 4. Zip6 can also form a homodimer or a heterodimer with another ZIP family member.

[0018] The dysregulation of zinc homeostasis has been implicated in the progression of cancer and other diseases. Many cancers exhibit aberrant expression of Zip6. As an example, Zip6 is overexpressed in breast cancers and has been demonstrated to play a role in the epithelial-mesenchymal transition (EMT) which can lead to malignant progression and metastasis of breast cancers. Zip6 is thus a promising target for the treatment of cancers and other diseases.

[0019] NKp46 is a natural cytotoxicity receptor expressed on most or all mature NK (natural killer) cells. NK cells can be weakly activated by engagement of NKp46 alone. However, co-stimulation of NKp46 and other activating co-receptors (particularly 2B4, DNAM1, or CD2) enhances NK cell activation greatly. NKp46 does not appear to be downregulated in tumor-infiltrated NK cells in several cancers (e.g., lung carcinoma, acute myeloid leukemia and breast cancer) and, as such, NKp46 may be a promising target for an NK cell engager (NKCE), such as a BiKE (bispecific killer cell engager) or TriKE (bispecific killer cell engager). NKCEs hold NK and cancer cells together and activate the NK cells while they are attached to the cancer cells. NK cells can also attack virally-infected cells.

[0020] EGER

[0021] The epidermal growth factor receptor (EGFR) belongs to a family of proteins harboring intrinsic tyrosine kinase function. EGFR, like other members of its family, plays key roles in the growth and proliferation of both normal and malignant cells. Overexpression of EGFR has been observed in over 70% of human cancers, including many solid tumors. EGFR thus presents an attractive pharmacological target for treating a variety of cancers. Tyrosine kinase inhibitors (Tikis) have been previously used to target EGFR, but resistance to TKIs frequently arise in tumors due to mutations in EGFR.

[0022] GIPR

[0023] Glucose-dependent Insulinotropic Polypeptide, also named Gastric Inhibitory Polypeptide (or GIP), has long been known as one of the incretins stimulating insulin secretion in response to food intake. However, in the context of diabetes the insulinotropic action of GIP is markedly diminished. In contrast, effects of GIP on fat deposition and lipid metabolism in adipose tissue are not impaired, thus promoting the development of insulin resistance and obesity. Furthermore, GIP stimulates the secretion of glucagon, which might contribute to the lack of postprandial glucagon suppression and hyperglycemia seen in patients with type 2 diabetes.

[0024] GIP, a 42 -amino acid peptide, is released into circulation from K cells in the duodenum and small intestine upon nutrient ingestion. GIP exerts is activity via its receptor, GIPR. GIPR is expressedAtty. Dkt: CRYS-029WO

[0025] primarily in the pancreas, adipose tissue, stomach, small intestine, bone and central nervous system. The GIP receptor is a member of the Class B (Secretin) family of G protein-coupled receptors and activation results in the stimulation of adenylyl cyclase and Ca(2+)-independent phospholipase A(2) and activation of protein kinase (PK) A and PKB. The GIP receptor is coupled to Gas, and activation of the receptor leads to an increase of the second messenger cAMP. GIPR is characterized by a large extracellular loop (extracellular domain - ECD) that serves as the site of specific interaction with its ligand, binding with low affinity (pM range). The ECD confers the selectivity of the receptor to its ligand, and upon binding a conformational change leads to receptor activation with potency in the pM range.

[0026] Various groups have shown that GIPR antagonism has a beneficiary impact on disease phenotype in rodent models. Under a high fat diet, GIPR knock-out mice show an increased insulin-sensitivity, a resistance against diet-induced obesity, suppression of liver steatosis, and reduced plasma cholesterol and triglyceride levels. Similar effects are seen with a variety of antagonistic peptides, and recently with antagonistic antibodies raised in a phage display campaign.

[0027] GPCRs, however, are difficult targets for antibody campaigns. Often, GPCRs occur in low density on the cell surface and are very unstable when purified from the cellular membrane, presenting a challenge in obtaining sufficient amounts of immunogen in which native epitopes are maintained for antibody recognition. Furthermore, a particular therapeutic concept may require an antibody that does not just bind the GPCR but acts as an agonist or antagonist, which may necessitate the recognition of particular, potentially ligand-sensitive, epitopes. These additional requirements may further reduce the effective hit rate in antibody generation campaigns.

[0028] There is a clear need for new polypeptides that specifically recognize Kvl.3, Zip6, NKp46, EGFR, and GIPR.

[0029] SUMMARY

[0030] The present disclosure provides polypeptides comprising an antibody binding domain comprising: i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a heavy chain variable domain selected from Table 1 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of a heavy chain variable domain selected from Table 1 except for up to 10 amino acid substitutions in the collective CDR regions, and ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 2 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 2 except for up to 10 amino acid substitutions in the collective CDR regions, wherein the antibody bindingAtty. Dkt: CRYS-029WO

[0031] domain binds to Kvl.3. In some embodiments, the antibody binding domain comprises: a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain selected from Table 3 and a light chain variable domain that is at least 90% identical to the light chain variable domain selected from Table 4. In some cases, the antibody binding domain comprises:a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain selected from Table 3 and a light chain variable domain that is at least 95% identical to the light chain variable domain selected from Table 4.

[0032] The present disclosure also provides polypeptides comprising a) an antibody binding domain comprising: i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 9 or are otherwise identical to the CDR1, CDR2. and CDR3 regions of an antibody selected from Table 9 except for up to 10 amino acid substitutions in the collective CDR regions, and ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 10 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of an antibody selected from Table 10, except for up to 10 amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to Zip6; or b) an antibody binding domain comprising: i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 8 or are otherwise identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 8 except for up to 10 amino acid substitutions in the collective CDR regions, and ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of the selected common light chain antibody or are otherwise identical to the light chain CDR1, CDR2, and CDR3 regions of the selected common light chain antibody except for up to 10 amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to Zip6. In some embodiments, the antibody binding domain comprises: a) a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain selected from Table 12 and a light chain variable domain that is at least 90% identical to the light chain variable domain selected from Table 13; or b) a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of a common light antibody selected from Table 11 and a light chain variable domain that is at least 90% identical to the light chain variable domain of the selected common light chain antibody. In some cases, the antibody binding domain comprises: a) the antibody binding domain comprises a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain selected from Table 12 and a light chain variable domain that is at least 95% identical to the light chain variable domain selected from Table 13; or b) the antibody binding domain comprises a heavy chain variable domain that is at least 95% identical to the heavy chain variable domainAtty. Dkt: CRYS-029WO

[0033] of a common light antibody selected from Table 11 and a light chain variable domain that is at least 95% identical to the light chain variable domain of the selected common light chain antibody.

[0034] The present disclosure also provides polypeptides comprising a knob domain comprising an amino acid sequence that is the same as or comprises up to 10 amino acid substitutions relative to the knob domain of an ultralong CDR3 domain selected from Table 19, wherein the knob domain binds to NKp46. In some embodiments, the knob domain comprises an amino acid sequence that is at least 90% identical to the sequence of the knob domain of an ultralong CDR3 domain selected from Table 19. In some cases, the knob domain comprises an amino acid sequence that is at least 95% identical to the sequence of the knob domain of an ultralong CDR3 domain selected from Table 19. In some cases, the polypeptide comprises an amino acid sequence that is at least 90%> identical to the sequence of an ultralong CDR3 selected from Table 19. In some embodiments, the polypeptide comprises an antibody binding domain, and wherein the antibody binding domain comprises a heavy chain variable domain that is at least 90% identical to the sequence of a heavy chain variable domain selected from Table 23, and a light chain variable domain that is at least 90% identical to the sequence of a light chain variable domain selected from Table 22.

[0035] The present disclosure also provides a polypeptide comprising: a) a knob domain comprising an amino acid sequence that is the same as or comprises up to 10 amino acid substitutions relative to the knob domain of an ultralong CDR3 domain selected from Table 24 or Table 37, wherein the knob domain binds to EGFR; b) a VHH domain comprising: i) CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a VHH antibody selected from Table 25 or Table 38, or ii) CDR1, CDR2, and CDR3 regions that are otherwise identical to the CDR1, CDR2, and CDR3 regions of a VHH antibody selected from Table 25 or Table 38 except for up to 10 amino acid substitution in the collective CDR regions, wherein the VHH domain binds to EGFR; c) an antibody binding domain comprising: i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 39 or are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 39 except for up to 10 amino acid substitutions in the collective CDR regions, and ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 40 or are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 40 except for up to 10 amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to EGFR; or d) an antibody binding domain comprising: i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that arc identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 36 or are otherwise identical to the heavy chain CDR1, CDR2Atty. Dkt: CRYS-029WO

[0036] and CDR3 regions of a common light chain antibody selected from Table 36 except for up to 10 amino acid substitutions in the collective CDR regions, and ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody or are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody except for up to 10 amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to EGFR. In some embodiments, a) the knob domain comprises an amino acid sequence that is at least 90% identical to the sequence of the knob domain of an ultralong CDR3 domain selected from Table 24 or Table 37; b) the amino acid sequence of the VHH domain is at least 90% identical to a VHH sequence selected from Table 28 or Table 43; c) an antibody binding domain comprising a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain selected from Table 44 and a light chain variable domain that is at least 90% identical to the light chain variable domain selected from Table 45; or d) an antibody binding domain comprising a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of a common light antibody selected from Table 41 and a light chain variable domain that is at least 90% identical to the light chain variable domain of the selected common light chain antibody. In some cases, a) the knob domain comprises an amino acid sequence that is at least 95% identical to the sequence of the knob domain of an ultralong CDR3 domain selected from Table 24 or Table 37; b) the amino acid sequence of the VHH domain is at least 95% identical to a VHH sequence selected from Table 28 or Table 43; c) an antibody binding domain comprising a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain selected from Table 44 and a light chain variable domain that is at least 95% identical to the light chain variable domain selected from Table 45; or d) an antibody binding domain comprising a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain of a common light antibody selected from Table 41 and a light chain variable domain that is at least 95% identical to the light chain variable domain of the selected common light chain antibody. In some embodiments, wherein the knob domain comprises an amino acid sequence that is at least 90% identical to the sequence of an ultralong CDR3 selected from Table 24 or 37.

[0037] In varying embodiments, the polypeptides described herein comprise a VHH domain that is humanized. In varying embodiments, the polypeptides described herein comprise an antibody binding domain that is humanized. In varying embodiments, the polypeptides described herein are an antibody. In varying embodiments, the polypeptides described herein comprise an antibody binding domain that is a scFv or Fab. In varying embodiments, the polypeptides described herein are fused to an albumin or Fc domain.

[0038] Also provided in the present disclosure are multispecific binding molecule that comprises the knob domain, the VHH domain, or the antibody binding domain of a polypeptide described herein and atAtty. Dkt: CRYS-029WO

[0039] least one other binding domain. In some cases, the at least one other binding domain recognizes a cancer antigen, an immune cell antigen, or a viral antigen. In some embodiments, the antibody binding domain binds to Zip6, and wherein the immune cell antigen is CD2 CD16, NKp30, NKp44, NKp46, NKG2C, NKG2D, DNAM1, SLAMF7, 2B4, KIR2DS, or KIR3DS. In some embodiments, the knob domain binds to NKp46, and wherein the cancer antigen is CD19, CD20, BCMA, ALPP, CS1 (SLAMF7), CLDN18.2, AXL, R0R2, TM4SF1, ICAM-1, L1CAM (CD171), CD4, CD5, CD7, CD10, CD38, CEA, FLT3, CD70, CD30, CD37, or CD147. In some embodiments, the knob domain, VHH domain, or antibody binding domain binds to EGFR, and wherein the cancer antigen is EGFR, HER2, VEGFR2, DLL4, ANG2, c-MET, PD-L1, LGR5, ZIP6, or MET. In some embodiments, the knob domain, VHH domain, or antibody binding domain binds to EGFR, and wherein the immune cell antigen is CD2 CD 16, NKp30, NKp44, NKp46, NKG2C, NKG2D, DNAM1, SLAMF7, 2B4, KIR2DS, KIR3DS, CD3, or CD28. In certain embodiments, a subject polypeptide is a bispecific binding molecule that comprises (i) the knob domain and (ii) a binding domain that recognizes a cancer antigen or a viral antigen, wherein the knob domain binds to NKp46. In some embodiments, the subject polypeptide is a bispecific binding molecule that comprises (i) the knob domain, (ii) a binding domain that recognizes a cancer antigen or a viral antigen and (iii) either a binding domain that recognizes a co-stimulatory receptor on NK cells or a binding domain that recognizes a second cancer antigen or a second viral antigen. In some cases, the costimulatory receptor is 2B4, DNAM1, or CD2. In some cases, the cancer antigen is a blood cancer antigen. In some cases, the cancer antigen is a solid tumor antigen.

[0040] In addition, the present disclosure provides the polypeptides described herein, wherein the polypeptide is conjugated to a pharmacologically active agent. In some embodiments, the polypeptide comprises, or is conjugated to, a radioactive agent. In some embodiments, the polypeptide is in the extracellular domain of an engineered immune cell receptor.

[0041] Also provided in the present disclosure are pharmaceutical compositions comprising: a) the polypeptide of any prior claim or a polynucleotide, e.g., an RNA, encoding the same, and b) a pharmaceutically acceptable carrier. In some embodiments, the polypeptide of the pharmaceutical composition is a multispecific binding molecule that comprises the VHH domain, the antibody binding domain, or the knob domain and at least one other binding domain that binds to a cancer antigen, an immune cell antigen, or a viral antigen.

[0042] The present disclosure also provides methods for modulating Kvl.3 activity, the method comprising contacting a cell comprising Kvl.3 with a polypeptide comprising an antibody binding domain that binds Kvl.3. In some embodiments, the cell is a T cell. In some cases, the T cell is an effector memory T cell. In some cases, the T cell is an auto-reactive T cell. In some embodiments, the cell is in vivo.Atty. Dkt: CRYS-029WO

[0043] The present disclosure also provides methods of treating an inflammatory disease in a subject, the method comprising administering an effective amount of the pharmaceutical composition as described herein to a subject in need thereof to heat the inflammatory disease in the subject, wherein the antibody binding domain of the polypeptide binds to Kvl.3. In some embodiments, the treatment comprises modulating Kvl.3 activity in a T cell of the subject. In some embodiments, the treatment comprises reducing activation of a T cell in the subject. In some cases, the T cell is an effector memory T cell. In some cases, the T cell is an auto-reactive T cell. In some embodiments, the inflammatory disease is selected from: allergy, alopecia areata, Alzheimer's disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), contact-mediated dermatitis, delayed type hypersensitivity, fibrosis, glomerulonephritis, inflammatory bone resorption, inflammatory bowel disease, graft-versus host disease, hepatic fibrosis, multiple sclerosis, obesity, Parkinson’s disease, psoriasis, psoriatic arthritis, restenosis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, systemic sclerosis, type-1 diabetes mellitus, type-2 diabetes mellitus, transplant rejection, ulcerative colitis, and vasculitis.

[0044] The present disclosure additionally provides methods of killing or inhibiting the proliferation of a target cell, the method comprising contacting a cell comprising Zip6 with a polypeptide comprising and antibody binding domain that binds to Zip6. In some embodiments, the target cell is a cancer cell. In some cases, the cancer cell is a cell of a solid tumor.

[0045] The present disclosure also provides methods of treating a cancer in a subject, the method comprising administering an effective amount of the pharmaceutical composition as described herein to a subject in need thereof to treat the cancer in the subject, wherein the antibody binding domain of the polypeptide binds to Zip6. In some embodiments, the method comprises killing or inhibiting the proliferation of a target cancer cell in the subject. In some cases, the cancer cell is a cell of a solid tumor. In some cases, the cancer is selected from breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, melanoma, cervical cancer, and uterine cancer.

[0046] In addition, the present disclosure provides methods of increasing binding between an NK cell and a cancer cell, comprising incubating the NK cell and a cancer cell with a polypeptide comprising a knob domain that binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell.

[0047] The present disclosure also provides methods of killing a cancer cell, comprising contacting the cancer cell with an NK cell and a polypeptide comprising a knob domain that binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell.Atty. Dkt: CRYS-029WO

[0048] Also provided are methods of treatment comprising administering a polypeptide comprising a knob domain that binds to NKp46 or a polynucleotide encoding the same (e.g., an RNA) to a subject that has cancer, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell.

[0049] The present disclosure additionally provides methods of increasing binding between an NK cell and a virally-infected cell, comprising incubating the NK cell and a virally-infected cell with a polypeptide comprising a knob domain that binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a viral antigen on the virally-infected cell.

[0050] The present disclosure also provides methods of killing a virally-infected cell, comprising contacting the virally-infected cell with an NK cell and a polypeptide comprising a knob domain that binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a viral antigen on the virally-infected cell.

[0051] Also provided are methods of treatment comprising administering a polypeptide comprising a knob domain that binds to NKp46 or a polynucleotide encoding the same (e.g., an RNA), to a subject that has a viral infection, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a viral antigen on a virally-infected cell. Also provided are methods of killing or inhibiting the proliferation of a target cell, the method comprising contacting a cell comprising EGFR with a polypeptide comprising a knob domain, VHH domain, or an antibody binding domain that binds to EGFR. In some embodiments, the target cell is a cancer cell. In some cases, the cancer cell is a cell of a solid tumor.

[0052] In addition, the present disclosure provides methods of treating and / or imaging cancer in a subject, the method comprising administering an effective amount of the pharmaceutical composition described herein to a subject in need thereof to treat and / or image the cancer in the subject, wherein the knob domain, the VHH domain, or the antibody binding domain of the polypeptide binds to EGFR. In some embodiments, the method comprises killing or inhibiting the proliferation of a target cancer cell in the subject. In some cases, the method comprises imaging a target cancer cell in the subject. In some cases, the target cancer cell comprises EGFR. In some cases, the cancer cell is a cell of a solid tumor. In some embodiments, the cancer is selected from brain cancer, breast cancer, esophageal cancer, gastrointestinal cancer, anal cancer, pancreatic cancer, cervical cancer, uterine cancer, ovarian cancer, lymphoid cancer, lung cancer, thyroid cancer, head and neck cancer, pharyngeal cancer, skin cancer, prostate cancer, kidney cancer, liver cancer, or hematological cancer.Atty. Dkt: CRYS-029WO

[0053] BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1A-1C: Depict exemplary data of subject anti-EGFR knob domain binding kinetics using varying surface plasmon resonance (SPR) assay formats.

[0054] FIG. 2: Depicts target epitope network clustering of select subject polypeptides and benchmark anti-EGFR antibodies.

[0055] FIG. 3: Heavy and light chain sequences for antibody 11271pl. A3.

[0056] FIG. 4: cAMP functional assay data This figure shows representative activity data from an Alphascreen cAMP assay. The natural agonist of the GIPR GIP (top row, left) was used a positive control. As expected, it increases the cAMP signal in a concentration dependent manner. Antagonistic antibodies are able to reduce the cAMP production stimulated by GIP (11271pl. Fl, 11271pl. A3), whereas non-antagonists (1127 lp2.2. A8, 1127 lp3. D9) have no impact on the amount of cAMP.

[0057] FIGs. 5A-5B: (A) depicts specific surface binding of an exemplary subject anti-Zip6 antibody (CL-30676: ‘79830-M9-A05’) to MCF-7 cells expressing Zip6 on their surface compared to a known anti-Zip6 antibody (‘Ladiratizumab’). (B) shows quantification of surface binding of subject anti-Zip6 antibodies to MCF-7 cells expressing Zip6 on their surface. Surface binding is quantified relative to Ladiratizumab.

[0058] FIGs.6A-6D: each depict specific surface binding of subject anti-Zip6 antibodies to MCF-7 cells expressing Zip6 on their surface (top) and the EC50 and Emax of binding of the subject anti-Zip6 antibodies to MCF-7 cells expressing Zip6 on their surface as measured relative to Ladiratizumab (bottom).

[0059] FIG. 7: shows internalization of subject anti-Zip6 antibodies into MCF-7 cells at two timepoints (15 min, overnight) following application of the antibodies to the MCF-7 cells compared to Ladiratizumab.

[0060] FIGs.8A-8B: (A) depicts quantification of the internalization of subject anti-Zip6 antibodies into MCF-7 cells over time (top). The EC50 values of the tested clones are also shown (bottom). (B) depicts quantified internalization of subject anti-Zip6 antibodies in MCF-7 cells after 15 min (top, left) or overnight (top, right) and quantified surface binding of anti-Zip6 antibodies to MCF-7 cells after 15 min (bottom, left) or overnight (bottom, right)

[0061] FIG. 9: depicts lineage data of subject anti-Zip6 antibodies.

[0062] DEFINITIONS

[0063] The terms "antibodies" and “immunoglobulin” include antibodies or immunoglobulins of any isotypc, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, andAtty. Dkt: CRYS-029WO

[0064] fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. The antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the term are Fab’, Fv, F(ab’)2, and or other antibody fragments that retain specific binding to antigen, and monoclonal antibodies. An antibody may be monovalent or bivalent.

[0065] " 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 (Zapata et al.. Protein Eng. 8(10): 1057-1062 (1995)); singlechain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called " Fab" fragments, each with a single antigen-binding site, and a residual " Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[0066] " Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRS of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the enure binding site.

[0067] The “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CHi) of the heavy chain. Fab fragments differ from Fab’ fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHi domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0068] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins:Atty. Dkt: CRYS-029WO

[0069] IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.

[0070] " Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0071] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0072] As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U. S. Dept, of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et al.,.1. Mol. Biol. 196:901-917 (1987); and MacGallum et al., J. Mol. Biol. 262:732-745 (1996), and Lefranc, M.-P. et al., Dev. Comp. Immunol., 200327: 55-77, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein.

[0073] As used herein, the term “collectively” in the context of a variant of an immunoglobulin variable domain that is otherwise identical to the immunoglobulin variable domain except for a defined number of amino acid substitutions “in the collective CDR regions”, indicates that the number of amino acid substitutions is counted using all six CDRs. Explained by example, if the variant has 5 amino acid substitutions relative to the antibody variable domain, then the six CDRs of the variant, combined, have a total of 5 amino acid substitutions relative to the antibody variable domain. This phrase is not intended to mean that each CDR has a defined number of amino acid substitutions.

[0074] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd). Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater,Atty. Dkt: CRYS-029WO

[0075] at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences. Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0076] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. By way of example, an anti-Kvl.3 polypeptide binds specifically to an epitope within a Kvl.3 polypeptide. As another example, an anti-Zip6 polypeptide binds specifically to an epitope within a Zip6 polypeptide. As another example, an anti-NKp46 polypeptide binds specifically to an epitope within a NKp46 polypeptide. As yet another example, an anti-EGFR polypeptide binds specifically to an epitope within a EGFR polypeptide. Non-specific binding would refer to binding with an affinity of less than about 107M, e.g., binding with an affinity of 106M, 105M, IO'4M, etc.

[0077] The terms “domain” and “motif’, used interchangeably herein, refer to both structured domains having one or more particular functions and unstructured segments of a polypeptide that, although unstructured, retain one or more particular functions. For example, a structured domain may encompass but is not limited to a continuous or discontinuous plurality of amino acids, or portions thereof, in a folded polypeptide that comprise a three-dimensional structure which contributes to a particular function of the polypeptide. In other instances, a domain may include an unstructured segment of a polypeptide comprising a plurality of two or more amino acids, or portions thereof, that maintains a particular function of the polypeptide unfolded or disordered. Also encompassed within this definition are domains that may be disordered or unstructured but become structured or ordered upon association with a target or binding partner. Non-limiting examples of intrinsically unstructured domains and domains of intrinsically unstructured proteins are described, e.g., in Dyson & Wright. Nature Reviews Molecular Cell Biology 6:197-208.

[0078] The terms “synthetic”, “chimeric” and “engineered” as used herein generally refer to artificially derived polypeptides or polypeptide encoding nucleic acids that are not naturally occurring. Synthetic polypeptides and / or nucleic acids may be assembled de novo from basic subunits including, e.g., single amino acids, single nucleotides, etc., or may be derived from pre-existing polypeptides orAtty. Dkt: CRYS-029WO

[0079] polynucleotides, whether naturally or artificially derived, e.g., as through recombinant methods. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids will generally be constructed by the combination, joining or fusing of two or more different polypeptides or polypeptide encoding nucleic acids or polypeptide domains or polypeptide domain encoding nucleic acids. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids include where two or more polypeptide or nucleic acid “parts” that are joined are derived from different proteins (or nucleic acids that encode different proteins) as well as where the joined parts include different regions of the same protein (or nucleic acid encoding a protein) but the parts are joined in a way that does not occur naturally.

[0080] The terms “chimeric antigen receptor” and “CAR”, used interchangeably herein, refer to artificial multi-module molecules capable of triggering or inhibiting the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand / aniigen binding domain), a transmembrane domain and one or more intracellular signaling domains. The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled (e.g., as described in PCT publication no. WO 2014 / 127261 Al and US Patent Application No.

[0081] 2015 / 0368342 Al, the disclosures of which are incorporated herein by reference in their entirety). CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013);5(215):215ra 172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2): 151-5; Riddell et al. Cancer J (2014) 20(2):141-4;

[0082] Pegram et al. Cancer J (2014) 20(2): 127-33; Cheadle et al. Immunol Rev (2014) 257(1):91 - 106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al.,.1 Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Useful CARs also include the anti-CD19 — 4-1BB — CD3 CAR expressed by lentivirus loaded CTL019 (Tisagenlecleucel-T) CAR-T cells as commercialized by Novartis (Basel, Switzerland).

[0083] The terms “T cell receptor” and “TCR” are used interchangeably and will generally refer to a molecule found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR complexAtty. Dkt: CRYS-029WO

[0084] is a disulfide -linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (|3) chains expressed as part of a complex with CD3 chain molecules. Many native TCRs exist in heterodimeric aP or y8 forms. The complete endogenous TCR complex in heterodimeric a form includes eight chains, namely an alpha chain (referred to herein as TCRa or TCR alpha), beta chain (referred to herein as TCRp or TCR beta), delta chain, gamma chain, two epsilon chains and two zeta chains. In some instance, a TCR is generally referred to by reference to only the TCRa and TCRP chains, however, as the assembled TCR complex may associate with endogenous delta, gamma, epsilon and / or zeta chains an ordinary skilled artisan will readily understand that reference to a TCR as present in a cell membrane may include reference to the fully or partially assembled TCR complex as appropriate.

[0085] Recombinant or engineered individual TCR chains and TCR complexes have been developed. References to the use of a TCR in a therapeutic context may refer to individual recombinant TCR chains. As such, engineered TCRs may include individual modified TCRa or modified TCRP chains as well as single chain TCRs that include modified and / or unmodified TCRa and TCRP chains that are joined into a single polypeptide by way of a linking polypeptide.

[0086] A “therapeutically effective amount” or “efficacious amount” refers to the amount of a polypeptide (e.g., an anti-Kvl.3, anti-Zip6, anti-NKp46, or anti-EGFR polypeptide ) that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the polypeptide (e.g., an anti-Kvl.3, anti-Zip6, anti-NKp46, or anti-EGFR polypeptide ), the disease and its severity and the age, weight, etc., of the subject to be treated.

[0087] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. " Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0088] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.Atty. Dkt: CRYS-029WO

[0089] DETAILED DESCRIPTION

[0090] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0091] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0092] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0093] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a binding domain” includes a plurality of binding domains. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0094] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0095] In the following description, the CDRs are defined by the IMGT system of Lefranc (Dev. Comp. Immun. 2003 27: 55-77).

[0096] Knob DomainsAtty. Dkt: CRYS-029WO

[0097] Cattle (i.e., cows or bovines) produce antibodies that have a cysteine -rich ultralong CDR H3 that can be in the range of 30 to 70 amino acids in length and folds into a unique “stalk and knob” domain, with the knob protruding far out of the antibody surface (see, e.g., Huang et al Proc. Natl. Acad. Sci. 2023 120: e2303455120). These knobs retain their ability to bind to the antigen when they are expressed on their own, i.e., independently from the rest of the antibody. In these embodiments, the knob can be expressed as a fusion protein which is then cleaved to release the knob (see, e.g., Huang et al Proc. Natl. Acad. Sci. 2023 120: e2303455120). Alternatively, the knob can be cleaved from an antibody or Fab scaffold (Macpherson et al. PLoS Biol 2020 18: e3000821) or synthesized chemically (Macpherson et al ACS Chem. Biol. 2021 16, 9, 1757-1769). These knobs (which are referred to as “knob domains” herein and may be referred to as PICOBODIES™ elsewhere) are the smallest known fragment of an antibody that can bind to an antigen independently. A knob domain may be expressed on its own or as a fusion with another protein (referred to as a “fusion partner” herein), wherein the knob domain may be at the N-terminus, C-terminus or within the fusion partner. If the fusion partner is an antibody, the knob domain may be fused to the N- or C- terminus of the heavy or light chain (in which case the antibody could potentially be bispecific since the variable domain may be intact), or it could positioned in the variable domain of the antibody (e.g., in the heavy chain CDR3). In embodiments in which the knob domain is fused to another protein, the knob domain may in some cases additionally comprise a stalk and / or a flexible linker that connects the knob domain to the protein. For example, if the knob domain is in the heavy chain CDR3, then it may contain a stalk to allow it to protrude from the rest of the antibody. If the knob domain is expressed in conjunction with a stalk, then an almost unlimited number of sequences are available for the ascending and descending regions of the stalk. Specifically, in the context of bovine antibodies the stalk has a purely structural function (i.e., it does not participate in binding). In bovine antibodies, the stalk is a beta sheet (composed of two anti -parallel beta strands that are hydrogen bonded). Stalk sequences should be interchangeable between antibodies because, functionally, they only serve to distance the knob from the rest of the antibody while keeping the ends of the knob in proximity with one another. Since the sequences of thousands of bovine antibodies are publicly available or could be readily obtained, a large number of options would be available. Moreover, methods for designing beta sheets (composed of two anti-parallel beta strands) de novo are well known (see, e.g., Hecht et al (Proc Natl Acad Sci U S A. 199491: 8729-8730), Marcos (Nat Struct Mol Biol. 201825: 1028-1034) and Pan et al (J. Biol. Chem. 2021 296, 100558), among many others)) and, as such, stalks can be readily designed.

[0098] As such, a knob domain is typically in the range of 15 to 50 amino acids in length, cysteine -rich (i.e., may contain 4-10 cysteines, e.g., 4, 6, 8 or 10 cysteines) and may be based on an ultralong-CDR3 antibody (which, e.g., may be made by a cow or another species that naturally makes such antibodies, or by another species that has been engineered to produce such antibodies (e.g., a chicken)). Knob domainsAtty. Dkt: CRYS-029WO

[0099] have the potential to bind antigens with concave epitopes and, as such, may have particular value for certain targets. Further details of knob domains may be found in, e.g., Svilenov et al (Nature Com. 2021 12: 6737), Huang et al (Proc. Natl. Acad. Sci 2023 120 (39) e2303455120) and Passon et al (Biotechnol Adv. 2023:64: 108120).

[0100] Anti-NKp46 Knob Domains

[0101] Table 19 of the present disclosure provides the sequences of a number of ultralong CDR H3s from cow antibodies that bind to NKp46. These sequences have stalk sequences at the ends and an internal knob domain. The knob domains of the ultralong CDR H3s are underlined in the sequences of Table 19. In some embodiments, a subject polypeptide comprises a knob domain underlined in this table, where the knob domain may comprise an amino acid sequence that is the same as or comprises up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions relative to the knob domain of an ultralong CDR3 domain selected from 'fable 19, wherein the knob domain and / or the polypeptide comprising the knob domain binds to NKp46. In some cases, the knob domain of the ultralong CDR H3s in the sequences of Table 19 may be considered to include 1, 2, 3, 4, or 5 contiguous additional amino acids N-terminal and / or C-terminal to the underlined amino acids in the sequences of Table 19 and / or exclude the first and / or last 1, 2, 3, 4, or 5 contiguous underlined amino acids in the sequences of Table 19. In some embodiments, the knob domain may be associated with stalk sequences (e.g., the sequences that are already associated with that domain in fable 19 or another pair of sequences that form an anti -parallel beta strand). In some embodiments, a knob domain may comprise an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a knob domain or ultralong CDR3 selected from Table 19.

[0102] In some embodiments, the knob domain may be grafted into the heavy chain CDR3 region of an antibody along with stalk sequences, i.e., to replace the heavy chain CDR3 region of an antibody. In these embodiments, the antibody may be a human antibody, a humanized antibody, or a variant of the same that has been modified to accommodate an ultralong heavy chain CDR. As such, in some embodiments, some embodiments provide an antibody that has a heavy chain CDR3 that comprises a knob domain, wherein the knob domain has been grafted onto the antibody. In some embodiments, a subject polypeptide comprises an antibody binding domain, the antibody binding domain comprising a heavy chain variable domain that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a heavy chain variable domain selected from Table 21, and a light chain variable domain that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a light chain variable domain selected from Table 22.Atty. Dkt: CRYS-029WO

[0103] In addition, Table 20 provides the CDR sequences of a number of antigen-binding polypeptides that bind to NKp46, and comprise an ultralong CDR H3 selected from Table 19.

[0104] In some embodiments, a knob domain and / or a polypeptide comprising a subject knob domain may bind to NKp46 (e.g., human NKp46) with an affinity in the range of 106M1to 1012M1(e.g., an affinity in the range of 106M1to 1011M1, in the range of 107M1to 1010M1, in the range of 108M1to 109M'1).

[0105] Anti-EGFR Knob Domains

[0106] Tables 24, 37, and 62 of the present disclosure provides the sequences of a number of ultralong CDR H3s from antibodies that bind to EGFR. These sequences have stalk sequences at the ends and an internal knob domain. The knob domains of the ultralong CDR H3s are underlined in the sequences of Table 24, 37, and 62. In some embodiments, a subject polypeptide comprises a knob domain underlined in these tables, where the knob domain may comprise an amino acid sequence that is the same as or comprises up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions relative to the knob domain of an ultralong CDR3 domain selected from Table 24, 37, and 62, wherein the knob domain and / or the polypeptide comprising the knob domain binds to EGFR. In some cases, the knob domain of the ultralong CDR H3s in the sequences of Tables 24, 37, and 62 may be considered to include 1, 2, 3, 4, or 5 contiguous additional amino acids N-terminal and / or C -terminal to the underlined amino acids in the sequences of '1’ables 24, 37, and 62 and / or exclude the first and / or last 1, 2, 3, 4, or 5 contiguous underlined amino acids in the sequences of Tables 24, 37, and 62. In some embodiments, the knob domain may be associated with stalk sequences (e.g., the sequences that are already associated with that domain in Tables 24, 37, and 62 or another pair of sequences that form an anti -parallel beta strand). In some embodiments, a knob domain may comprise an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a knob domain of an ultralong CDR3 selected from Table 24, 37, or 62. In some embodiments, the subject polypeptide comprises a knob domain, where the knob domain may comprise an amino acid sequence that is the same as or comprises up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions relative to a knob domain selected from Table 61, wherein the knob domain binds to EGFR. In some cases, the knob domain of the subject polypeptide may comprise an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a knob domain selected from Table 61.

[0107] In some embodiments, the knob domain may be grafted into the heavy chain CDR3 region of an antibody along with stalk sequences, i.c., to replace the heavy chain CDR3 region of an antibody. In these embodiments, the antibody may be a human antibody, a humanized antibody, or a variant of the same thatAtty. Dkt: CRYS-029WO

[0108] has been modified to accommodate an ultralong heavy chain CDR. As such, some embodiments provide an antibody that has a heavy chain CDR3 that comprises a knob domain, wherein the knob domain has been grafted onto the antibody. In some of these embodiments, the subject polypeptide may comprise an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of an ultralong CDR3 selected from Table 24, 37, or 62.

[0109] Tables 26, 42, and 63 provide the sequences of a number of heavy chain variable domains from antibodies comprising an ultralong CDR H3. Tables 27 and 64 provide the sequences of a number of light chain variable domains from antibodies comprising an ultralong CDR H3. In some embodiments, a subject polypeptide comprises an antibody binding domain, the antibody binding domain comprising a heavy chain variable domain that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a heavy chain variable domain selected from Table 26, 42, or 63. In some embodiments, a subject polypeptide comprises an antibody binding domain, the antibody binding domain comprising a heavy chain variable domain that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a heavy chain variable domain selected from Table 26, and a light chain variable domain that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a light chain variable domain selected from Table 27. In some embodiments, a subject polypeptide comprises an antibody binding domain, the antibody binding domain comprising a heavy chain variable domain that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a heavy chain variable domain selected from Table 63, and a light chain variable domain that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the sequence of a light chain variable domain selected from Table 64.

[0110] In some embodiments, a knob domain and / or a polypeptide comprising a subject knob domain may bind to EGFR (e.g., human EGFR) with an affinity in the range of 106M1to 1012M1(e.g., an affinity in the range of 106M1to 1011M1, in the range of 107M1to 1010M1, in the range of 108M1to 109M'1).

[0111] VHH Domain

[0112] VHH antibodies are known in the art and have a heavy chain variable domain that can fold and bind to epitopes autonomously, i.e., without an associated light chain and without significantlyAtty. Dkt: CRYS-029WO

[0113] aggregating. These antibodies may be referred to as “heavy chain-only”, “HCO” or single domain antibodies (sdAbs), shark antibodies, camelid antibodies and nanobodies in other publications. VHH antibodies occur naturally in shark, camel and llama. However, there are several strategies for producing such antibodies from VH antibodies. See, e.g., Janssens et al (Proc. Natl. Acad. Sci. 2006 103:15130-5), Briiggemann et al (Crit. Rev. Immunol. 200626:377-90), Zou et al (J. Immunol. 2005 175:3769-79) and Nguyen et al (Immunology 2003 109: 93-101), for example. A VHH variable domain can be made by introducing substitutions into the variable domain of a VH antibody. Such stabilized or ‘camelized’ VH antibodies are referred to as VHH antibodies herein and, as may be apparent, the term “VHH” could be substituted with the term “heavy chain-only”, “HCO”, “autonomous heavy chain” or “single domain” since these terms are intended to refer to the same thing. In the present disclosure, VHH antibodies were made by chickens that have been genetically engineered to produce VHH antibodies. Advantageous features of VHH antibodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. VHH antibodies can readily be linked genetically to, e.g., Fc-domains, other nanobodies or single chain antibodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles, etc. The binding domain of a VHH antibody does not require a light chain for correct folding or binding to an antigen. See, e.g., Bever et al (Anal Bioanal Chem. 2016 Sep; 408: 5985-6002). As with conventional VH antibodies, VHH antibodies have three CDRs (CDR1, CDR2 and CDR3) that are flanked by framework. The structure of the binding domain (which may be referred to as a ‘variable domain’) of a VHH antibody is as follows: FW1-CDR1- FW2-CDR2-FW3-CDR3-FW4 (see, e.g., Noel et al, Biochimie 2016 131:11-19 for a detailed description of VHH structure).

[0114] Anti-EGFR VHH domains

[0115] Using Tables 25, 28, 38, and 43 as a reference, the present disclosure provides a polypeptide that comprises a VHH domain comprising: (a) CDR1, CDR2 and CDR3 regions that are identical to the CDR1, CDR2 and CDR3 regions of a VHH antibody selected from Tables 25, 28, 38, or 43; or (b) CDR1, CDR2 and CDR3 regions that are otherwise identical to the CDR1, CDR2 and CDR3 regions of a VHH antibody selected from Tables 25, 28, 38 or 43 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions, wherein the VHH domain binds to EGFR.

[0116] In some embodiments, the amino acid sequence of the VHH domain may be at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the variable domain of the selected VHH antibody. These sequences are shown in Tables 28 and 43.

[0117] In any embodiment, the polypeptide that comprises a VHH domain may comprise at least the FR2 (i.e., the sequence between CDR1 and CDR2) of the selected VHH antibody, or a FR2 that is otherwiseAtty. Dkt: CRYS-029WO

[0118] identical to the FR2 of the selected VHH antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the FR2.

[0119] In some embodiments, the VHH may be humanized, i.e., modified so that it becomes more like a human antibody and therefore less immunogenic, methods for which are known.

[0120] In these embodiments, the VHH domain of a subject polypeptide may bind to EGFR (e.g., human EGFR) with an affinity in the range of 106M1to 1012M1(e.g., an affinity in the range of 106M1to 1011M1, in the range of 107M1to 1010M1, in the range of IO8M1to IO9M1).

[0121] In certain embodiments, a subject VHH domain may comprise one or more amino acid substitutions that improve the biophysical properties, e.g., improve the solubility, of the VHH domain. In some embodiments, the amino acid substitutions may comprise L46P, I100V, L3Q, W33Y, D59E, or any combination thereof, as shown in Table 57. In some cases, a subject VHH domain may comprise a L46P amino acid substitution, as shown in Table 57. In some cases, a subject VHH domain may comprise an I100V amino acid substitution, as shown in Table 57. In some cases, a subject VHH domain may comprise a L3Q amino acid substitution, as shown in Table 57. In some cases, a subject VHH domain may comprise a L46P and I100V amino acid substitution, as shown in Table 57. In some cases, a subject VHH domain may comprise a L46P, I100V, and L3Q amino acid substitution, as shown in Table 57. In some cases, a subject VHH domain may comprise a W33Y amino acid substitution, as shown in Table 57. In some cases, a subject VHH domain may comprise a W33Y, L46P, I100V, and L3Q amino acid substitution, as shown in Table 57. In some cases, a subject VHH domain may comprise a D59E, L46P, I100V, and L3Q amino acid substitution, as shown in Table 57. In some embodiments, a subject VHH domain may be at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to a variant VHH sequence selected from Table 57.

[0122] In some embodiments, a subject polypeptide may comprise one or more VHH domains, as described herein, fused to an Fc domain. For example, in some cases, a subject polypeptide may comprise two VHH domains (e.g., two identical VHH domains), as described herein, fused to an Fc domain. In some embodiments, a subject polypeptide may be at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to a VHH-Fc polypeptide selected from Table 58. In some embodiments, a subject polypeptide may be at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to a VHH- VHH-Fc polypeptide selected from Table 60.Atty. Dkt: CRYS-029WO

[0123] Common Light Chain Antibodies

[0124] Common light chain antibodies are antibodies that are composed of a heavy and light chain that are made in an animal that has a ‘fixed’ fight chain, i.e., a light chain that has a diminished capacity to diversify. The antibodies produced by such an animal have a diversified heavy chain and the same light chain (i.e., a ‘common’ light chain). In these animals the heavy chain sequence is diversified and capable of high-affinity antigen-specific binding and broad epitope diversity when paired with a fight chain (which, in many cases, will be a human light chain). The light chain provides the proper structure for assembly of the full antibody molecule but is a passive partner for antigen binding. Transgenic animals that produce common light chain antibodies include rats (see, e.g., Harris et al (Front Immunol. 2018 24:9:889)) and chickens (Ching et al ( Abs. 2021; 13(1): 1862451)), among others.

[0125] Common light chain antibodies find particular use in producing multispecific antibodies, since it is advantageous if the light chain is common to all branches of the multispecific antibody, with the binding specificity determined solely by the heavy chain. For example, expression of a bispecific antibody is simplified because it only requires the two heavy chains and the one, common light chain.

[0126] Common light chain antibodies have a conventional “VH” structure and have both a heavy chain sequence and a light chain sequence. Both the heavy and light chains of a common light chain antibody have the following structure: FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4.

[0127] Anti-Zip6 Common Light Chain Antibody Binding Domains

[0128] Using Tables 8 and 11 as a reference, the present disclosure provides a polypeptide comprising an antibody binding domain that comprises a heavy chain variable domain and a light chain variable domain (which may be both present in a single chain or present in two chains (e.g., as in an scFv or Fab format)), where the antibody binding domain comprises (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 8 or are otherwise identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 8 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of the selected common light chain antibody or are otherwise identical to the light chain CDR1, CDR2, and CDR3 regions of the selected common light chain antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to Zip6.

[0129] In some embodiments, the antibody binding domain may comprise: (a) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the heavy chain CDR1, CDR2Atty. Dkt: CRYS-029WO

[0130] and CDR3 regions of a common light chain antibody selected from Table 8; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody; or (b) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 8 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; wherein the antibody binding domain binds to Zip6.

[0131] In some embodiments, the antibody binding domain may have one chain that has CDRs that are identical to the CDRs of an antibody from Table 8, where the CDRs of the other chain are otherwise identical to the other chain of the selected antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDRs. In these embodiments, the binding domain may comprise: (a) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 8; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions or (b) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 8 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody; wherein the antibody binding domain binds to Zip6.

[0132] In some embodiments, the antibody binding domain comprises a heavy chain variable domain that is 90% or more (e.g., 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the heavy chain variable domain of the selected antibody and a light chain variable domain that is 90% or more (e.g., 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the light chain variable domain of the selected antibody. The heavy chain variable domain sequences of said selected antibodies are shown in Table 11.

[0133] In some embodiments, the antibody binding domain may be humanized, i.c., modified so that it becomes more like a human antibody and, in theory, less immunogenic, methods for which are known.Atty. Dkt: CRYS-029WO

[0134] In these embodiments, the antibody binding domain of a subject polypeptide may bind to Zip6 with an affinity in the range of 106M1to 1012M1(e.g., an affinity in the range of 106M1to 1011M1, in the range of 107M1to 1010M1, in the range of 108M1to 109M-1).

[0135] In any embodiment, the subject anti-Zip6 polypeptide is capable of being internalized by a cell comprising Zip6 (e.g., expressing Zip6 on its surface) following contacting of the cell with the subject anti-Zip6 polypeptide.

[0136] Anti-EGFR Common Light Chain Antibody Binding Domains

[0137] Using Tables 36 and 41 as a reference, the present disclosure provides a polypeptide comprising an antibody binding domain that comprises a heavy chain variable domain and a light chain variable domain (which may be in a single chain or two chains (e.g., in the scFv or Fab formats)), where the binding domain comprises: (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 36 or are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 36 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody or are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to EGFR.

[0138] In some embodiments, the antibody binding domain may comprise: (a) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 36; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody; or (b) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 36 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; where the antibody binding domain binds to EGFR.Atty. Dkt: CRYS-029WO

[0139] In some embodiments, the antibody binding domain may have one chain that has CDRs that are identical to the CDRs of an antibody from table 36, where the CDRs of other chain are otherwise identical to the other chain of the selected antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDRs. In these embodiments, the binding domain may comprise: (a) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 36; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions or (b) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 36 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the light chain CDR 1, CDR2 and CDR3 regions of the selected common light chain antibody; where the antibody binding domain binds to EGFR.

[0140] In some embodiments, the antibody binding domain comprises a heavy chain variable domain that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the heavy chain variable domain of the selected antibody and a light chain variable domain that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the light chain variable domain of the selected antibody. These sequences are shown in Table 41.

[0141] In some embodiments, the antibody binding domain may be humanized, i.e., modified so that it becomes more like a human antibody and, in theory, less immunogenic, methods for which are known.

[0142] In these embodiments, the antibody binding domain of a subject polypeptide may bind to EGFR (e.g., human EGFR) with an affinity in the range of 106M'1to 1012M1(e.g., an affinity in the range of 106M1to 1011M1, in the range of 107M-1to 1010M1, in the range of 108M1to 109M1).

[0143] Antibodies with Diversified Light Chains

[0144] Antibodies with diversified light chains comprise antibody binding domains composed of diversified heavy chain and diversified light chain variable domains. These antibodies may include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The polypeptides comprising antibody binding domains describedAtty. Dkt: CRYS-029WO

[0145] below may be monomeric (e.g., having the heavy chain variable domain and the light chain variable in a single polypeptide chain) or multimeric (e.g., having the heavy chain variable domain and the light chain variable in separate polypeptide chains).

[0146] Anti-Kyl.3 Antibody Binding Domains

[0147] Using Tables 1 and 2 as a reference, the antibody binding domain of a subject polypeptide comprises (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a heavy chain variable domain selected from Table 1, or are otherwise identical to the CDR1, CDR2, and CDR3 regions of a heavy chain variable domain selected from Table 1 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 2 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 2 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds Kvl.3.

[0148] In some embodiments, the amino acid sequence of the heavy chain variable domain may be at least 90% (e.g., 90% or more, 91% or more, 92% or more. 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the heavy chain variable domain of the selected antibody. The heavy chain variable domains of said selected antibodies are shown in Table 3. In some embodiments, the amino acid sequence of the light chain variable domain may be at least 90% (e.g., 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the light chain variable domain of the selected antibody. The light chain variable domains of said selected antibodies are shown in Table 4.

[0149] For example, in some cases, a polypeptide of the present disclosure comprises an antibody binding domain comprising: (i) a heavy chain variable domain that is at least 90% (e.g., 90% or more, 91 % or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the heavy chain variable domain of the selected antibody (i.e., any of the heavy chain variable domains shown in Table 3); and (ii) a light chain variable domain that is at least 90% (e.g., 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the light chain variable domain of the selected antibody (i.e., any of the light chain variable domains shown in Table 4).

[0150] A polypeptide of the present disclosure may be multimeric, i.e., comprised of two or more polypeptide chains. For example, in some embodiments, the heavy chain variable domain and light chain variable domain of a subject antibody binding domain may be present in separate polypeptides as further described herein (e.g., as in a Fab). A polypeptide of the present disclosure may be monomeric, i.e.,Atty. Dkt: CRYS-029WO

[0151] comprised of a single polypeptide chain. For example, in some embodiments, the heavy chain variable domain and light chain variable domain of a subject antibody binding domain may be present in a single polypeptide as further described herein (e.g., as in an scFv).

[0152] In some embodiments, the antibody binding domain may be humanized, i.e., modified so that it becomes more like a human antibody and, in theory, less immunogenic, methods for which are known.

[0153] The antibody binding domain of a subject polypeptide may bind to a mammalian Kvl.3. In some embodiments, the antibody-binding domain of a subject polypeptide binds to human Kvl.3. In certain embodiments, the antibody-binding domain of a subject polypeptide may bind to human Kvl.3 with an affinity of at least 10-7M, e.g., 10'7M or more, 10'8M or more, IO-9M or more, IO40M or more, 101M or more, or 1042M or more. In some cases, the antibody binding domain of a subject polypeptide may bind to an epitope present on human Kvl.3 with an affinity of at least 10’7M, e.g., 10'7M or more, 10’8M or more, 10‘9M or more, IO-10M or more, 101M or more, or 1042M or more.

[0154] A polypeptide of the present disclosure comprising an antibody binding domain that binds Kvl.3 may modulate the activity of Kvl.3. For example, the antibody binding domain of the subject polypeptide may modulate the activity of Kvl.3. By “modulate the activity of Kvl.3” it is meant modulation of one or more of the activities of Kvl.3 required for voltage-gated permeability of K+. For example, in some cases, the antibody binding domain of a subject polypeptide may occlude or otherwise modulate the permeability of the ion pore of Kvl.3. In some cases, the antibody binding domain of a subject polypeptide may bind to the voltage-sensing domain and / or the pore domain of Kvl.3 and modulate the ability of Kvl.3 to enter into or maintain the active, ion-permeable, conformation (i.e., interfere with the voltage mediated gating of Kvl.3).

[0155] In some embodiments, a subject polypeptide may inhibit the activity (e.g., inhibit the K+permeability) of Kv.1.3. In certain embodiments, a subject polypeptide may inhibit the activity (e.g., K+ion-permeability) of Kvl.3 by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, compared to the degree of Kvl.3 activity (e.g., K+ion-permeability) in the absence of the subject polypeptide. In some cases, the antibody binding domain of a subject polypeptide may inhibit the activity (e.g., K+ion-permeability) of human Kvl.3 by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, compared to the degree of Kvl.3 activity (e.g., K+ion-permeability) in the absence of the subject polypeptide.

[0156] Anti-Zip6 Antibody Binding Domains

[0157] Using Tables 9-10 and Tables 12-13 as a reference, the present disclosure provides a polypeptide comprising an antibody binding domain that comprises a heavy chain variable domain and a light chainAtty. Dkt: CRYS-029WO

[0158] variable domain (which may be both present in a single chain or present in two chains (e.g., as in an scFv or Fab format)), where the antibody binding domain comprises: (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 9 or are otherwise identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 9 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 10 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 10 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to Zip6.

[0159] In some embodiments, the antibody binding domain may comprise: (a)(i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 9; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 10; or (b) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected form Table 9 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 10 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; wherein the antibody binding domain binds to Zip6.

[0160] In some cases, the antibody binding domain may comprise: (a)(i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 9; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of the selected antibody or (b) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected form Table 9 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; wherein the antibody binding domain binds to Zip6.Atty. Dkt: CRYS-029WO

[0161] In some embodiments, the antibody binding domain comprises (i) a heavy chain variable domain that is 90% or more (e.g., 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the heavy chain variable domain of the selected antibody; and (ii) a light chain variable domain that is 90% or more (e.g., 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the light chain variable domain of the selected antibody. The heavy chain variable domain sequences of said selected antibodies are shown in Table 12. The light chain variable domain sequences of said selected antibodies are shown in Table 13.

[0162] In some embodiments, the antibody binding domain may be humanized, i.e., modified so that it becomes more like a human antibody and, in theory, less immunogenic, methods for which are known.

[0163] In these embodiments, the antibody binding domain of a subject polypeptide may bind to Zip6 with an affinity in the range of 106M1to 1012M1(e.g., an affinity in the range of 106M1to 1011M1, in the range of 107M1to 1010M1, in the range of 108M1to 109M1).

[0164] In any embodiment, the subject anti-Zip6 polypeptide is capable of being internalized by a cell comprising Zip6 (e.g., expressing Zip6 on its surface) following contacting of the cell with the subject anti-Zip6 polypeptide.

[0165] Anti-EGFR Antibody Binding Domains

[0166] Using Tables 39 and 40 as a reference, the present disclosure provides a polypeptide comprising an antibody binding domain that comprises a heavy chain variable domain and a light chain variable domain (which may be both present in a single chain or present in two chains (e.g., as in an scFv or Fab format)), where the antibody binding domain comprises: (i) a heavy chain variable domain comprising CDR1, CDR2. and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 39 or are otherwise identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 39 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 40 or are otherwise identical to the light chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 40 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; wherein the antibody binding domain binds to EGFR.

[0167] In some embodiments, the antibody binding domain may comprise: (a)(i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 39; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3Atty. Dkt: CRYS-029WO

[0168] regions of an antibody selected from Table 40; or (b) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected form Table 39 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 40 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; wherein the antibody binding domain binds to EGFR.

[0169] In some cases, the antibody binding domain may comprise: (a)(i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 39; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of the selected antibody or (b) (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 39 except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions; wherein the antibody binding domain binds to EGFR.

[0170] In some embodiments, the antibody binding domain comprises (i) a heavy chain variable domain that is 90% or more (e.g., 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the heavy chain variable domain of the selected antibody; and (ii) a light chain variable domain that is 90% or more (e.g., 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the light chain variable domain of the selected antibody. The heavy chain variable domain sequences of said selected antibodies are shown in Table 44. The light chain variable domain sequences of said selected antibodies are shown in Table 45.

[0171] In some embodiments, the antibody binding domain may be humanized, i.e., modified so that it becomes more like a human antibody and, in theory, less immunogenic, methods for which are known.

[0172] In these embodiments, the antibody binding domain of a subject polypeptide may bind to EGFR (e.g., human EGFR) with an affinity in the range of 106M1to 1012M1(e.g., an affinity in the range of 106M1to 1011M1, in the range of 107M-1to 1010M1, in the range of 108M1to 109M1).

[0173] Anti-GIPR antibodiesAtty. Dkt: CRYS-029WO

[0174] In some embodiments, the antibody may comprise: (a) a variable domain comprising: i. heavy chain CDR1, CDR2 and CDR3 regions that are identical to SEQ ID NOs. 3241, 3242 and 3243, respectively; and ii. light chain CDR1, CDR2 and CDR3 regions that are identical to SEQ ID NOs. 3245, 3246 and 3247, respectively; or (b) a variant of said variable domain of (a) that is otherwise identical to the antibody variable domain except for up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions of the variable domain of (a). In some embodiments, the antibody may comprise: a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to SEQ ID NO: 3240; and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to SEQ ID NO: 3244.

[0175] In some embodiments, the antibody may contain only a heavy chain variable domain described herein. In these embodiments, the antibody may be a “heavy chain only” antibody.

[0176] In some embodiments, a subject antibody (e.g., a subject antibody that specifically binds GIPR may comprises: a) a light chain region comprising: i) one, two, or three complementarity determining regions (CDRs) from a light chain variable region sequence of a selected anti-GIPR antibody; and ii) a light chain framework region, e.g., a framework region from a human immunoglobulin light chain; and b) a heavy chain region comprising: i) one, two, or three CDRs from the heavy chain variable region sequence of a selected antibody; and ii) a heavy chain framework region, e.g., a framework region from a human immunoglobulin heavy chain.

[0177] A subject antibody can comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3240. A subject antibody can comprise a heavy chain variable region comprising one, two, or three of the heavy chain complementarity determining regions (CDRs) of a selected anti-GIPR antibody.

[0178] A subject antibody can comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3244. A subject antibody can comprise a light chain variable region comprising one, two, or three of the light chain CDRs of a selected anti-GIPR antibody.

[0179] In some embodiments, a subject antibody comprises anti-GIPR antibody heavy chain CDRs and anti-GIPR antibody light chain CDRs in a single polypeptide chain, e.g., in some embodiments, a subject antibody is a scFv. In some embodiments, a subject antibody comprises, in order from N-terminus to C-terminus: a first amino acid sequence of from about 5 amino acids to about 25 amino acids in length; an light chain CDR1 of a selected anti-GIPR antibody; a second amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a light chain CDR2 of a selected anti-GIPR antibody; a third amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a light chain CDR3Atty. Dkt: CRYS-029WO

[0180] of a selected anti-GIPR antibody; a fourth amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a heavy chain CDR1 of a selected anti-GIPR antibody; a fifth amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a heavy chain CDR2 of a selected anti-GIPR antibody; a sixth amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a heavy chain CDR3 of a selected anti-GIPR antibody; and a seventh amino acid sequence of from about 5 amino acids to about 25 amino acids in length.

[0181] In some embodiments, a subject antibody may comprise, in order from N-terminus to C-terminus: a light chain FR1 region; a light chain CDR1 of a selected anti-GIPR antibody; a light chain FR2 region; a light chain CDR2 of a selected anti-GIPR antibody; a light chain FR3 region; a light chain CDR3 of a selected anti-GIPR antibody; optionally a light chain FR4 region; a linker region; optionally a heavy chain FR1 region; a heavy chain CDR1 of a selected anti-GIPR antibody; a heavy chain FR2 region; a heavy chain CDR2 of a selected anti-GIPR antibody; a heavy chain FR3 region; a heavy chain CDR3 of a selected anti-GIPR antibody; and a heavy chain FR4 region. In some of these embodiments, each of the FR regions is a human FR region. The linker region can be from about 5 amino acids to about 50 amino acids in length, e.g., from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa in length.

[0182] In some embodiments, a subject antibody specifically binds GIPR from humans and other mammals, e.g., monkey and mouse.

[0183] For example, a subject antibody may bind to human, monkey and / or mouse GIPR with an affinity of at least about 10-7M, at least about 10’8M, at least about 10’9M, at least about IO10M, at least about 1011M, or at least about 1012M, or greater than 1012M. A subject antibody binds to an epitope present on human, monkey and / or mouse GIPR with an affinity of from about 10‘7M to about 10‘8M, from about 10'8M to about 10'9M, from about 10'9M to about 10-10M, from about 10-10M to about 10-11M, or from about 10-11M to about 10-12M, or greater than 10-12M.

[0184] A subject antibody can in some embodiments reduce binding of GIPR to a GIP. For example, in some embodiments a subject antibody can reduce binding of GIPR to GIP by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the degree of binding between GIPR and GIP in the absence of the antibody.

[0185] In some embodiments a subject antibody may reduce GIPR activity, i.e., signaling in response to GIP. For example, in some embodiments a subject antibody may reduce GIPR signaling by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, atAtty. Dkt: CRYS-029WO

[0186] least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to GIPR signaling in the absence of the antibody.

[0187] In some embodiments, a subject antibody is linked (e.g., covalently linked) to a polymer (e.g., a polymer other than a polypeptide). Suitable polymers include, e.g., biocompatible polymers, and water-soluble biocompatible polymers. Suitable polymers include synthetic polymers and naturally-occurring polymers. Suitable polymers include, e.g., substituted or unsubstituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymers or branched or unbranched polysaccharides, e.g. a homo- or hetero-polysaccharide. Suitable polymers include, e.g., ethylene vinyl alcohol copolymer (commonly known by the generic name EVOH or by the trade name EVAL); polybutylmethacrylate; poly (hydroxy valerate); poly(L-lactic acid); poly caprolactone; poly(lactide-co-glycolide);

[0188] poly (hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester; polyanhydride; poly(glycolic acid); poly(D, L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphoester; polyphosphoester urethane; poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(iminocarbonate); copoly(ether-esters) (e.g., polyethylene oxide)-poly(lactic acid) (PEO / PLA) co-polymers); polyalkylene oxalates; polyphosphazenes; biomolecules, such as fibrin, fibrinogen, cellulose, starch, collagen and hyaluronic acid; polyurethanes; silicones; polyesters; polyolefins; polyisobutylene and ethylene-alphaolefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; polyamides, such as Nylon 66 and polycaprolactam; alkyd resins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous Teflon; polyethylene glycol); and carboxymethyl cellulose.

[0189] Suitable synthetic polymers include unsubstituted and substituted straight or branched chain poly (ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol), and derivatives thereof, e.g., substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol), and derivatives thereof. Suitable naturally-occurring polymers include, e.g., albumin, amylose, dextran, glycogen, and derivatives thereof.

[0190] Suitable polymers can have an average molecular weight in a range of from 500 Da to 50000 Da, e.g., from 5000 Da to 40000 Da, or from 25000 to 40000 Da. For example, in some embodiments, where a subject antibody comprises a poly(ethylene glycol) (PEG) or methoxypoly(ethyleneglycol) polymer, theAtty. Dkt: CRYS-029WO

[0191] PEG or methoxypoly(ethyleneglycol) polymer can have a molecular weight in a range of from about 0.5 kiloDaltons (kDa) to 1 kDa, from about 1 kDa to 5 kDa, from 5 kDa to 10 kDa, from 10 kDa to 25 kDa, from 25 kDa to 40 kDa, or from 40 kDa to 60 kDa.

[0192] As noted above, in some embodiments, a subject antibody is covalently linked to a PEG polymer. In some embodiments, a subject scFv multimer is covalently linked to a PEG polymer. See, e.g., Albrecht et al. (2006) J. Immunol. Methods 310: 100. Methods and reagents suitable for PEGylation of a protein are well known in the art and may be found in, e.g., U. S. Pat. No. 5,849,860. PEG suitable for conjugation to a protein is generally soluble in water at room temperature, and has the general formula R(O-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. Where R is a protective group, it generally has from 1 to 8 carbons.

[0193] The PEG conjugated to the subject antibody can be linear. The PEG conjugated to the subject protein may also be branched. Branched PEG derivatives such as those described in U. S. Pat. No.

[0194] 5,643,575, “star-PEG's” and multi-armed PEG'S such as those described in Shearwater Polymers, Inc. catalog “Polyethylene Glycol Derivatives 1997-1998.” Star PEGs are described in the art including, e.g., in U. S. Patent No. 6,046,305.

[0195] A subject antibody can be glycosylated, e.g., comprise a covalently linked carbohydrate or polysaccharide moiety. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5 -hydroxylysine may also be used.

[0196] Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). Similarly, removal of glycosylation sites can be accomplished by amino acid alteration within the native glycosylation sites of an antibody.

[0197] Antibodies & Antigen-binding fragments thereof

[0198] Any subject polypeptide of the present disclosure may be an antibody. For example, a subject polypeptide may be multimeric and the antibody binding domain of the subject polypeptide may compriseAtty. Dkt: CRYS-029WO

[0199] one or more (e.g., one or two) heavy chain polypeptides (each heavy chain polypeptide comprising a heavy chain variable domain, VH) and / or one or more (e.g., one or two) light chain polypeptides (each light chain polypeptide comprising a light chain variable domain, VL). In these embodiments, the heavy chain polypeptide may, in addition to a heavy chain variable domain, include all or part of a heavy chain constant region to thereby form an immunoglobulin heavy chain and the light chain polypeptide may, in addition to a light chain variable domain, include all or part of a light chain constant region to thereby form an immunoglobulin light chain. A heavy chain constant region may be comprised of three domains, CHI, CH2, and CH3. A light chain constant region may be comprised of one domain, CL. The constant regions of the antibody can mediate binding of the antibody to host tissues and factors, including various cells of the immune system and the first component of the complement system. In some embodiments, a subject antibody may comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain. In certain embodiments, said antibody may be a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains. In some cases, a subject polypeptide may be an immunoglobulin of type IgA, IgG, IgE, IgD, IgM and subtypes thereof. In certain embodiments, the subject polypeptide may be an immunoglobulin of type IgG. In some cases, the subject polypeptide may be a heavy chain-only antibody (e.g., polypeptides comprising a subject VHH domain described herein).

[0200] In some embodiments, a subject polypeptide does not comprise a full-length heavy immunoglobulin chain and a full-length light immunoglobulin chain, and instead the subject polypeptide comprises antigen-binding fragments of a full-length immunoglobulin heavy chain and / or a full-length immunoglobulin light chain. In some cases, the antigen-binding fragments (e.g., the heavy and light variable domains) are present in separate polypeptides. In other cases, the antigen-binding fragments (e.g., the heavy and light variable domains) are present in a single polypeptide. In these embodiments, the antigen binding domain of a subject polypeptide may comprise (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHI domains), (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment (consisting of the VH and CHI domains); (iv) an Fv fragment (consisting of the VH and VL domains of a single arm of an antibody); (v) a dAb fragment (consisting of the VH domain); (vi) an isolated CDR; (vii) a single chain Fv (scFv) (consisting of the VH and VL domains of a single arm of an antibody joined by a synthetic linker using recombinant means such that the VH and VL domains pair to form a monovalent molecule); or (viii) diabodies (consisting of two scFvs in which the VH and VL domains are joined such that they do not pair to form a monovalent molecule; the VH of each one of the scFv pairs with the VL domain of the other scFv to form a bivalent molecule). In some embodiments, the antibody binding domain of a subject polypeptide is a Fab fragment. In some embodiments, the antibody binding domain of a subject polypeptide is a single-chain antibody (scFv).Atty. Dkt: CRYS-029WO

[0201] In certain embodiments, a subject polypeptide is a recombinant or modified antibody, e.g., a chimeric, humanized, deimmunized or an in vitro generated antibody. The term "recombinant" or "modified" antibody as used herein is intended to include all antibodies that are prepared, expressed, created, or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell; (ii) antibodies isolated from a recombinant, combinatorial antibody library; (iii) antibodies isolated from an animal (e.g. a chicken) that is transgenic for human immunoglobulin genes; or (iv) antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include humanized, CDR grafted, chimeric, deimmunized, and in vitro generated antibodies; and can optionally include constant regions derived from human germline immunoglobulin sequences.

[0202] Multispecific Antibodies

[0203] In some embodiments, the subject polypeptide is a multispecific binding molecule that comprises the antibody binding domain, VHH domain, or knob domain, as described above, and at least one other binding domain. These domains are connected by a suitable linker, many of which are known in the art. The at least one other binding domain may be another antibody binding domain, VHH domain, a scFv, a ligand for a cell surface receptor, or it may be based on an alternative scaffold. In varying embodiments, the at least one other binding domain may recognize, e.g., a cancer antigen, an immune cell antigen (i.e., an antigen found on the surface of an immune cell), or a viral antigen. The at least one other binding domain may recognize any desired or suitable cancer antigen, immune cell antigen, or viral antigen. A multitude of cancer antigens, immune cell antigens, and viral antigens are known in the art, many of which are described in, e.g., in Yi, Xinpei, et al. Iscience 24.10 (2021), Vita, Randi, et al. Nucleic acids research 47. DI (2019): D339-D343, and Ansari, Hifzur Rahman, Darren R. Flower, and G. P. S.

[0204] Raghava. Nucleic acids research 38.suppl_l (2010): D847-D853, the disclosures of which are incorporated by reference herein.

[0205] In certain embodiments, where the antibody binding domain binds to Zip6, at least one of the other binding domains may recognize an immune cell antigen (i.e., an antigen found on the surface of an immune cell) and / or a cancer antigen. In some cases, at least one of the other binding domains may recognize a T cell antigen or a natural killer (NK) cell antigen (i.e., an antigen found on the surface of a T cell or NK cell). Such antigens include, without limitation, CD2, CD16, NKp30, NKp44, NKp46, NKG2C, NKG2D, DNAM1, SLAMF7, 2B4, KIR2DS, and KIR3DS among others. In some embodiments, at least one of the other binding domains may recognize another cancer antigen (i.e., another antigen found on the surface of cancer cells). Such cancer antigens can include, e.g., CD19,Atty. Dkt: CRYS-029WO

[0206] CD20, BCMA, ALPP, CS1 (SLAMF7), CLDN18.2, AXL, ROR2, TM4SF1, ICAM-1, L1CAM (CD171), CD4, CD5, CD7, CD10, CD38, CEA, FLT3, CD70, CD30, CD37, CD147, HER2, MUC1, CEA, WT1, although there are many others.

[0207] In certain embodiments, where the knob domain binds to NKp46, at least one of the other binding domains may recognize a cancer antigen (i.e., an antigen found on the surface of cancer cells). Such cancer antigens include e.g., CD19, CD20, BCMA, ALPP, CS1 (SLAMF7), CLDN18.2, AXL, ROR2, TM4SF1, ICAM-1, L1CAM (CD171), CD4, CD5, CD7, CD10, CD38, CEA, FLT3, CD70, CD30, CD37, or CD 147, although there are many others. The cancer antigen may be a blood cell antigen or a solid tumor antigen, depending on how the polypeptide is going to be used. In alternative embodiments, at least one of the other binding domains may recognize a viral antigen on a cell that is infected by a virus.

[0208] In certain embodiments, where the knob domain, VHH domain, or antibody binding domain binds to EGFR, at least one of the other binding domains may recognize a cancer antigen (i.e., an antigen found on the surface of cancer cells) and / or an immune cell antigen (i.e., an antigen found on the surface of immune cells). Such cancer antigens include EGFR, HER2, VEGFR2, DLL4, ANG2, c-MET, PD-L1, LGR5, ZIP6, or MET, although there are many others. The cancer antigen may be a blood cell antigen or a solid tumor antigen, depending on how the polypeptide is going to be used. In embodiments where the at least one other binding domain also binds to EGFR, the at least one other binding domain may comprise the antigen binding domain of a suitable known anti-EGFR antibody. Examples of suitable anti-EGFR antibodies include, without limitation, panitumumab, cetuximab, necitumumab, and nimotuzumab. In such embodiments, the at least one other binding domain may bind to an epitope on EGFR that is distinct from the epitope recognized by the subject anti-EGFR VHH domain, knob domain, or antibody binding domain. In some embodiments, the first and second antigens can be different epitopes in the same protein. In these embodiments, the bispecific antibodies may be biparatopic. In some cases, a bispecific antibody may comprise two arms, wherein each arm comprises a first binding domain and a second binding domain. This antibody is considered “tetravalent” because it has a total of four binding domains, “symmetrical” in that both arms bind to the same antigens and “2+2” because each arm binds to two antigens. As such, this antibody can be referred to as a “symmetrical 2+2 tetravalent” bispecific antibody. In some embodiments, the subject polypeptide may be a biparatopic polypeptide, meaning that the symmetrical arms may bind to different epitopes in the same target. In some embodiments, the at least one other binding domain recognizes an immune cell antigen. In some cases, the at least one of the other binding domains may recognize a T cell antigen or a natural killer (NK) cell antigen (i.e., an antigen found on the surface of a T cell or NK cell). Such antigens include, without limitation, CD2 CD16, NKp30, NKp44, NKp46, NKG2C, NKG2D, DNAM1, SLAMF7, 2B4, KIR2DS, KIR3DS, CD3, or CD28.Atty. Dkt: CRYS-029WO

[0209] In the varying embodiments where at least one of the binding domains recognizes an antigen on the surface of an NK cell (e.g., NKp46, NKG2C), the polypeptide may be an NK cell engager (i.e., an NKCE, alternatively known as a killer cell engager) where, in some embodiments, the polypeptide may be a bispecific NK cell engager (referred to as a “BiKE” in some publications) or a trispecific K cell engager (referred to as a “TriKE” in some publications. BiKEs and TriKEs are reviewed in Felices et al (Methods Mol Biol. 2016; 1441: 333-346). Such molecules tether NK cells to a tumor cell (e.g., a tumor cell comprising EGFR) and induce their activation at that site. BiKEs and TriKEs are molecules that contain a single variable portion of an antibody linked to one (BiKE) or two (TriKE) variable portions from other antibodies of different specificity. Similarly, the polypeptide could be a T cell engager (e.g., a BiTE).

[0210] As such, in embodiments where the antibody binding domain binds Zip6, the polypeptide may be a bispecific binding molecule that comprises (i) the anti-Zip6 antibody binding domain, and (ii) a binding domain that recognizes an antigen found on the surface of an immune cell. In other embodiments where the antibody binding domain binds Zip6, the polypeptide may be a trispecific binding molecule that comprises (i) the anti-Zip6 antibody binding domain, (ii) a binding domain that that recognizes a first antigen on the surface of an immune cell, and (iii) either a binding domain that recognizes a second antigen on the surface of an immune cell (e.g., a co-stimulatory receptor) or a binding domain that recognizes an antigen (e.g., other than Zip6) on the surface of a cancer cell. In embodiments where the knob domain binds NKp46, the polypeptide may be a bispecific binding molecule that comprises (i) the anti-NKp46 knob domain and (ii) a binding domain that recognizes a first antigen found on the surface of cancer cells or virally infected cells. In other embodiments, the polypeptide may be a bispecific binding molecule that comprises (i) the anti-NKp46 knob domain, (ii) a binding domain that recognizes a first antigen on the surface of cancer cells or virally infected cells and (iii) either a binding domain that recognizes a co-stimulatory receptor or a binding domain that recognizes a second antigen on the surface of cancer cells or virally infected cells. In these latter embodiments, it has been noted that NKp46 synergizes with other immune receptors (referred as co-stimulatory receptors or co-receptors herein), e.g., 2B4, DNAM1 and CD2 (see, e.g., Zmai et al Cells 2020 9: 753) and, as such, a bispecific binding molecule may bind to NKp46 and one or more of 2B4, DNAM1, or CD2. NKp46 provides a better alternative to CD 16 (another NK cell-specific stimulatory immune receptor) in some embodiments, because CD 16 levels are downregulated by NK cells in the tumor microenvironment. In embodiments where the knob domain, VHH domain, or antibody binding domain binds EGFR, the polypeptide may be a bispecific binding molecule that comprises (i) the anti-EGFR knob domain, VHH domain, or antibody binding domain, and (ii) a binding domain that recognizes an antigen found on the surface of an immune cell. In other embodiments, the polypeptide may be a trispecific binding molecule that comprises (i) theAtty. Dkt: CRYS-029WO

[0211] anti-EGFR knob domain, VHH domain, or antibody binding domain, (ii) a binding domain that that recognizes a first antigen on the surface of an immune cell, and (iii) either a binding domain that recognizes a second antigen on the surface of an immune cell (e.g., a co-stimulatory receptor) or a binding domain that recognizes an antigen (e.g., other than EGFR, or another epitope on EGFR) on the surface of a cancer cell.

[0212] Multispecific antibodies can be in a variety of different formats, including, but not limited to IgG-like antibody formats (including an Fc domain) and non-IgG-like antibody formats (without an Fc domain). Multispecific antibodies with IgG-like antibody formats can be in a variety of different formats, including, but not limited to knob-into-hole (KIH), TrioMab, Duobody, KZ body, CrossMab, common light chain, strand exchange engineered domain bodies (SEEDBodies), Azymetric heterodimeric Fc, dual action Fab (DAF), dual-variable-domain immunoglobulin (DVD-Ig), IgG-scFv, Fab-Fab-Fc, DutaMab, and DutaFab.

[0213] Non-IgG-like antibody formats may lack an Fc region entirely. For example, Fab, Fv and VHH antibody regions may be genetically engineered and combined in various orientations and pairings.

[0214] Multispecific antibodies in non-IgG-like formats can be in a variety of different formats, including, but not limited to bivalent dual -affinity re-targeting protein (DART), tetravalent DART, half-life extended bispecific T-cell engager (HLE-BiTE), bispecific T-cell engager (BiTE), immune mobilizing monoclonal T-cell receptor (ImmTAC), tandem diabody (TandAb), bispecific killer T-cell engager (BiKE), trispecific killer T-cell engager (TRiKE), multispecific scFV single-chain variable fragment, trispecific T-cell activation construct (TriTAC), bispecific nanobody, and cross-over dual variable region (CODV).

[0215] Multispecific antibodies may be bifunctional, trifunctional or even tetrafunctional. Multispecific antibodies may be IgG-fusion proteins. These antibody formats have been reviewed in a variety of publications, including Elshiaty et al. (2021), International Journal of Molecular Science, 22(11):5632; Jin et al. (2022), Signal Transduction Targeted Therapy, 7(39); Weidle et al. (2013), Cancer Genomics & Proteomics 10:1-18).

[0216] Linkers

[0217] In some embodiments, a subject polypeptide may comprise one or more polypeptide linkers interposed between the components of a subject polypeptide. For example, in some cases, a subject polypeptide may comprise a linker interposed between a subject knob domain, VHH domain, or antibody binding domain and another protein (e.g., an Fc domain or an albumin domain). As another example, in some cases, the antibody binding domain of a subject polypeptide may be an scFv comprising a linker interposed between the heavy chain variable domain and the light chain variable domain present in a single polypeptide chain.Atty. Dkt: CRYS-029WO

[0218] Linkers suitable for use a subject in a subject polypeptide include “flexible linkers”. If present, the linker molecules are generally of sufficient length to permit some flexible movement between linked regions. The linker molecules are generally about 6-50 atoms long. The linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Other linker molecules which can bind to polypeptides may be used in light of this disclosure. Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0219] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO: 3260) and GGGSn (SEQ ID NO: 3261), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components. Glycine polymers are of particular interest since glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev.

[0220] Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited GGSG (SEQ ID NO: 3262), GGSGG (SEQ ID NO: 3263), GSGSG (SEQ ID NO: 3264), GSGGG (SEQ ID NO: 3265), GGGSG (SEQ ID NO: 3266), GSSSG (SEQ ID NO: 3267), and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.

[0221] Modified Fc domains

[0222] A polypeptide of the present disclosure may have a modified Fc domain (i.e., an Fc domain with enhanced or decreased functionality) (see, e.g., van der Horst et al Cancers (Basel) 2020 12: 3041 and Wilkinson et at (PLoS One. 2021; 16: e0260954) or no Fc domain. In some embodiments, a polypeptide may have an Fc region that has been modified to abolish or reduce binding to the Fc receptor. Amino acid substitutions that abolish or reduce Fc binding to the Fc receptor include, but are not limited to:

[0223] L234A / L235A (LALA), L234F / L235E / P331S (FES), L234F / L235Q / K322Q (FQQ), L234A / G237A, L234A / L235A / G237A, L234A / L235A / G237A / P238S / H268A / A330sS / P330S, L234A / L235E, G236R / L328R, and L234A / L235A / K322A. Sec, e.g., Wilkinson et at (PLoS One. 2021; 16: c0260954).Atty. Dkt: CRYS-029WO

[0224] Albumin domains

[0225] A polypeptide of the present disclosure may comprise an albumin domain (i.e., an albumin protein or derivative thereof). In some embodiments, the albumin domain may increase the half-life or decrease the immunogenicity of the subject polypeptide (see, e.g., Rogers, Brian, et al. Current pharmaceutical design 21.14 (2015): 1899-1907). In some cases, the albumin may be a human serum albumin (e.g., a wild-type human serum albumin), a recombinant albumin, and / or a genetically engineered human serum albumin. Suitable albumin domains for use in the subject polypeptides are known in the art and described, e.g., in W02013075066A2, WO2011051489A2, and WO2014072481A1, the disclosures of which are incorporated by reference herein.

[0226]

[0227] In any embodiment, a subject polypeptide described herein can be conjugated to a drug. In these embodiments, the polypeptide may be an antibody drug conjugate. Antibody drug conjugates (ADCs) are targeted medicines that deliver pharmacologically active agents to cells, e.g., deliver chemotherapeutic agents to cancer cells. ADCs deliver the drug via a linker attached polypeptide (typically an antibody) that binds to a specific target expressed on the cells. After binding to the target (cancer protein or receptor), the ADC releases a drug into or on the cell. In some embodiments, the agent is not released and, rather, the polypeptide holds the drug close to the site at which it is believed to be effective (e.g., near to its binding site on a receptor). Antibody-drug conjugates are reviewed in several publications, including Peters (Biosci Rep. 2015 35: e00225).

[0228] ADC Payloads

[0229] An ADC of the present disclosure may comprise one or more of any pharmacologically active agent, e.g., an enzyme inhibitor, channel inhibitor, a peptide, a nucleic acid, or a cytotoxic and / or cytostatic agent, which may be referred to as the “payload” of the ADC. The payload of an ADC of the present disclosure may be any pharmacologically active agent or drug suitable to achieve the intended physiological effect, e.g., modulating the expression of a gene, modulating the activity of an enzyme, modulating the activity of a signaling pathway, inhibiting cell division. In varying embodiments, the pharmacologically active agent may exert a physiological effect relevant for treating, by way of example, cancers, inflammatory disorders, cardiovascular disorders, gastrointestinal disorders, Genito-urological disorders, hematological disorders, hormonal disorders, infectious disease, metabolic disorders, muscular disorders, neurological disorders, ophthalmologic disorders, and respiratory disorders. The payload can be, e.g., an anti-inflammatory agent, a cytotoxic agent, a ligand for a receptor (e.g., a cytokine), or a nucleic acid (e.g., an oligonucleotide) that effects alters gene expression of the cell to which the polypeptide binds.Atty. Dkt: CRYS-029WO

[0230] In some embodiments, the payload of an ADC of the present disclosure may be a cytostatic and / or cytotoxic agent. Cytotoxic agents are drugs that result in cell death, whereas cytostatic agents are drugs that inhibit cellular proliferation. In practice many drugs act as cytotoxic and cytostatic agents depending on dosage or biological context. Accordingly, the designation “cytotoxic” and “cytostatic” are used interchangeably herein. Both cytotoxic and cytostatic activity are advantageous in chemotherapeutic agents, e.g., to shrink and / or limit growth of a tumor. Cytotoxic and / or cytostatic agents that are suitable for use as payloads in ADCs have been generally reviewed in several publications, including:

[0231] Theocharopoulos et al. 2021 (Theocharopoulos, Charalampos, et al. " Antibody-drug conjugates:

[0232] Functional principles and applications in oncology and beyond." Vaccines 9.10 (2021): 1111) and Anand et al. 2023 (Anand, Uttpal, et al. " Cancer chemotherapy and beyond: Current status, drug candidates, associated risks and progress in targeted therapeutics." Genes & Diseases 10.4 (2023): 1367-1401), the entirety of each being incorporated herein by reference.

[0233] Microtubule Inhibitors

[0234] In some embodiments, the cytotoxic payload of an ADC of the present disclosure may be a microtubule inhibitor or microtubule disrupting agent. Microtubule assembly plays a critical role in cell division and cellular transport, among other key cellular functions, and inhibition of microtubule assembly leads to arrest of cell division and / or cell death. In some cases, the microtubule inhibitor may be an auristatin or analogues and derivatives thereof. Auristatins are symbolic analogues of the natural molecule Dolostatin 10, isolated from Dolabella Auricularia. Suitable auristatins and auristatin derivatives for use as a payload include, without limitation, monomethyl auristatin E (MMAE; CAS Registry No. 474645-27-7), monomethyl auristatin F (MMAE; CAS Registry No. 745017-94-1), and monomethyl auristatin D (MMAD; CAS Registry No. 203849-91-6). The auristatin MMAE, for example, has been used as the cytotoxic payload in the clinically approved ADCs brentuximab vedotin, inotuzumab ozogamicin, gemtuzumab ozogamicin, polatuzumab vedotin. In some cases, the microtubule inhibitor may be a maytansinoid or derivatives thereof. Maytansinoids are derived from the natural molecule maytansine, isolated from Maytenus ovatus. Suitable maytansinoids and maytansinoid derivatives for use as a payload include, without limitation, maytansine (CAS Registry No. 35846-53-8) and mertansine (DM1; CAS Registry No. 139504-50-0). The maytansinoid DM1, for example, has been used as the cytotoxic payload in the clinically approved ADC trastuzumab emtansine. Other suitable microtubule inhibitors include, by way of example and without limitation, colchicine (CAS Registry No. 64868), halichondrin B (CAS Registry No. 103614-76-2), rhizoxin (CAS Registry No. 90996-54-6), paclitaxel (CAS Registry No. 33069-62-4), and vinca alkaloids such as vindesine (CAS Registry No. 53643-48-4). Microtubule inhibitors / disrupting agents arc known in the ait and described, for example, in Wang ct al.

[0235] 2023 (Wang, Xingyu, et al. " Microtubule-targeting agents for cancer treatment: Seven binding sites andAtty. Dkt: CRYS-029WO

[0236] three strategies." MedComm-Oncology 2.3 (2023): e46), the entirety of which is incorporated herein by reference.

[0237] DNA-damaging agents

[0238] In some embodiments, the cytotoxic payload of an ADC of the present disclosure may be a DNA-damaging agent. DNA-damaging agents act through a variety of mechanisms and include, for example, alkylating agents, DNA intercalators, and topoisomerase inhibitors.

[0239] In some cases, the DNA-damaging agent may be a calicheamicin or derivatives thereof.

[0240] Calicheamicins are anticancer antiobiotics isolated from the actinomycete Micromonospora echinospora spp. Calichensis. Calicheamicins bind the minor groove of DNA in a site-specific manner and reductive cleavage by thiols present in a cell generates reactive diradical species that lead to strand scission of the DNA and, subsequently, cell death. Suitable calicheamicins and calicheamicin derivatives for use as a payload include, without limitation, calicheamicin y 1 (CAS Registry No. 108212-75-5) and

[0241] N-acetyl-gamma calicheamicin 1,2-dimethyl hydrazine. Calicheamicins have, for example, been used as the cytotoxic payload in the clinically approved ADCs inotuzumab ozogamicin and gemtuzumab ozogamicin. Calicheamicin and derivatives thereof are known in the art and described, for example, in Maiese at al. 1989 (Maiese, William M., et al. " Calicheamicins, a novel family of antitumor antibiotics: taxonomy, fermentation and biological properties." The Journal of antibiotics 42.4 (1989): 558-563), the entirety of which is incorporated herein by reference.

[0242] In some cases, the DNA-damaging agent may be a pyrrolobenzodiazepine (PBD) or derivatives thereof. PBDs can alkylate and cross-link opposite DNA strands, preventing strand separation during genomic replication and inducing cell death. Suitable PBDs for use as a payload include, without limitation, tesirine (SG3249; CAS Registry No. 1595275-62-9) and SJG-136 (CAS Registry No. 232931-57-6). The PBD tesirine, for example, has been used as the cytotoxic payload in the clinically approved ADC loncastuximab tesirine. PBDs and derivatives thereof are known in the art and are described, for example, in: Mantaj et al. 2017 (Mantaj, Julia, et al. " From anthramycin to pyrrolobenzodiazepine (PBD)-containing antibody-drug conjugates (ADCs)." Angewandte Chemie International Edition 56.2 (2017): 462-488), the entirety of which is incorporated herein by reference.

[0243] In some cases, the DNA-damaging agent may be a topoisomerase inhibitor, e.g., a topoisomerase I or topoisomerase II inhibitor. Topoisomerases are necessary for regulating DNA topology and torsional stresses during DNA replication, repair and transcription. In certain embodiments, the topoisomerase inhibitor may be a topoisomerase I inhibitor, e.g., a camptothecin or a derivative thereof. Camptothecin and its derivates inhibit topoisomerase I, inhibiting replication and causing cleavage of the DNA. Suitable camptothccins and camptothecin derivatives for use as a payload include, without limitation, camptothecin (CAS Registry No. 7689-03-4), topotecan (CAS Registry No. 123948-87-8), irinotecanAtty. Dkt: CRYS-029WO

[0244] (CAS Registry No. 97682-44-5), belotecan (CAS Registry No. 256411-32-2), exatecan (CAS Registry No. 171335-80-1), deruxtecan (CAS Registry No. 1599440-13-7), and SN-38 (CAS Registry No. 86639-52-3). The camptothecin analog deruxtecan, for example, has been used as the payload in the clinically approved ADC trastuzumab deruxtecan. In certain embodiments, the topoisomerase inhibitor may be a topoisomerase II inhibitor, e.g., an anthracycline, anthracenedione, acridine, epipodophyllotoxin, or derivatives thereof. Anthracyclines intercalate DNA and poison DNA-topoisomerase II complexes, inhibiting DNA replication and causing cell death. Additionally, anthracyclines generate free -radicals in an iron-dependent manner, further enhancing their cytotoxic effects. Suitable anthracycline and anthracycline derivatives for use as a payload include, without limitation, doxorubicin (CAS Registry No.

[0245] 23214-92-8), epirubicin (CAS Registry No. 56420-45-2), valrubicin (CAS Registry No. 56124-62-0), daunorubicin (CAS Registry No. 20830-81-3), idarubicin (CAS Registry No. 58957-92-9), and PNU-159682 (Cas Registry No. 202350-68-3). Anthracenediones, acridines, epipodophyllotoxins, and derivatives thereof intercalate DNA and poison DNA-topoisomerase II complexes, inhibiting DNA replication and causing cell death. Suitable anthracenediones and anthracenedione derivatives for use as a payload include, without limitation, mitoxantrone (CAS Registry No. 65271-80-9) and pixantrone (CAS Registry No. 144510-96-3). Suitable epipodophyllotoxins and epipodophyllotoxin derivatives include, without limitation, etoposide (CAS Registry No. 33419-42-0) and teniposide (CAS Registry No. 29767-20-2). Various classes of topoisomerase inhibitors are known in the art and described, for example, in Yakkala et al. 2023 (Yakkala PA, Penumallu NR, Shall S, Kamal A. “Prospects of Topoisomerase Inhibitors as Promising Anti-Cancer Agents”. Pharmaceuticals (Basel). 2023 Oct 13; 16(10): 1456), the entirety of which is incorporated herein by reference.

[0246] In some cases, the DNA-damaging agent may be an alkylating agent. Alkylaling agents interact with DNA and form covalent adducts to cross-link DNA or cross-link DNA with protein, leading to DNA cleavage, inhibition of replication, and cell death. Suitable alkylating agents for use as a payload include, without limitation, adozelesin (CAS Registry No. 110314-48-2) carboplatin (CAS Registry No. 41575-94-4), chlorambucil (CAS Registry No. 305-03-3), cisplatin (CAS Registry No. 15663-27-1), cyclophosphamide (CAS Registry No. 50-18-0), lomustine (CAS Registry No. 13010-47-4), melphalan (CAS Registry No. 148-82-3), mitomycin C (CAS Registry No. 50-07-7) and temozolomide (CAS Registry No. 85622-93-1).

[0247] Other Cytotoxic / Cytostatic Agents

[0248] In some embodiments, the cytotoxic payload of an ADC of the present disclosure may be an antimetabolite, a protein-synthesis inhibitor, a mitochondrial inhibitor, a histone deacetylase (HD AC) inhibitor, or a cell-cycle disruptor. Antimetabolites inhibit metabolic pathways, c.g., nucleic acid or amino acid synthesis, necessary for cell proliferation and survival. Exemplary antimetabolites include 5-Atty. Dkt: CRYS-029WO

[0249] fluorouracil (5-FU; CAS Registry No. 51-21-8) and fludarabine (CAS Registry No. 21679-14-1), among others. Various classes of antimetabolites are known in the art and described, for example, in: Cole et al.

[0250] 2005 (Cole, Peter D., John A. Zebala, and Barton A. Kamen. " Antimetabolites: A new perspective." Drug Discovery Today: Therapeutic Strategies 2.4 (2005): 337-342), the entirety of which is incorporated herein by reference. Protein-synthesis inhibitors disrupt translation of mRNAs through different mechanisms. Exemplary protein-synthesis inhibitors include tomivosertib (eFT-508; CAS Registry No.

[0251] 1849590-01-7), zotatifin (CAS Registry No. 2098191-53-6), and silvestrol (CAS Registry No.

[0252] 697235-38-4). Various classes of protein-synthesis inhibitors are known in the art and described, for example, in Kovalski et al. 2022 (Kovalski, Joanna R., Duygu Kuzuoglu-Ozturk, and Davide Ruggero. " Protein synthesis control in cancer: selectivity and therapeutic targeting." The EMBO Journal 41.8 (2022): el09823), the entirety of which is incorporated herein by reference. Mitochondrial inhibitors disrupt mitochondrial function and or cellular respiration. Exemplary mitochondrial inhibitors include tamoxifen (CAS Registry No. 10540-29-1), gamitrinib (CAS Registry No. 1131626-46-4), and tigecycline (CAS Registry No. 220620-09-7). HDAC inhibitors disrupt the regulation of gene-expression by HDAC mediated remodeling of chromatin. Exemplary HDAC inhibitors include trichostatin A (CAS Registry No. 58880-19-6), and SK-7041 (CAS Registry No. 617690-98-9). Various classes of HDAC inhibitors are known in the art and described, for example, in Shanmugam et al. 2022 (Shanmugam, Geetha, Sudeshna Rakshit, and Koustav Sarkar. " HDAC inhibitors: Targets for tumor therapy, immune modulation and lung diseases." Translational Oncology 16 (2022): 101312), the entirety of which is incorporated herein by reference. Cell-cycle disruptors interfere with the regulation of cell division, e.g., interfering with cyclin-CDK function. Exemplary cell-cycle disruptors include flavopiridol (CAS Registry No. 146426-40-6), dinacicilib (CAS Registry No. 779353-01-4), and roniciclib (CAS Registry No. 1223498-69-8). Various classes of cell-cycle disruptors are known in the art and described, for example, in: Zhang et al. 2021 (Zhang, Mengna, et al. " CDK inhibitors in cancer therapy, an overview of recent development." American journal of cancer research 11.5 (2021): 1913) and Bai et al. 2017 (Bai, Jingwen, Yaochen Li, and Guojun Zhang. " Cell cycle regulation and anticancer drug discovery." Cancer biology & medicine 14.4 (2017): 348), the entirety of each being incorporated herein by reference.

[0253] Nucleic Acid Payloads

[0254] In some embodiments, the payload of an ADC of the present disclosure may be a nucleic acid, e.g., an RNA or DNA oligonucleotide. A key feature of nucleic acid payloads is the capability to offer highly selective modulation of, in theory, any gene of interest by designing to the nucleic acid payload to bind to a target nucleic acid through relatively simple Watson-Crick base pairing mechanisms. Nucleic acid payloads can be used to, for example, modulate the expression of a target gene through a variety of mechanisms that involve base-pairing to the target, e.g., base-pairing to a complementary sequence of aAtty. Dkt: CRYS-029WO

[0255] transcript of the target gene. Pharmacologically active nucleic acids, and uses thereof in ADCs, are known in the art and described, for example, in Dugal-Tessier et al. 2021 (Dugal -Tessier, Julien, Srinath Thirumalairajan, and Nareshkumar Jain. " Antibody-oligonucleotide conjugates: a twist to antibody-drug conjugates." Journal of Clinical Medicine 10.4 (2021): 838) and Moumne et al. 2022 (Moumne, Lara, Anne-Celine Marie, and Nicolas Crouvezier. " Oligonucleotide therapeutics: from discovery and development to patentability." Pharmaceutics 14.2 (2022): 260), of which the entirety of each are incorporated herein by reference.

[0256] Anti-sense oligonucleotides (ASOs)

[0257] In some embodiments, the nucleic acid payload of an ADC of the present disclosure may be an anti-sense oligonucleotide (ASO). ASOs are designed to be complementary (i.e., anti-sense) to a target nucleic acid RNA transcript (e.g., an mRNA). In some cases, an ASO can modulate expression of a desired gene (e.g., a disease associated gene) by triggering degradation of the target gene transcript. For example, an DNA, or DNA-like, ASO can be designed to be complementary to a target RNA transcript and hybridize to form a DNA / RNA complex with the transcript. The ASO / transcript complex is then susceptible to the activity of RNAse H, which selectively degrades the RNA strands of DNA / RNA complexes, thereby degrading the hybridized target transcript.

[0258] In some cases, the ASO can be designed to alter the expression of a specific isoform of a gene through modulation of RNA splicing. For example, an ASO can be designed to bind the intron-exon junction of the pre -mRNA of a target gene and sterically interfere with the spliceosome machinery and thereby alter splicing events.

[0259] Suitable ASOs for use as a payload in an ADC of the present disclosure can include an ASO specific to, in principle, any conceivable gene of interest. In some embodiments, the ASO may be a known or clinically approved ASO specific to a known target. Exemplary known target gene-expression inhibiting ASOs suitable for use as a nucleic acid payload include, without limitation, TTR-targeting inotersen, TTR-targeting eplontersen, APOB targeting mipomersen, APOC3 targeting volanesorsen, APOC3 -targeting olezarsen, CMV IE2-targeting fomivirsen, IRS 1 -targeting aganirsen, ICAM1 -targeting alicaforsen, LPA-targeting, pelacarsen, SOD 1 -targeting tofersen, HTT-targeting tominersen, TGFB2-targeting trabedersen, GFAP-targeting zilganersen, GHR-targeting atesidorsen, GHR-targeting cimderlirsen, PCSK9-targeting cepadacursen, HBV-targeting bepirovirsen, STAT3-targeting danvatirsen, KLKB1 -targeting donidalorsen, GRB2-targeting prexigebersen, and ANGPTL3 -targeting vupanorsen. Exemplary known splice-modulating ASOs suitable for use as a nucleic acid payload include, without limitation, CEP290-targeting sepofarsen, DMD-targeting eteplirsen, DMD-targeting viltolarsen, DMD-targeting casimcrscn, DMD-targeting golodirscn, DMD-targeting rcnadirscn, and SMN2-targeting nusinersen.Atty. Dkt: CRYS-029WO

[0260] Short-interfering RNAs (siRNAs)

[0261] In some embodiments, the nucleic acid payload of an ADC of the present disclosure may be a short-interfering RNA (siRNA). An siRNA is a double-stranded RNA molecule, a strand of which can hybridize to a complementary target RNA, that can induce degradation of the target RNA through recruitment of and incorporation into an RNA-induced silencing complex (RISC) in a target cell. Suitable siRNAs for use as a payload in an ADC of the present disclosure can include an siRNA specific to, in principle, any conceivable gene of interest. In some embodiments, the siRNA may be a known or clinically approved siRNA. Exemplary known siRNAs suitable for use as a nucleic acid payload include, without limitation, TTR-targeting patisiran, TTR-targeting vutrisiran, ALAS 1 -targeting givosiran, PCSK9-targeting inclisiran, HAO I -targeting lumasiran, SERPINC1 -targeting fitusiran, LDHA-targeting nedosiran, TP53-targeting teprasiran, TRPV1 -targeting tivanisiran, ADRB2-targeting bamosiran, SERP1NA1 -targeting belcesiran, SERPINAl-targeting fazisiran, C5-targeting cemdisiran, LPA-targeting olpasiran, and AGT-targeting zilebesiran.

[0262] Nucleic Acid Modifications

[0263] In some embodiments, the nucleic acid payload of an ADC of the present disclosure comprises one or more modifications (e.g., a nucleobase modification, a sugar modification, a backbone modification) to improve, for example, affinity or the stability of the nucleic acid payload in circulating plasma. Suitable nucleic acid modifications include, by way of example, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, 2’-O-Methyl modified nucleotides, 2’ fluoro modified nuclolides, and phosphorothioate linkages, among others. Nucleic acid modifications useful for nucleic acids (e.g., RNA or DNA oligonucleotides) with therapeutic applications are known in the art and described, for example, in WO 2007 / 047913, Ochoa et al. 2020 (Ochoa, Steven, and Valeria T. Milam. " Modified nucleic acids: Expanding the capabilities of functional oligonucleotides." Molecules 25.20 (2020): 4659), Kulkarni et al. 2021 (Kulkarni, Jayesh A., et al. " The current landscape of nucleic acid therapeutics." Nature nanotechnology 16.6 (2021): 630-643) and Moumne et al. 2022 (Moumne, Lara, Anne-Celine Marie, and Nicolas Crouvezier. " Oligonucleotide therapeutics: from discovery and development to patentability." Pharmaceutics 14.2 (2022): 260), of which the entirety of each is incorporated herein by reference.

[0264] Peptide Payloads

[0265] In some embodiments, the payload of an ADC of the present disclosure may be a peptide, e.g., a therapeutic peptide or a labeled peptide. In such embodiments, the ADC may be alternately referred to as an antibody-peptide conjugate (APC). The conjugated peptide may chosen and / or designed to deliver a therapeutic agent, enhance targeting of the antibody, or provide additional funchons, e.g., for aiding in imaging applications or for enhancing cellular uptake of the APC. In some cases, the conjugated peptideAtty. Dkt: CRYS-029WO

[0266] itself may be the therapeutic agent. In other cases, the peptide may be linked to another therapeutic agent (e.g., a cytotoxic drug). In some cases, the peptide may be functionally labeled with, e.g., fluorescent dyes or radioactive agents isotopes to enable the use of the APC in imaging applications.

[0267] G-protein Couple Receptor (GPCR) Modulators

[0268] In some embodiments, the payload of an ADC of the present disclosure may be a modulator of a G-protein coupled receptor (GPCR). GPCRs encompass the largest protein family encoded in the human genome and are involved in diverse physiological processes and have been implicated in many diseases ranging from type 2 diabetes to schizophrenia. GPCRs transduce extracellular signals in the form of varying ligands which induce a conformational change in the GPCR upon binding, subsequently leading to activation of G proteins and other downstream intracellular molecular pathways. GPCRs can be grouped into four classes based on their amino acid sequences: class A (rhodopsin-type), class B (secretin / adhesion-type), class C (glutamate -type), and class F (frizzled-type) GPCR subfamilies. The class A type GPCRs can be further subdivided into aminergic, peplidc. lipid, protein, nucleotide, and steroid receptor GPCRs. Detailed investigation of the structure and function of GPCR activation has led to the development of numerous molecular modulators for hundreds of GPCRs across each class described above.

[0269] Suitable aminergic GPCR modulators for use as a payload may include any known or clinically approved aminergic GPCR modulator. Exemplary aminergic GPCR modulators suitable as payloads include, by way of example: Acebutolol, Acetophenazine, Acetylcholine, Aclidinium, Acrivastine, Benperidol, Benzphetamine, Benztropine, Bepotastine, Betahistine, Cabergoline, Carbachol, Carbinoxamine, Cariprazine, Carteolol, Dapiprazole, Darifenacin, Desipramine, Desloratadine, Dexbrompheniramine, Eletriptan, Emedastine, Ephedrine, Epinastine, Epinephrine, Famotidine, Fenoldopam, Fenoterol, Fesoterodine, Fexofenadine, Gilteritinib, Glycopyrronium, Guanabenz, Guanfacine, Haloperidol, Hexocyclium, Histamine, Homatropine methylbromide, Hydroxyzine, Iloperidone, Indacaterol, Indoramin, Ipratropium, Isoetharine, Ketotifen, Eabetalol, Easmiditan, Levobunolol, Levocabastine, Levocetirizine, Meclizine, Mepenzolate, Mephentermine, Mesoridazine, Metaproterenol, Nadolol, Naphazoline, Naratriptan, Nebivolol, Nefazodone, Olanzapine, Olodaterol, Olopatadine, Orphenadrine, Oxprenolol, Paliperidone, Pemirolast, Penbutolol, Pergolide, Perphenazine, Quetiapine, Ranitidine, Remoxipride, Revefenacin, Risperidone, Ritodrine, Salbutamol, Salmeterol, Scopolamine, Silodosin, Solifenacin, Tamsulosin, Tegaserod, Terazosin, Terbutaline, Terfenadine, Umeclidinium, Vilanterol, Vilazodone, Vortioxetine, Xylometazoline, Ziprasidone, Zolmitriptan, and Zuclopenthixol.

[0270] Suitable peptide GPCR modulators for use as a payload may include any known or clinically approved peptide GPCR modulator. Exemplary peptide GPCR modulators suitable as payloads include,Atty. Dkt: CRYS-029WO

[0271] by way of example: Angiotensin II, Azilsartan medoxomil, Candesartan cilexetil, Eprosartan, Forasartan, Irbesartan, Losartan, Olmesartan medoxomil, Telmisartan, Valsartan, Icatibant, Ramipril, Captopril, Enalaprilat, Zinc, Zinc chloride, Zinc acetate, Zinc sulfate, Pentagastrin, Ambrisentan, Bosentan, Macitentan, Sitaxentan, Acetylsalicylic acid, Nedocromil, Macimorelin, Abarelix, Buserelin, Cetrorelix, Danazol, Degarelix, Ganirelix acetate, Gonadorelin, Goserelin, Histrelin, Leuprolide, Nafarelin, Triptorelin, Elagolix, Afamelanotide, Cosyntropin, Bremelanotide, Erythromycin, Cysteamine, Levocabastine, Alfentanil, Alvimopan, Anileridine, Buprenorphine, Butorphanol, Cocaine, Codeine, Dezocine, Difenoxin, Dihydrocodeine, Diphenoxylate, Eluxadoline, Ethylmorphine, Fentanyl, Hydrocodone, Hydromorphone, Ketobemidone, Levallorphan, Levorphanol, Levomethadyl acetate, Loperamide, Meperidine, Methadone, Methadyl acetate, Methylnaltrexone, Morphine, Nalbuphine, Naldemedine, Nalmefene, Naloxegol, Naloxone, Naltrexone, Oxycodone, Oxymorphone, Pentazocine, Propoxyphene, Remifentanil, Sufentanil, Tapentadol, Tramadol, Suvorexant, Lemborexant, Vorapaxar, Thrombin, Serelaxin, Gallium 68 DOTATOC, Lutetium Lu 177 dotatate, Pasireotide, Lanreotide, Octreotide, Vapreotide, Aprepitant, Netupitant, Rolapitant, Fosnetupitant and palonestron, Protirelin, Atosiban, Conivaptan, Desmopressin, Oxytocin, Terlipressin, Tolvaptan, and Vasopressin.

[0272] Suitable lipid GPCR modulators for use as a payload may include any known or clinically approved lipid GPCR modulator. Exemplary lipid GPCR modulators suitable as payloads include, by way of example: Dronabinol, Marinol, Nabilone, Tetrahydrocannabinol, Cannabidiol, Icosapent, Montelukast, Nedocromil, Pranlukast, Zafirlukast, Asfotase alfa, Fingolimod, Siponimod, Ozanimod, Rupatadine, Alprostadil, Bupivacaine, Bimatoprost, Carboprost tromethamine, Dinoprost tromethamine, Dinoprostone, Epoprostenol, Iloprost, Latanoprostene bunod, Latanoprost, Misoprostol, Ridogrelum, Selexipag, Tafluprost, Travoprost, Treprostinil, Gemeprost, Nedocromil, Indomethacin, and Sulindac.

[0273] Suitable nucleotide GPCR modulators for use as a payload may include any known or clinically approved nucleotide GPCR modulator. Exemplary nucleotide GPCR modulators suitable as payloads include, by way of example: Adenosine, Caffeine, Regadenoson, Theophylline, Pentoxifylline, Defibrotide, Istradefylline, Tramadol, Gabapentin, Aminophylline, Oxtriphylline, Mefloquine, Lamotrigine, Fostamatinib, Cangrelor, Clopidogrel, Prasugrel, Suramin, Ticagrelor, Ticlopidine, Treprostinil, Epoprostenol, and Promethazine.

[0274] Suitable class B GPCR modulators for use as a payload may include any known or clinically approved class B GPCR modulator. Exemplary class B GPCR modulators suitable as payloads include, by way of example: Calcitonin, Ubrogepant, Rimegepant, Pramlintide acetate, Secretin, Sermorelin, Tesamorelin, Albiglutide, Dulaglutide, Exenatide, Liraglutide, Lixisenatide, Semaglutide, Teduglutide, Glucagon, Abaloparatide, and Teriparatide.Atty. Dkt: CRYS-029WO

[0275] Suitable class C GPCR modulators for use as a payload may include any known or clinically approved class C GPCR modulator. Exemplary class C GPCR modulators suitable as payloads include, by way of example: Acamprosate, Baclofen, Cinacalcet, Etelcalcetide, Gamma hydroxybutyric acid, Progabide, and Vigabatrin.

[0276] Suitable class F GPCR modulators for use as a payload may include any known or clinically approved class F GPCR modulator. Exemplary class F GPCR modulators suitable as payloads include, by way of example: Itraconazole, Sonidegib, Vismodegib, Glasdegib, Halcinonide, and Fluocinonide.

[0277] Hundreds of known and clinically approved GPCR modulators are known in the art and reviewed extensively in several publications, including Hauser et al. 2017 (Hauser, Alexander S., et al. " Trends in GPCR drug discovery: new agents, targets and indications." Nature reviews Drug discovery 16.12 (2017): 829-842) and Yang et al. 2021 (Yang, Dehua, et al. " G protein-coupled receptors: structure-and functionbased drug discovery." Signal transduction and targeted therapy 6.1 (2021): 7), of which the entirety of each is incorporated herein by reference.

[0278] Ion-channel Modulators

[0279] In some embodiments, the payload of an ADC of the present disclosure may be a modulator (e.g., an activator or inhibitor) of an ion-channel. Ion-channels are pore-forming trans-membrane proteins that allow the passage of ions across cell and organelle membranes. Ion-channels play key roles in diverse physiological processes such as regulating ion homeostasis, muscle contraction, nervous system signaling, and T cell activation, among others. Ion-channels may be grouped by the ion-species that the channel is permeable to, e.g., calcium, sodium, potassium, and proton channels. The ion-permeability of many ion-channels is conditionally dependent on surrounding physiological signals or conditions (i.e., the ion channels are “gated”). Ion-channels may be further, or alternatively, grouped and classified by their gating mechanisms, e.g., voltage-gated, ligand-gated, and mechanosensitive ion channels.

[0280] In some cases, the payload of an ADG of the present disclosure may be a calcium channel modulator. Calcium channels play important roles in a variety of physiological processes including muscle contraction, hormone release, neurotransmitter release, and gene transcription. Suitable calcium channel modulators for use as a payload include any known or clinically approved calcium channel inhibitor. Exemplary calcium channel modulators suitable for use as a payload include, without limitation, isradipine, nimodipine, cilnidipine, gabapentin, pregabalin, lamotrigine, topiramate, zonisamide, ethosuximide, ziconotide, valproate, nifedipine, SNX-482, agatoxins (e.g., co-agatoxin IVA), conotoxins (e.g., (B-conotoxin MVIIA), calciseptine, and calcicludine. Calcium channel modulators are known in the art and described, for example, in Zamponi 2016 (Zamponi, Gerald W. " Targeting voltage-gated calcium channels in neurological and psychiatric diseases." Nature reviews Drug discovery 15.1 (2016): 19-34) and Pringos et al. 2011 (Pringos, Emilie, et al. " Peptide neurotoxins that affect voltage-gated calciumAtty. Dkt: CRYS-029WO

[0281] channels: a close-up on co-agatoxins." Toxins 3.1 (2011): 17-42), of which the entirety of each is incorporated herein by reference.

[0282] In some cases, the payload of an ADC of the present disclosure may be a potassium channel modulator. Potassium channels can repolarize or hyperpolarize the membrane of excitable cell following potential firing, and thus work in conjunction with calcium and sodium channels in diverse physiological processes. Suitable potassium channel modulators for use as a payload include any known or clinically approved potassium channel modulator. Exemplary potassium channel modulators suitable for use as a payload include, without limitation, amiodarone, dofetilide, sotalol, azimilide, bretylium, clofilium, tedisamil, sematilide, astemizole, imipramine, verapamil, anemone BDS toxins, and mallotoxin.

[0283] Potassium channel modulators are known in the art and described, for example, in Wolff et al. 2009 (Wolff, H., N. A. Castle, and L. A. Pardo. " Voltage-gated potassium channels as therapeutic drug targets." Nat Rev Drug Discov 8 (2009): 982-1001) and Hopkins et al. 1996 (Hopkins, W. F., J. L. Miller, and G. P. Miljanich. " Voltage-gated potassium channel inhibitors." Current Pharmaceutical Design 2.4 (1996): 389-396).

[0284] In some cases, the payload of an ADC of the present disclosure may be a sodium channel inhibitor. Sodium channels and channels are critical for the propagation of action potentials in excitable cells and, like calcium channels, play key roles in diverse physiological processes such as nerve signaling, muscle contraction, melanogenesis, and immune cell maturation, among others. Suitable sodium channel modulators for use as a payload include any known or clinically approved sodium channel modulator. Exemplary sodium channel modulators suitable for use as a payload include, without limitation, ciguatoxins, saxitoxins, gonyautoxins, batrachotoxins, tetrodotoxins, veratridine, grayanotoxin, aconitine, μ-conotoxins, μO-conotoxins, δ-conotoxins, ι-conotoxins, ProTx-I, ProTx-II, ProTx-III, HwTx-IV, ATX-II, and scorpion peptide toxins (ScTxs). Sodium channel modulators are known in the art and have been described, for example, in Cardoso et al. 2018 (Cardoso, Fernanda C., and Richard J. Lewis. " Sodium channels and pain: From toxins to therapies." British journal of pharmacology 175.12 (2018): 2138-2157), the entirety of which is incorporated herein by reference.

[0285] In some embodiments, the payload of an ADC may be a zinc transporter (e.g., a ZIP family transporter) inhibitor. Zinc transporters are key for regulating zinc levels in cells and cell organelles. Zinc is essential for diverse physiological processes including cell growth and proliferation. Suitable zinc transporter inhibitors can include any known zinc channel inhibitors. In some cases, a suitable zinc transporter inhibitor may be a small molecule, a peptide, or an antibody binding domain (e.g., an scFv) that can block or disrupt zinc transport. Exemplary suitable zinc transporter inhibitors are described, for example, in Anzilotti, Consuelo, et al. " An essential role for the Zn2+ transporter ZIP7 in B cell development." Nature immunology 20.3 (2019): 350-361, Woodruff, Grace, et al. " The zinc transporterAtty. Dkt: CRYS-029WO

[0286] SLC39A7 (ZIP7) is essential for regulation of cytosolic zinc levels." Molecular pharmacology 94.3 (2018): 1092-1100, of which the entirety of each is incorporated herein by reference.

[0287] Kvl modulators

[0288] In some embodiments, the payload of an ADC of the present disclosure may be a Kvl family ion channel modulator, e.g., a Kvl.3 modulator. In some embodiments, the Kvl.3 modulator may be a small molecule. Suitable small molecule modulators of Kvl.3 include, without limitation, PAP-1, curcumin, bergapten, and PAPTP. In some cases, the Kvl.3 modulator is a peptide toxin. Suitable Kvl.3 peptide toxin modulators include, without limitation, BmKTX, ShK, OSK1, mokal, HsTxl, and derivatives thereof. BmKTX is a toxin derived from the scorpion Buthus martensi. Shk is a toxin derived from the sea anemone Stichodactyla helianthus. OSK1 is a toxin derived from the scorpion Orthochirus scrobiculosus. Mokal is a toxin derived from the scorpion Centruroides elegans. HsTxl is a toxin derived from the scorpion Heterometrus spinnifer. Peptide toxin Kvl.3 channel inhibitors are known in the art and described, for example, in Gubic, Spela, et al. " Discovery of Kvl. 3 ion channel inhibitors: Medicinal chemistry approaches and challenges." Medicinal research reviews 41.4 (2021): 2423-2473, the disclosure of which is incorporated by reference herein.

[0289] ADC Linkers

[0290] The ADCs of the present disclosure comprise an antibody that is covalently linked to the payload via a linker. Linkers link payloads to the antibody through a covalent bond to the antibody at a first location of the linker and a covalent bond to the payload at a second location of the linker. Depending on the choice of linker, payload and antibody attachment site, various conjugation chemistries can be used to covalently attach linkers to the payload and antibody.

[0291] Linkers may be broadly grouped into cleavable and non-cleavable linkers. The stability of the linker during circulation is critical for the controlled and targeted release of the payload to target cells and prevent off-target activity. Non-cleavable linkers require proteolysis of the antibody for release of the payload (e.g., lysosomal proteolysis of the antibody following internalization). Cleavable linkers enable the payload to be released from the ADC without proteolytic cleavage of the antibody, the cleavage of the linker being triggered by specific chemical or enzymatic cues (e.g., a chemical or enzymatic cue characteristic of a tumor microenvironment) to release the payload at its intended site of action.

[0292] Suitable linkers can vary in length, hydrophilicity / hydrophobicity, and flexibility or can be comprised of distinct segments that each vary in one or more of the aforementioned properties. The properties of the linker can be varied in accordance with the properties of the payload. A hydrophilic linker, for example, may be chosen to pair with a hydrophobic payload to improve solubility and reduce aggregation of ADCs. In addition, a linker may be monovalent (i.e., link a single payload molecule to aAtty. Dkt: CRYS-029WO

[0293] single site on the antibody) or polyvalent / branched (i.e., link more than one payload molecule to a single site on the antibody).

[0294] ADC linkers have been generally reviewed in several publications, including: Jain et al. 2025 (Jain, Nareshkumar, et al. " Current ADC linker chemistry." Pharmaceutical research 32.11 (2015): 3526-3540), Maecker et al. 2023 (Maecker, Heather, et al. " Exploration of the antibody-drug conjugate clinical landscape." MAbs. Vol. 15. No. 1. Taylor & Francis, 2023), Sasso et al. 2023 (Sasso, Janet M., et al. " The Evolving Landscape of Anlibody-Drug Conjugates: In Depth Analysis of Recent Research Progress." Bioconjugate Chemistry 34.11 (2023): 1951-2000), Sheyi et al. 2022 (Sheyi, Rotimi, Beatriz G. de la Torre, and Fernando Albericio. " Linkers: An assurance for controlled delivery of antibody-drug conjugate." Pharmaceutics 14.2 (2022): 396), and Theocharopoulos et al. 2021 (Theocharopoulos.

[0295] Charalampos, et al. " Antibody-drug conjugates: Functional principles and applications in oncology and beyond." Vaccines 9.10 (2021): 1111), the entirety of each being incorporated herein by reference.

[0296] Native and non-specific Attachment Sites

[0297] In some embodiments the payload may be chemically conjugated to a native amino acid residue of the antibody (i.e., an amino acid residue that was not engineered into the antibody for the attachment of a linker) via a linker moiety. Advantageously, the use of native amino acid residues as attachment sites requires no additional modification of the amino acid sequence of the antibody to enable attachment of the linker. In some cases, the conjugation of the linker and payload to native residues of the antibody is heterogeneous. In other words, the site of attachment and the total number of conjugated linkers with payloads can vary across ADCs in a given batch.

[0298] In certain embodiments, the native amino acid residue is lysine, where the linker is covalently attached via the primary amine of the lysine residue. In such cases, the linker and payload may be reacted with the lysine via amide coupling using an activated carboxylic ester in the linker. For example, the primary amine of the lysine can be reacted with N-hydroxy succinimide (NHS) esters introduced into the linker, forming a stable amide bond. Exemplary linker types that may be conjugated to a lysine residue include, without limitation, N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) linkers, sulfo-SPDB linkers, maleimidomethyl cyclohexane- 1 -carboxylate (MCC), 4-(4-acetylphenoxy)butanoic acid (AcBut) linkers, and derivatives thereof.

[0299] In certain embodiments, the native amino acid residue is cysteine, where the linker is covalently attached via the thiol group of the cysteine residue. In such cases, under controlled conditions, the antibody may be interchain disulfide-bonds can be selectively reduced to generate reactive thiol groups in surface cysteine residues, while intrachain-disulfides remain intact. The free thiol groups may then serve as reactive attachment sites to conjugate the linker via a variety of chemical reactions. For example, the linker may be reacted with the free thiol group by Michael addition, a-halo carbonyl alkylation, orAtty. Dkt: CRYS-029WO

[0300] disulfide formation. Exemplary linker types that may be conjugated to a cysteine residue include, without limitation, maleimidocaproyl (MC) linkers, maleimidomethyl cyclohexane- 1 -carboxylate (MCC) linkers, and derivatives thereof.

[0301] Available conjugation chemistries for attaching linkers to lysine and cysteine residues of an antibody are known in the art and reviewed, for example, in Lu et al. 2016 (Lu, Jun, et al. " Linkers having a crucial role in antibody-drug conjugates." International journal of molecular sciences 17.4 (2016): 561), McDonagh et al. 2006 (McDonagh, Charlotte F., et al. " Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment." Protein Engineering, Design and Selection 19.7 (2006): 299-307), and Sun et al. 2005 (Sun, Michael MC, et al. " Reduction- alkylation strategies for the modification of specific monoclonal antibody disulfides." Bioconjugate chemistry 16.5 (2005): 1282- 1290). In addition, methods for conjugating linkers to cysteine residues of an antibody are described, for example, in WO 2014 / 197612.

[0302] Engineered and Site-Specific Attachment Sites

[0303] In some embodiments the payload may be conjugated via a linker to an engineered amino acid residue. In certain embodiments the engineered amino acid may be a cysteine residue, which can be introduced into specific sites of the antibody via recombinant methods. Site-specificity may be achieved, for example, by substituting a cysteine residue in a particular location to ensure that the cysteine is unpaired (i.e., not available to form an intra- or inter-chain disulfide with a spatially adjacent cysteine). Exemplary sites for insertion of cysteine substitutions include the constant regions and / or Fc regions of an antibody. Methods for inserting cysteine substitutions in an antibody are known in the art and described, for example in Lyons et al. 1990 (Lyons, Alan, et al. " Site-specific attachment to recombinant antibodies via introduced surface cysteine residues." Protein Engineering, Design and Selection 3.8 (1990): 703-708), WO 2011 / 005481, WO2014 / 124316, and WO 2015 / 138615, of which the entirety of each is incorporated herein by reference.

[0304] In certain embodiments, the engineered amino acid may be a selenocysteine, a cysteine analogue, which may be introduced into specific sites of the antibody via recombinant and / or co-translational methods. The selenol group is more nucleophilic than a thiol group and does not require reduction of the antibody. In such cases, linkers may be attached to engineered cysteines or selenocysteines via the conjugation chemistries for cysteine residues discussed above. In certain embodiments, the engineered amino acid may be a tyrosine.

[0305] In some embodiments, the engineered amino acid may be an unnatural or non-canonical (NCAA) amino acid. By virtue of their orthogonal chemistry, unnatural amino acids enable site-specific conjugation of linkers. Unnatural amino acids include, without limitation, p-acetylphenylalanine (pAcF), p-azidomethyl-L-phenylalanine (pAMF), azido-lysine (AzK). As an example, pAcF residues can beAtty. Dkt: CRYS-029WO

[0306] conjugated specifically to linkers comprising an oxime moiety. As another example, azide containing pAMF residues can be specifically conjugated to an alkyne -containing linker via Click chemistry.

[0307] Chemical conjugation of linkers to unnatural amino acids, among other methods, are known in the art and described, for example, in Dennler et al. 2015 (Dennler, Patrick, Eliane Fischer, and Roger Schibli. " Antibody conjugates: from heterogeneous populations to defined reagents." Antibodies 4.3 (2015): 197-224) and Zimmerman et al. 2014 (Zimmerman ES, Heibeck TH, Gill A, Li X, Murray CJ, Madlansacay MR, et al. Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug Chem. 2014; 25:351-61).

[0308] Cleavable Linkers

[0309] In some embodiments, the ADC linker is selectively cleavable in vivo. Cleavable linkers comprise a selectively cleavable, unstable, or degradable moiety that enables release of the payload at its intended site of action upon a specific stimulus or process, e.g, a stimulus or process characteristic of a tumor microenvironment or an intracellular compartment. In some cases, cleavage of the linker may be triggered by a chemical stimulus, e.g., a change in pH. In other cases, cleavage of the linker may be triggered by an enzyme. As such, cleavable linkers may be utilized to add an additional dimension of target selectivity in addition to the target-specific antibody. Cleavable linkers generally incorporate one or more chemically or enzymatically cleavable moieties, whereas the rest of the linker may remain uncleavable.

[0310] In some embodiments, the cleavable linker may be a pH sensitive (i.e., acid-labile) linker or comprises a pH sensitive moiety. pH sensitive cleavable linkers and pH sensitive cleavable moieties are stable under alkaline conditions but are sensitive to hydrolysis under acidic conditions. For example, the release of a payload from an acid-labile linker is facilitated by the acidic conditions of the endosome and lysosome, encountered by an ADC following internalization via endocytosis. In addition, tumor microenvironments are often acidic, and pH sensitive moieties can offer an additional dimension of target-selectivity for delivery of the cytotoxic payload to tumors. Suitable pH sensitive moieties for incorporation into an acid-labile linker include hydrazone moieties, which are hydrolyzed under acidic conditions. Additional acid-labile moieties that may be incorporated into a pH sensitive include carbonate and cis-aconityl moieties. Exemplary cleavable linker types that incorporate pH sensitive moieties include, without limitation, AcBut-like linkers incorporating a hydrazone moiety, MC-like linkers incorporating a hydrazone moiety, and MCC-like linkers incorporating a hydrazone moiety. For example, suitable acid-labile linkers incorporating an acid-labile hydrazone moiety include the acid-labile linkers utilized in gemtuzumab ozogamicin, inotuzumab ozogamicin, PF-06647263, CMD-193, CMB-401, SGN-15, milatuzumab doxorubicin.Atty. Dkt: CRYS-029WO

[0311] In some embodiments, the cleavable linker may be a reducible or glutathione sensitive linker or comprises a reducible or glutathione sensitive moiety. For example, the release of a payload from a reducible linker into the cytosol of a cell is facilitated by the reducing environment and high glutathione concentration of the cytosol, encountered following internalization of an ADC into the cell. Glutathione is present at much higher concentrations intracellularly than in plasma, thus a marked difference in reduction potential exists intracellularly in comparison to plasma in circulation. In addition, glutathione is released during cell replication and thus proliferating cancer cells exhibit high concentrations of glutathione, offering an additional dimension of target-selectivity for delivery of the cytotoxic payload to proliferating cancer cells. A suitable glutathione sensitive moiety for incorporation into a reducible linker is a disulfide moiety. Exemplary cleavable linker types that incorporate glutathione sensitive moieties include, without limitation, AcBut-like linkers incorporating a disulfide moiety, MC-like linkers incorporating a disulfide moiety, SPDB-like linkers, and sulfo-SPDB-like linkers. For example, suitable reducible linkers incorporating an reducible moiety include the reducible linkers utilized in gemtuzumab ozogamicin, inotuzumab ozogamicin, cantuzumab mertansine, bivatuzumab mertansine, lorvotuzumab mertansine, MLN2704, SAR566658, cantuzumab ravtansine, IMGN388, HKT288, BIIB015, LY3076226, SAR428926, coltuximab ravtansine, AVE9633, DHES0815A, PF-06647263, CMD-193, and CMB-401.

[0312] In some embodiments, the cleavable linker may be a proteolytically-cleavable linker or comprises a proteolytically-cleavable moiety. Proteolytically-cleavable linkers comprise peptide motifs that are specifically targeted and hydrolyzed by intracellular proteolytic enzymes (e.g., proteases). Notably, intracellular proteases, e.g., lysosomal proteases, generally have poor activity in the unfavorably alkaline conditions of circulating plasma in comparison to their activity in the acidic conditions of the endosome and lysosome. For example, the release of a payload from a proteolytically-cleavable linker is facilitated by the high concentration of proteases (e.g., cathepsin B) in the acidic endosomal and lysosomal compartments encountered by an ADC following internalization by endocytosis. In addition, proteases, such as cathepsin B, are often overexpressed in or secreted by tumor cells, offering an additional dimension of target-selectivity for delivery of the cytotoxic payload to cancerous cells. In certain embodiments, the proteolytically-cleavable moiety is a dipeptide moiety. Suitable dipeptide moieties for incorporation into a proteolytically-cleavable linker include, without limitation, valine-citrulline (Val-Cit), valine-alanine (Val-Ala), phenylalanine-valine (Phe-Val), and phenylalanine-lysine (Phe-Lys) dipeptide moieties. The Val-Cit dipeptide moiety, for example, can be hydrolyzed by the cathepsin B protease. In certain embodiments, the proteolytically-cleavable peptide motif is a tetrapeptide. Suitable tetrapeptide moieties for incorporation into a proteolytically-cleavable linker include glycine-phenylalanine-leucine-glycine (Gly-Phe-Leu-Gly) and alanine-leucine-alanine-leucine (Ala-Leu-Ala-Leu)Atty. Dkt: CRYS-029WO

[0313] tetrapeptide moieties. For example, suitable linkers that incorporate proteolytically-cleavable moieties include, without limitation, the dipeptide incorporating linkers utilized in brentuximab vedotin, polatuzumab vedotin, loncastuximab tesirine, enapotamab vedotin, BAY79-4620, losatuxizumab vedotin, indusatumab vedotin, glembatumumab vedotin, samrotamab vedotin, DLYE5953A, sofituzumab vedotin, DMOT4039A, DMUC4064A, lifastuzumab vedotin, sirtratumab vedotin, vandortuzumab vedotin, CDX-014, pinatuzumab vedotin, AGS67E, iladatuzumab vedotin, DFRF4539A, azintuxizumab vedotin, AbGn-107, PF-06650808, PF-06664178, BMS-986183, SC-004, rovalpituzumab tesirine, SC-002, tamrintamab pamozirine, ADCT-502, rolinsatamab talirine, ADCT-401, SC-006, SGN-CD70A, SGN-CD19B, MEDI7247, MEDI2228, vadastuximab talirine, SGN-CD123A, SGN-CD352A, BMS-986148, and MDX- 1203.

[0314] In some embodiments, the cleavable linker may be a glycosidase-cleavable linker or comprise a glycosidase-cleavable moiety. Various glycosidase enzymes, such as β-glucuronidase or β-galactosidase, are localized to the acidic lysosomal compartment of a cell. For example, the release of a payload from a glycosidase-cleavable linker is facilitated by the presence of glycosidase enzymes, such as P-glucuronidase or p-galactosidase, in the lysosomal compartment of a cell encountered by an ADC following internalization by endocytosis. In addition, glycosidase enzymes are often overexpressed in or secreted by tumor cells, offering an additional dimension of target-selectivity for delivery of the cytotoxic payload to cancerous cells. In certain embodiments, the glycosidase-cleavable linker is a P-glucuronidase-cleavable linker or comprises a P-glucuronidase-cleavable moiety. The P-glucuronidase-cleavable moiety includes a P-glucuronide moiety that is specifically targeted and hydrolyzed by P-glucuronidase. For example, a suitable linker that incorporates a P-glucuronidase-cleavable moiety includes, without limitation, the P-glucuronidase-cleavable linker utilized in the ADC SGN-CD48A. In certain embodiments, the glycosidase-cleavable linker is a p-galactosidase-cleavable linker or comprises a P-galactosidase-cleavable moiety. P-galactosidase-cleavable linkers are similar to P-glucuronidase-cleavable linkers, but, instead of a P-glucuronide moiety, utilize a P-galactoside moiety that is hydrolyzed by P-galactosidase.

[0315] In some embodiments, the cleavable linker is a phosphatase-cleavable linker or comprises a phosphatase-cleavable moiety. Acid pyrophosphatases and acid phosphatases are present in the lysosome and hydrolyze pyrophosphates and terminal phosphates, respectively, into alcohols. For example, the release of a payload from a phosphatase-cleavable linker is facilitated by the presence of pyrophosphatase and phosphatase enzymes in the lysosomal compartment of a cell encountered by an ADC following internalization by endocytosis. In certain embodiments, the phosphatase-cleavable moiety is pyrophosphate or phosphate moiety. In some instances, a suitable phosphatase-cleavable linker may be covalently linked to the payload via a pyrophosphate or phosphate moiety.Atty. Dkt: CRYS-029WO

[0316] In certain embodiments, a cleavable linker may incorporate two or more cleavable moieties that are cleaved in response to different stimuli. For example, a cleavable linker may incorporate an acid-labile and a glutathione-sensitive moiety (e.g., a hydrazone and a disulfide). As another example, the cleavable linker may incorporate a proteolytically-cleavable moiety and a phosphatase-cleavable moiety (e.g., a Val-Cit dipeptide and a phosphate moiety). Cleavable linkers are known in the art and described, for example, in Bargh et al. 2019 (Bargh, Jonathan D., et al. " Cleavable linkers in antibody-drug conjugates." Chemical Society Reviews 48.16 (2019): 4361-4374), Sheyi et al. 2022 (Sheyi, Rotimi, Beatriz G. de la Torre, and Fernando Albericio. " Linkers: An assurance for controlled delivery of antibody-drug conjugate." Pharmaceutics 14.2 (2022): 396), and Jain et al. 2025 (Jain, Nareshkumar, et al. " Current ADC linker chemistry." Pharmaceutical research 32.11 (2015): 3526-3540), the entirety of each being incorporated herein by reference.

[0317] Non-Cleavable Linkers

[0318] In some embodiments, the ADC is non-cleavable or substantially non-cleavable in vivo.

[0319] Non-cleavable linkers comprise stable moieties that prevent enzymatic or chemical degradation and ensure higher stability in circulating plasma than cleavable linker counterparts. Release of payloads from ADCs utilizing non-cleavable linkers relies on lysosomal degradation of the antibody following internalization of the ADC. Exemplary non-cleavable linkers include maleimidocaproyl (MC) linkers, maleimidomethyl cyclohexane- 1 -carboxylate (MCC) linkers, N-succinimidyl-4-(maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC) linkers, and derivatives thereof. For example, suitable linkers that incorporate non-cleavable moieties include, without limitation, the non-cleavable linkers utilized in trastuzumab emtansine, PF-06263507, depatuxizumab mafodotin, AGS16F, MEDI547, vorsetuzumab mafodotin, denintuzumab mafodotin, lupartumab amadotin, aprutumab ixadotin, AMG 172, LOP628, laprituximab emtansine, AMG 595, PCA062, AMG 224, BAT8001, and BAT8003. Non-cleavable linkers are known in the art and described, for example, in Sheyi et al. 2022 (Sheyi, Rotimi, Beatriz G. de la Torre, and Fernando Albericio. " Linkers: An assurance for controlled delivery of antibody-drug conjugate." Pharmaceutics 14.2 (2022): 396), and Jain et al. 2025 (Jain, Nareshkumar, et al. " Current ADC linker chemistry." Pharmaceutical research 32.11 (2015): 3526-3540), the entirety of each being incorporated herein by reference.

[0320] Spacers

[0321] In some embodiments, the linker (e.g., a cleavable or non-cleavable linker) may comprise one or more spacer moieties. In some cases, due to the size and / or structure of the payload a spacer may be required to avoid having the payload sterically interfere with the function of a cleavable moiety in the linker, e.g., a cleavable linker where an enzymatically cleavable moiety must remain accessible to the corresponding enzyme. Exemplary spacers for use in a linker include polyethylene glycol (PEG)Atty. Dkt: CRYS-029WO

[0322] polymeric moieties. In some instances, cleavage of a cleavable linker may result in the release of undesirable payload adducts when the payload is directly attached to the linker. In such cases a self-immolative spacer moiety may be used to link the payload to the cleavable linker. Self-immolative spacers can spontaneously decompose following disruption of an adjacently linked cleavable-moiety and allowing the release of a chemically unmodified (i.e., lacking undesirable adducts) payload. An exemplary self-immolative spacer for use in the presently described linkers is the p-aminobenzylcarbamate (PABC) spacer. As an example, a PABC linker may be linked to an adjacent cleavable dipeptide moiety through an amide bond. Following proteolytic cleavage of the amide bond, the PABC linker spontaneously decomposes via a 1,6-elimination reaction to release CO2 and aza-quinone methide and the desired payload. Self-immolative spacers are known in the art and reviewed, for example, in Alouane et al. 2015 (Alouane, Ahmed, et al. " Self-immolative spacers: kinetic aspects, structureproperty relationships, and applications." Angewandte Chemie International Edition 54.26 (2015): 7492-7509), Bargh et al. 2019 (Bargh, Jonathan D., et al. " Cleavable linkers in antibody-drug conjugates." Chemical Society Reviews 48.16 (2019): 4361-4374), Sheyi et al. 2022 (Sheyi, Rotimi, Beatriz G. de la Torre, and Fernando Albericio. " Linkers: An assurance for coni rolled delivery of antibody-drug conjugate." Pharmaceutics 14.2 (2022): 396), of which the entirety of each is incorporated herein by reference.

[0323] Branched / Polyvalent Linkers

[0324] In some embodiments, the linker is a branched or polyvalent linker (i.e., link more than one payload molecule to a single site on the antibody). In some cases, the polyvalent linker is selectively cleavable in vivo. Methods of making and using a variety of polyvalent and branched linkers in ADCs are known in the art and described, for example, in WO 2009 / 073445, WO 2010 / 068795, WO 2010 / 138719, WO 2011 / 120053, WO 2011 / 171020, WO 2013 / 096901, WO 2014 / 008375, WO 2014 / 093379, WO 2014 / 093394, WO 2014 / 093640, WO / 2015 / 054659, WO 2018 / 098269, WO 2018 / 237262, WO 2021 / 142199, of which the entirety of each is incorporated herein by reference.

[0325] Non-covalent linkers

[0326] In some embodiments, the linker may be a non-covalent linker. For example, in some cases, the non-covalent linker may comprise a first member of a specific binding pair which can non-covalently bind to a second member of the specific binding pair that is attached to or incorporated into the payload molecule. Exemplary linkers comprising a first and second member of a specific binding pair, suitable for use in an ADC of the present disclosure, are avidin-biotin linkers. The avidin linker comprises an avidin polypeptide e.g., an avidin, streptavidin, neutravidin) which can bind a biotin moiety attached to or incorporated into the payload molecule (i.e., a biotinylated payload molecule) with high-affinity (e.g., a KD ~ IO10to IO15M). Avidin-based linkers for linking biotinylated payloads to antibodies are known inAtty. Dkt: CRYS-029WO

[0327] the art and described, for example, in Xia et al. 2009 (Xia, Chun-Fang, Ruben J. Boado, and William M. Pardridge. " Antibody-mediated targeting of siRNA via the human insulin receptor using avidin- biotin technology." Molecular pharmaceutics 6.3 (2009): 747-751), the entirety of which is incorporated herein by reference.

[0328] In some cases, the linker may be an ionic or electrostatic linker. For example, where the payload molecule exhibits a sufficient charge under the relevant physiological conditions, an oppositely charged linker may be used to link the charged payload to the antibody. For instance, where the payload molecule is negatively charged (e.g., as in cases where the payload is a nucleic acid) the linker may be a positively charged linker. Exemplary positively charged linkers for linking negatively charged payload molecules (e.g., nucleic acid payloads) include, without limitation, poly-arginine linkers, poly-lysine linkers, and protamine linkers. Ionic and electrostatic linkers for linking negatively charged payloads (e.g., nucleic acids) to antibodies are known in the art and described, for example, in Song et al. 2005 (Song, Erwei, et al. " Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors." Nature biotechnology 23.6 (2005): 709-717), Chandela et al. 2019 (Chandela, Akash, and Yoshihito Ueno.

[0329] " Systemic delivery of small interfering RNA therapeutics: Obstacles and advances." Reviews in Agricultural Science 7 (2019): 10-28), Shi et al. 2019 (Shi, Sheng-Jia, et al. " Therapeutic effects of human monoclonal PSMA antibody -mediated TRIM24 siRNA delivery in PSMA-positive castrationresistant prostate cancer." Theranostics 9.5 (2019): 1247), and Lu et al. 2013 (Lu, Hua, et al. " Sitespecific antibody-polymer conjugates for siRNA delivery." Journal of the American Chemical Society 135.37 (2013): 13885-13891), of which the entirety of each is incorporated herein by reference.

[0330] In some cases, where the payload is a nucleic acid (e.g., an RNA or DNA oligonucleotide), the linker may be an oligonucleotide designed to specifically hybridize with all, or a portion of, the nucleic acid payload, thereby non-covalently linking the nucleic acid payload to the antibody. Oligonucleotide linkers for linking nucleic acid payloads to an antibody via hybridization are known in the art and described, for example, in Hsu et al. 2020 (Hsu, Nai-Shu, et al. " Development of a versatile and modular linker for antibody-drug conjugates based on oligonucleotide strand pairing." Bioconjugate Chemistry 31.7 (2020): 1804-1811) and Dovgan et al. 2020 (Dovgan, Igor, et al. " On the use of DNA as a linker in antibody-drug conjugates: synthesis, stability and in vitro potency." Scientific Reports 10.1 (2020): 7691), of which the entirety of each is incorporated herein by reference.

[0331] Masked / Prodrug ADCs

[0332] In some embodiments, an ADC of the present disclosure may comprise a masked (i.e., prodrug) antibody. Although the antibody of an ADC provides target-specificity to the associated payload, there is still a potential risk for off-target delivery of the payload, e.g., in cases where the antigen targeted by theAtty. Dkt: CRYS-029WO

[0333] antibody of the ADC is also expressed in non-target cells or non-target tissues (e.g., non-cancerous cells). In such cases, additional selectivity of the ADC may be achieved by masking, blocking, or otherwise preventing the paratope of the antibody from binding its antigen target (i.e., an inactive, prodrug form) when administered, and unmasking, unblocking, or otherwise allowing the paratope of the antibody to bind its cognate antigen (i.e., an active, drug form) when near the target tissue region (e.g., a tumor). In certain embodiments, the masked ADC may comprise an antibody with an anti-idiotypic mask (e.g., an epitope mimetic). In certain embodiments, the masked ADC may comprise an antibody with a steric mask. The un-masking of the antibody (i.e., conversion from the prodrug to the drug form) may be triggered by any suitable stimulus that differentiates the target tissue from non-target tissues.

[0334] Masked and / or prodrug antibodies for use in the ADCs of the present disclosure are known in the art and reviewed, for example, in Lucci et al. 2021 (Lucchi, Roberta, Jordi Bentanachs, and Benjamf Oiler-Salvia. " The masking game: design of activatable antibodies and mimetics for selective iherapeulics and cell control." ACS central science 7.5 (2021): 724-738), the entirety of which is incorporated herein by reference.

[0335] Mask Linkers

[0336] In certain embodiments, un-masking of the antibody may be triggered by a proteolytic enzyme acting on a cleavable linker polypeptide joining the mask to the antibody. In some cases, the cleavable polypeptide linker is a proteolytically cleavable polypeptide. Upon encountering a target-tissue environment characterized by high levels of active proteolytic enzymes, such as a tumor microenvironment, the proteolytically cleavable linker is cleaved and the mask is released, enabling the antibody to bind its target-antigen. The proteolytically cleavable polypeptide linker may comprise any suitable proteolytic enzyme substrate sequence. Suitable amino acid sequences for use in a proteolytically cleavable linker include substrates for matrix metalloproteinases (MMPs) such as MMP-2 or MMP-9. Cancer-associated proteolytic enzyme substrates are known in the art and described, for example, in Vasiljeva et al. 2019 (Vasiljeva, Olga, et al. " The multifaceted roles of tumor-associated proteases and harnessing their activity for prodrug activation." Biological Chemistry' 400.8 (2019): 965-977) and Sevenich et al. 2014 (Sevenich, Lisa, and Johanna A. Joyce. " Pericellular proteolysis in cancer." Genes & development 28.21 (2014): 2331-2347), of which the entirety of each is incorporated herein by reference Anti-Idiotypic Masks

[0337] In some embodiments, a masked ADC of the present disclosure comprises an antibody with an anti-idiotypic mask. An anti-idiotypic mask is specific to the paratope of the antibody and interacts with the paratope of the antibody specifically and thus prevents binding of the antibody to its cognate epitope.

[0338] In certain embodiments, the anti-idiotypic mask is an cpitopc-mimctic polypeptide, also referred to as a mimotope, that is tethered via a cleavable polypeptide linker to the N-terminus of the antibody andAtty. Dkt: CRYS-029WO

[0339] specifically binds the paratope of the antibody. A mimotope or epitope-mimetic polypeptide structurally mimics the cognate epitope and thereby competes with the cognate epitope for binding to the paratope of the antibody. In some cases, the cleavable polypeptide linker is a proteolytically cleavable polypeptide as described above. Upon encountering a target-tissue environment characterized by high levels of active proteolytic enzymes the proteolytically cleavable linker is cleaved and the mimotope polypeptide is released, enabling the antibody to bind its target-antigen. Mimotope polypeptides for use in a masked antibody are known in the art and described, for example, in WO 2009 / 025846, WO 2010 / 081173, WO 2013 / 163631, and Kavanaugh 2020 (Kavanaugh, W. Michael. " Antibody prodrugs for cancer." Expert opinion on biological therapy 20.2 (2020): 163-171), of which the entirety of each is incorporated herein by reference.

[0340] In certain embodiments, the anti-idiotypic mask is an antigen-binding polypeptide or may comprise an antigen-binding fragment polypeptide that is specific for the paratope or variable-fragment region of the antibody in such a way that the cognate epitope is occluded from binding the paratope of the antibody. In some cases, the antigen-binding polypeptide mask is bi-specific for a bulky serum protein (e.g., albumin) and for the antibody of the ADC, adding an additional steric hindrance to the binding of the ADC antibody to its cognate epitope. In certain embodiments, the antigen-binding polypeptide mask is tethered via a cleavable polypeptide linker to the N-terminus of the antibody. In some cases, the cleavable polypeptide linker is a proteolytically cleavable polypeptide as described above. Upon encountering a target-tissue environment characterized by high levels of active proteolytic enzymes the proteolytically cleavable linker is cleaved and the antigen-binding polypeptide mask is released, enabling the antibody to bind its target-antigen. Antigen-binding polypeptide masks are known in the art and described, for example, in WO 2019 / 222282, WO 2019 / 222283, WO 2023 / 064945, and Borras et al. 2023 (Borras, Anna Mestre, et al. " Generation of an anti-idiotypic affibody-based masking domain for conditional activation of EGFR-targeting." New Biotechnology 73 (2023): 9-18), of which the entirety of each is incorporated herein by reference.

[0341] Steric Masks

[0342] In some embodiments, a masked ADC of the present disclosure comprises an antibody with an steric mask. A steric mask is not specific to the antibody (e.g., it is not specific to tire paratope of variable domains of the antibody) and instead prevents binding of the antibody to its cognate epitope through steric hindrance and physical occlusion of the antibody paratope. In certain embodiments, the steric mask is a polypeptide that specifically binds a bulky serum protein, such that binding of the bulky serum protein sterically inhibits the binding of the antibody to its target antigen, wherein the steric binding mask itself docs not bind to the paratope or variable domain of the antibody. The bulky scrum protein may be any suitably bulky protein found in high abundance in serum. Exemplary bulky serum proteins that aAtty. Dkt: CRYS-029WO

[0343] steric mask polypeptide may specifically bind include, without limitation, albumin, fibrinogen, fibronectin, hemoglobin, transferrin, and immunoglobulins. In some embodiments, the steric mask polypeptide is tethered via a cleavable polypeptide linker to the N-terminus of the antibody. In some cases, the cleavable polypeptide linker is a proteolytically cleavable polypeptide as described above. Upon encountering a target-tissue environment characterized by high levels of active proteolytic enzymes the proteolytically cleavable linker is cleaved and the steric mask polypeptide is released, enabling the antibody to bind its target-antigen. Steric mask polypeptides are known in the art and described, for example, in WO 2013 / 192546 and WO 2014 / 197612A1, of which the entirety of each is incorporated herein by reference.

[0344] Radio-labeled Polypeptides

[0345] In some embodiments, a subject polypeptide comprises, or is conjugated to, a radioactive agent. The radioactive agent may be or may comprise a radionuclide (i.e., a radioactive isotope). In such embodiments, the subject polypeptide may be considered a radiopharmaceutical. The subject polypeptide comprising a radioactive agent may be a subject polypeptide that is labeled with a radionuclide. In some of these embodiments, the radio-labeled subject polypeptides enable targeted delivery of radionuclides to a target cell (e.g., a target cancer cell comprising EGFR) to effect targeted and / or localized radioactive emissions that can kill or inhibit the proliferation of the target cell. For example, a radio-labeled subject polypeptide may bind to a target cell, thus localizing the associated radionuclide to the target cell. Upon radioactive decay of the associated radionuclide, the particles emitted by the decay will be preferentially absorbed by the target cell, thus damaging the target cell. Additionally, or alternatively, the radio-labeled subject polypeptides may enable imaging of the target cell (e.g., a target cancer cell comprising EGFR). For example, a radio-labeled subject polypeptide may bind to a target cell, thus localizing the associated radionuclide to the target cell. Upon radioactive decay of the associated radionuclide, the particles emitted by the decay can be detected to image the target cell or cells (e.g., target cells of a cancerous tumor). In some cases, the subject polypeptide may be labeled directly (e.g., by labeling of an amino acid of the subject polypeptide) or labeled indirectly (e.g., by labeling of a moiety that is conjugated to the subject polypeptide). Radio-labeling of antibodies or other proteins for use as a radiopharmaceutical is described in, e.g., Salih, Suliman, et al. Molecules 27.16 (2022): 5231, and Dhoundiyal, Shivang, et al. European Journal of Medical Research 29.1 (2024): 26., the disclosures of which are incorporated by reference herein.

[0346] Radionuclides

[0347] Suitable radionuclides for labeling a subject polypeptide may be grouped by which particles arc emitted during radioactive decay. Depending on the particular application, the desired radionuclide mayAtty. Dkt: CRYS-029WO

[0348] be chosen based on the particles emitted by the radionuclide (e.g., the mean energy of the emitted particle, the mean distance traveled by the emitted particle), the half-life of the radionuclide, the affinity of the subject polypeptide for a target cell, and / or local tissue vascularity around the target cell (e.g., tissue vascularity around a tumor harboring a target cell).

[0349] Alpha particle emitters

[0350] In some embodiments, a suitable radionuclide may be an alpha particle emitter. Alpha particles are naked4He (helium) nuclei lacking any surrounding electrons (alpha particles are also denoted as He2+). Alpha particles relatively high energy (e.g., as compared to beta particles), usually in the range of 4-9 MeV and have an average path length of 40- 100pm (approximately 1-3 human cell diameters) in tissue. The combination of high energy per particle and short travel distances results alpha particles exhibiting a high amount of energy transferred per unit distance traveled to the surrounding tissue, this characterized as the linear energy transfer (LET) of the alpha particle. Alpha particles exhibit high LET, of around 80 KeV / pm. Due to their high LET, alpha particles exhibit high cellular toxicity. One mechanism of cellular toxicity mediated by alpha particles is the creation of double-strand breaks in genomic DNA. Due to the short distance traveled by alpha particles, cytotoxic damage in surrounding tissues is reduced. Exemplary alpha particle emitting radionuclides suitable for use in a subject radio-labeled polypeptide include, without limitation,211At,213Bi,223Ra, and225Ac. In addition, many radionuclides which produce alpha particles upon decay also produce gamma-ray emissions which can be utilized for imaging.

[0351] Beta particle emitters

[0352] In some embodiments, a suitable radionuclide may be a beta particle emiter. Beta particle emiters have been used in cancer radiation therapies for decades. Beta particles are high energy electrons, in the range of 50-2300 KeV. In contrast to alpha particles, beta particles can travel relatively far within tissues before being absorbed, in the range of 0.05 to 12 mm. The relatively high travel distances offer advantages when targeting solid tumors with higher volumes, as the beta particles can reach cancerous cells in the interior of said tumors. Beta particles have relatively a relatively low LET of around 0.2 KeV / pm, thus requiring more beta particles absorbed to achieve a similar dose of radiation compared to alpha particles. One mechanism of cellular toxicity mediated by beta particles is the creation of singlestrand breaks in DNA. Exemplary beta particle emitting radionuclides suitable for use in a subject radio-labeled polypeptide include, without limitation,131I,32P,89Sr,90Y,133Sm,169Er,177Lu,186Re, and188Re. In addition, some radionuclides which produce beta particles upon decay also produce gamma-ray emissions which can be utilized for imaging.

[0353] Auger electronsAtty. Dkt: CRYS-029WO

[0354] In some embodiments, a suitable radionuclide may emit Auger electrons. Auger electrons are generated from the Auger effect, the energy of the emitted electron depending on the energy of orbital transitions within the atom and ranging from around 4 to 26 Ke V / u m. Auger electrons have short travel distances, in the range of 1 to 1000 nm in tissue, and may be particularly effective at targeting a micro metastasis. Some mechanisms of cellular toxicity mediated by Auger electrons include damage to the plasma membrane and the creation of double-strand breaks in DNA. Exemplary Auger electron emitting radionuclides suitable for use in a subject radio-labeled polypeptide include, without limitation,77Br,111In,123I, and125I.

[0355] Radio-labeling methods

[0356] As discussed above, in some embodiments, a subject polypeptide may labeled directly (e.g., one or more amino acids of the subject polypeptide may be radiolabeled). Methods for radiolabeling proteins (e.g., such as antibodies) are known in the art and described, e.g., in WO1986006839A1, W02000052031 A2, and W01989009405A1, the disclosures of which are incorporated by reference herein. In other embodiments, a radio-labeled small molecule or other chemical moiety can be conjugated to the subject polypeptide by, e.g., the conjugation methods utilized for ADCs as described herein.

[0357] Radiolabeling of small molecules is known in the art and described, e.g., in Edelmann, Martin R. RSC advances 12.50 (2022): 32383-32400, the disclosure of which is incorporated by reference herein.

[0358] Imaging Radio-labeled polypeptides

[0359] In some embodiments, the subject radio-labeled polypeptide may be used to image a target cell (e.g., in vivo of the target cell or a target tumor in a subject). For example, the subject polypeptide can function as a radiotracer that localizes to a tumor and enables imaging of the tumor via a variety of imaging techniques known in the art, such as PET or SPECT (see, e.g., Salsano and Treglia, Research and Reports in Nuclear Medicine. 2013: 3: 9-17).

[0360] Engineered immune receptors

[0361] In some embodiments, a subject polypeptide described herein may be part of an engineered immune cell receptor, e.g., a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR) (including an HLA Independent TCR (HIT)). CAR-Tregs and TCR-Tregs are described in a number of publications, including: Arjomandnejad et al (Biomedicines 2022 10: 287), Skuljec et al (Front Immunol.

[0362] 2017 8: 1125) and Proics et al (Gene Therapy 202330: 309-322). In some cases, the subject polypeptide is in the extracellular domain of an engineered immune cell receptor. In these embodiments, the subject polypeptide may function as an antigen-binding domain of the engineered immune cell receptor. For example, the subject polypeptide may confer specificity for EGFR to a engineered immune cell receptor.Atty. Dkt: CRYS-029WO

[0363] CARs can be designed in several ways (see, generally, e.g., Guedan et al, Methods and Clinical Development 2019 12: 145-156) and may include an extracellular domain that contains an antigen binding domain such as a scFv or nanobody, a hinge, a transmembrane region (which may be derived from CD4, CD8a, or CD28), a costimulatory signaling domain (which may be derived from the intracellular domains of the CD28 family (e.g., CD28 and ICOS) CD2 (US9783591B2) or the tumor necrosis factor receptor (TNFR) family of genes (e.g., 4-1BB, 0X40, or CD27)), and an ITAM domain, e.g., the signaling domain from the zeta chain of the human CD3 complex (CD3zeta). In practice, any of these domains may be a variation of a wild type sequence. In practice, any of these sequences may be a variant of a wild type sequence, e.g., a sequence that is at least 90%, 95%, or 98% identical to a sequence described in WO2014127261, for example. For example, a CAR may have a signaling domain from CD3^ in which two of the three ITAM motifs (the second and third ITAM motifs) have been altered to be non-functional. More specifically, both tyrosine (Y) phosphorylation sites in the second and third ITAMs may be substituted by phenylalanine, thereby rendering those sites incapable of being phosphorylated. This altered CD3^ signaling domain is described in Feucht et al (Nat Med. 201925: 82-88). In any embodiment, the car may have signaling from CD3^, CD28 and 4-1BB.

[0364] The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled (e.g., as described in PCT publication no. WO 2014 / 127261 Al and US Patent Application No. 2015 / 0368342 Al, the disclosures of which are incorporated herein by reference in their entirety). CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013);5(215):215ral72; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2): 151-5; Riddell et al. Cancer.1 (2014) 20(2): 141-4; Pegram et al. Cancer J (2014) 20(2): 127-33; Cheadle et al. Immunol Rev (2014) 257(1):91 - 106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Useful CARs also include the anti-CD19 — 4-1BB —Atty. Dkt: CRYS-029WO

[0365] CD3^ CAR expressed by lentivirus loaded CTL019 (Tisagenlecleucel-T) CAR-T cells as commercialized by Novartis (Basel, Switzerland).

[0366] The terms “T cell receptor” and “TCR” are used interchangeably and will generally refer to a molecule found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR complex is a disulfide -linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (P) chains expressed as part of a complex with CD3 chain molecules. Many native TCRs exist in heterodimeric a or yS forms. The complete endogenous TCR complex in heterodimeric aP form includes eight chains, namely an alpha chain (referred to herein as TCRa or TCR alpha), beta chain (referred to herein as TCRp or TCR beta), delta chain, gamma chain, two epsilon chains and two zeta chains. In some instance, a TCR is generally referred to by reference to only the TCRa and TCRp chains, however, as the assembled TCR complex may associate with endogenous delta, gamma, epsilon and / or zeta chains an ordinary skilled artisan will readily understand that reference to a TCR as present in a cell membrane may include reference to the fully or partially assembled TCR complex as appropriate.

[0367] Recombinant or engineered individual TCR chains and TCR complexes have been developed. References to the use of a TCR in a therapeutic context may refer to individual recombinant TCR chains. As such, engineered TCRs may include individual modified TCRa or modified TCRp chains as well as single chain TCRs that include modified and / or unmodified TCRa and TCRP chains that are joined into a single polypeptide by way of a linking polypeptide

[0368] Any engineered TCR having immune cell activation function can be induced using a method of the present disclosure. Such TCRs include, e.g., antigen-specific TCRs, Monoclonal TCRs (MTCRs), Single chain MTCRs, High Affinity CDR2 Mutant TCRs, CD 1 -binding MTCRs, High Affinity NY-ESO TCRs, VYG HLA-A24 Telomerase TCRs, including e.g., those described in PCT Pub Nos. WO 2003 / 020763, WO 2004 / 033685, WO 2004 / 044004, WO 2005 / 114215, WO 2006 / 000830, WO 2008 / 038002, WO 2008 / 039818, WO 2004 / 074322, WO 2005 / 113595, WO 2006 / 125962; Strommes et al. Immunol Rev. 2014; 257(1): 145-64; Schmitt et al. Blood. 2013; 122(3):348-56; Chapuls et al. Sci Transl Med. 2013; 5(174):174ra27; Thaxton et al. Hum Vaccin Immunother. 2014; 10(11):3313-21 (PMID:25483644); Gschweng et al. Immunol Rev. 2014; 257(l):237-49 (PMID:24329801); Hinrichs et al. Immunol Rev. 2014; 257(l):56-71 (PMID:24329789); Zoete et al. Front Immunol. 2013; 4:268 (PMID:24062738); Marr et al. Clin Exp Immunol. 2012; 167(2):216-25 (PMID:22235997); Zhang et al. Adv Drug Deliv Rev. 2012; 64(8):756-62 (PMID:22178904); Chhabra et al. Scientific World Journal. 2011; 11:121-9 (PMID:21218269); Boulter et al. Clin Exp Immunol. 2005; 142(3):454-60

[0369] (PMID: 16297157); Sami et al. Protein Eng Des Sei. 2007; 20(8):397-403; Boulter et al. Protein Eng. 2003; 16(9):707-l 1; Ashfield et al. IDrugs. 2006; 9(8):554-9; Li et al. Nat Biotechnol. 2005; 23(3):349-Atty. Dkt: CRYS-029WO

[0370] 54; Dunn et al. Protein Sci. 2006; 15(4):710-21; Liddy et al. Mol Biotechnol. 2010; 45(2); Liddy et al. Nat Med. 2012; 18(6):980-7; Oates, et al. Oncoimmunology. 2013; 2(2):e22891; McCormack, et al. Cancer Immunol Immunother. 2013 Apr;62(4):773-85; Bossi et al. Cancer Immunol Immunother. 2014; 63(5):437-48 and Oates, et al. Mol Immunol. 2015 Oct;67(2 Pt A):67-74; the disclosures of which are incorporated herein by reference in their entirety. HLA-Independent TCRs (Eyquem et al. 2022 Feb;28(2):345-352)

[0371] Nucleic acids

[0372] A nucleic acid (e.g., expression vector) comprising a nucleotide sequence encoding a subject polypeptide is also provided. A subject nucleic acid may be produced by any method. Since the genetic code and recombinant techniques for manipulating nucleic acid are known, the design and production of nucleic acids encoding a subject fusion protein is well within the skill of an artisan. In certain embodiments, standard recombinant DNA technology (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N. Y.) methods are used.

[0373] Purification methods

[0374] The present disclosure provides methods of producing a subject polypeptide. The methods generally involve culturing, in a culture medium, a host cell that is genetically modified with one or more nucleic acids (e.g., one or more recombinant expression vectors) comprising nucleotide sequences encoding the polypeptide; and isolating the polypeptide from the genetically modified host cell and / or the culture medium. A host cell that is genetically modified with one or more nucleic acids (e.g., one or more recombinant expression vectors) comprising nucleotide sequences encoding the polypeptide is also referred to as an “expression host.” As noted above, in some cases, the polypeptide may be encoded in separate nucleic acids (e.g., separate recombinant expression vectors). In some cases, the polypeptide may be encoded in a single nucleic acid (e.g., a single recombinant expression vector).

[0375] Isolation of the polypeptide from the expression host cell (e.g., from a lysate of the expression host cell) and / or the culture medium in which the host cell is cultured, can be carried out using standard methods of protein purification.

[0376] For example, a lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. Alternatively, where the polypeptide is secreted from theAtty. Dkt: CRYS-029WO

[0377] expression host cell into the culture medium, the polypeptide can be purified from the culture medium using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some cases, the compositions which are used will comprise at least 80% by weight of the desired product (the polypeptide), at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. The percentages can be based upon total protein.

[0378] Pharmaceutical compositions

[0379] Also provided is a pharmaceutical composition comprising a polypeptide as described above or a polynucleotide encoding the same (e.g., an RNA) and a pharmaceutically acceptable earner. In any of these embodiments, the polypeptide in the composition may be a multispccific binding molecule that comprises the domain and at least one other binding domain that binds to a cancer antigen, an immune cell antigen or a viral antigen (e.g., as in any of the multispecific binding molecules described herein). In some embodiments, the polypeptide may be a BiKE or TriKE, as described above.

[0380] A wide variety of pharmaceutically acceptable ingredients are known in the art and hence are not discussed in detail herein. Moreover, pharmaceutically acceptable ingredients and compositions have been amply described in a variety of publications, including, but not limited to, A. Gennaro (2000) " Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7thed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rded. Amer. Pharmaceutical Assoc. Many other publications describing preparation of biopharmaceutical compositions may be consulted.

[0381] The composition may be formulated according to the various routes of administration described below. Generally speaking, a polypeptide of this disclosure will be an aqueous liquid and typically will be administered via an intravenous infusion. In some cases, the pharmaceutical composition comprising the polypeptide can be admixed with saline (e.g., 0.9% NaCl) prior to IV administration. Thus, the present disclosure provides a sterile composition comprising: a) a polypeptide of the present disclosure; and b) saline (e.g., 0.9% NaCl). Alternatively, it may be administered neat via an intravenous infusion, i.e., without further dilution. Alternatively, the pharmaceutical composition may be formulated so as to be administered by injection.

[0382] Methods of Use

[0383] The present disclosure provides various methods of use for the subject polypeptides. For instance, provided are methods of modulating Kvl.3 activity and methods of modulating Kvl.3 activity in aAtty. Dkt: CRYS-029WO

[0384] subject. Also provided are methods of treating inflammatory diseases in a subject in need thereof and methods of treating a Kvl.3-associated cancer in a subject in need thereof. As another example, provided are methods of killing or inhibiting the proliferation of a target cell and methods of treating cancer in a subject in need thereof. As another example, provided are methods of increase binding between an NK cell and a cancer cell or a virally infected cell. As yet another example, provided are methods of irealing and / or imaging cancer in a subject in need thereof.

[0385] Methods of modulating Kyl,3

[0386] The present disclosure provides methods for modulating Kvl.3 activity, the method comprising contacting a cell comprising Kvl.3 with a anti-Kvl.3 polypeptide of the present disclosure. In certain embodiments, the method comprises inhibiting the activity (e.g., inhibiting K+ permeability) of Kvl.3. In some embodiments, the methods comprise inhibiting Kvl.3 activity (e.g., inhibiting K+ permeability) by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, compared to the degree of Kvl.3 activity (e.g., K+ ion-permeability) in the absence of the subject polypeptide.

[0387] In certain embodiments, the methods comprise contacting a T cell comprising Kvl.3. As discussed herein, Kvl.3 is the only Kvl family ion channel expressed in T cells. In some cases, the T cell may be an effector memory T cell. Memory T cells can be sub-divided into two subsets: central memory T cells and effector memory T cells. In comparison to central memory T cells, have relatively shorter lives and proliferation potential but have a variety of effector functions. Consequently, effector memory T cells primed to respond to re-challenge from an antigen in comparison to central memory T cells. In some embodiments the T cell is an auto-reactive T cell. Autoreactive T cells are T cells that recognize and are activated by self-antigens, e.g., as opposed to a foreign antigen (e.g., a bacterial antigen). In some cases, the T cell is an auto-reach ve effector memory T cell. Auto-reactive effector memory T cells have been implicated as playing key roles in a myriad of autoimmune and inflammatory conditions. In some cases, such effector memory T cells can exhibit a several-fold increase in the expression of Kvl.3. Such autoreactive effector memory T cells are known in the ait and described, for example, in Devarajan, Priyadharshini, and Zhibin Chen. " Autoimmune effector memory T cells: the bad and the

[0388] good." Immunologic research 57 (2013): 12-22 and Cheng, Sixuan, et al. " Voltage-gated potassium channel 1.3: A promising molecular target in multiple disease therapy." Biomedicine & Pharmacotherapy 175 (2024): 116651, the disclosures of which are incorporated by reference herein.

[0389] In some embodiments, the contacting may be done in vitro. In other cases, the contacting may be done in vivo. In some embodiments, the cell is a mammalian cell. In some cases, the mammalian cell is a human cell.Atty. Dkt: CRYS-029WO

[0390] Methods of killing or inhibiting proliferation of a cell

[0391] The present disclosure provides methods for killing or inhibiting the proliferation of a target cell (e.g., a target cell comprising Zip6 or EGFR).

[0392] In certain embodiments, the subject methods comprise contacting a cell comprising Zip6 with an anti-ZIP6 polypeptide of the present disclosure as described above. In some cases, contacting the target cell comprising Zip6 with a subject anti-Zip6 polypeptide may kill the cell. By way of example, in cases where the subject anti-Zip6 polypeptide is a NK cell engager (as described herein), the subject anti-Zip6 polypeptide may bind to both the target cell comprising Zip6 and an NK cell, which may cause activation of the NK cell and subsequent death of the target cell. As another example, in cases where the subject anti-Zip6 polypeptide is an ADC (as described herein) and conjugated to a cytotoxic payload, the subject polypeptide can deliver the cytotoxic payload to a target cell comprising Zip6, which may cause the subsequent death of the target cell. As yet another example, in cases where the subject anti-Zip6 polypeptide comprises, or is conjugated to, a radioactive agent, the subject polypeptide can deliver the radioactive agent to a target cell comprising Zip6, causing the target cell to absorb radioactive emissions from the radioactive agent which may cause the subsequent death of the target cell. In some cases, contacting the target cell comprising Zip6 with a subject anti-Zip6 polypeptide may inhibit proliferation of the cell. By way of example, the anti-Zip6 subject polypeptide may bind to, and interfere with the function of, Zip6 on the target cell, which may subsequently inhibit proliferation of the target cell. As another example, in cases where the subject anti-Zip6 polypeptide is an ADC (as described herein) and conjugated to a cytostatic payload, the subject polypeptide can deliver the cytostatic payload to a target cell comprising Zip6, which may subsequently inhibit proliferation of the target cell.

[0393] In certain embodiments, the subject methods comprise coniacling a cell comprising EGFR with an anti-EGFR polypeptide of the present disclosure as described above. In some cases, contacting the target cell comprising EGFR with a subject anti-EGFR polypeptide may kill the cell. By way of example, in cases where the subject anti-EGFR polypeptide is a NK cell engager (as described herein), the subject polypeptide may bind to both the target cell comprising EGFR and an NK cell, which may cause activation of the NK cell and subsequent death of the target cell. As another example, in cases where the subject anti-EGFR polypeptide is an ADC (as described herein) and conjugated to a cytotoxic payload, the subject polypeptide can deliver the cytotoxic payload to a target cell comprising EGFR, which may cause the subsequent death of the target cell. As yet another example, in cases where the subject anti-EGFR polypeptide comprises, or is conjugated to, a radioactive agent, the subject polypeptide can deliver the radioactive agent to a target cell comprising EGFR, causing the target cell to absorb radioactive emissions from the radioactive agent which may cause the subsequent death of the target cell. In someAtty. Dkt: CRYS-029WO

[0394] cases, contacting the target cell comprising EGFR with a subject anti-EGFR polypeptide may inhibit proliferation of the cell. By way of example, the subject anti-EGFR polypeptide may bind to, and interfere with the function of EGFR on the target cell, which may subsequently inhibit proliferation of the target cell. As another example, in cases where the subject anti-EGFR polypeptide is an ADC (as described herein) and conjugated to a cytostatic payload, the subject polypeptide can deliver the cytostatic payload to a target cell comprising EGFR, which may subsequently inhibit proliferation of the target cell. In some embodiments of the methods, the subject and -EGFR polypeptide can bind to EGFR and block binding of EGFR to its native ligand, epidermal growth factor (EGF).

[0395] In some embodiments of these methods, the target cell is a cancer cell. The cancer cell may be a cell of a solid tumor (e.g., a sarcoma, a carcinoma). In varying embodiments, the cancer cell may be, without limitation, a cell of a(n): breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, melanoma, cervical cancer, uterine cancer, nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, choriocarcinoma, gastinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel- Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms’ tumor, liver cancer, Kaposi's sarcoma, lung cancer, testicular cancer, Hodgkin’s disease, non-Hodgkin’s lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

[0396] In some cases, the contacting may be done in vivo. In some embodiments, the target cell is a mammalian cell. In particular embodiments, the target cell is a human cell.

[0397] Methods of increasing binding between an NK cell and a target cell and methods related thereto The present disclosure provides methods for increasing binding between an NK cell and a target cell. In some embodiments, the method may increase binding between an NK cell (e.g., a CD3-Atty. Dkt: CRYS-029WO

[0398] / CD56+ cell that is also CD7+ / CD127- / NKp46+ / T-bet+ / Eomes+) and a cancer cell. These embodiments may comprise incubating the NK cell and a cancer cell with a subject anti-NKp46 polypeptide, wherein the polypeptide is a multispecific binding molecule that comprises the anti-NKp46 knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell. As noted above, in these embodiments, the polypeptide may bind to both the NK cell and the cancer cell, which may cause activation of the NK cell and subsequent death of the cancer cell. This reaction may occur in vitro, in vivo or ex vivo.

[0399] A method of increasing binding between an NK cell and a virally-infected cell is also provided. This method may comprise incubating the NK cell and a virally-infected cell with a subject anti-NKp46 polypeptide, wherein the polypeptide is a multispecific binding molecule that comprises the anti-NKp46 knob domain and at least one other binding domain that binds to a viral antigen on the virally-infected cell, in these embodiments, the polypeptide may bind to both the NK cell and the virally-infected cell, which may cause activation of the NK cell and subsequent death of the virally-infected cell. This reaction may occur in vitro, in vivo or ex vivo.

[0400] A method of killing a cancer cell is also provided. In these embodiments, the method may comprise contacting the cancer with an NK cell (e.g., a CD3- / CD56+ cell that is also CD7+ / CD127- / NKp46+ / T-bet+ / Eomes+) and a subject anti-NKp46 polypeptide, wherein the polypeptide is a multispecific binding molecule that comprises the anti-NKp46 knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell. As noted above, in these embodiments, the polypeptide may bind to both the NK cell and the cancer cell, which may cause activation of the NK cell and subsequent death of the cancer cell. This reaction may occur in vitro, in vivo or ex vivo.

[0401] A method of killing a virally-infected cell is also provided. In these embodiments, the method may comprise contacting the virally-infected cell with an NK cell and a subject anti-NKp46 polypeptide, wherein the polypeptide is a multispecific binding molecule that comprises the anti-NKp46 knob domain and at least one other binding domain that binds to a viral antigen on the virally-infected cell. In these embodiments, the polypeptide may bind to both the NK cell and the virally-infected cell, which may cause activation of the NK cell and subsequent death of the virally-infected cell. This reaction may occur in vitro, in vivo or ex vivo.

[0402] Methods of treatment

[0403] The present disclosure provides various methods of treatment, e.g., methods of treating inflammatory disease, methods of treating cancer, methods of treating viral infections, combination therapies, etc.

[0404] Methods of treating inflammatory diseaseAtty. Dkt: CRYS-029WO

[0405] The present disclosure provides methods for treating an inflammatory disease in a subject, the methods comprising administering an effective amount of a subject pharmaceutical composition (comprising an anti-Kvl.3 polypeptide as described herein, or a nucleic acid encoding the same) to a subject in need thereof.

[0406] An inflammatory disease may be any disease, disorder, or condition that is characterized or associated with inflammation. The inflammation may be acute or chronic. In some cases, the inflammatory disease is an autoimmune disease, where a subject’s immune system mistakenly targets the subject’s own tissues. Symptoms of inflammatory diseases are wide-ranging and can include pain, swelling, fatigue, gastrointestinal issues, and insomnia. Many other symptoms are more specific to the particular inflammatory disorder and the tissues and organs they affect.

[0407] Inflammatory diseases suitable for treatment with a subject pharmaceutical composition can include, without limitation, allergy, alopecia areata, Alzheimer's disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), contact-mediated dermatitis, delayed type hypersensitivity, fibrosis, glomerulonephritis, inflammatory bone resorption, inflammatory bowel disease, graft-versus host disease, hepatic fibrosis, multiple sclerosis, obesity, Parkinson’s disease, psoriasis, psoriatic arthritis, restenosis, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, systemic lupus erythematosus, systemic sclerosis, type-1 diabetes mellitus, type-2 diabetes mellitus, transplant rejection, ulcerative colitis, and vasculitis.

[0408] Inflammatory conditions may, in some cases, be triggered or exacerbated by hyperactive immune cells (e.g., T cells). As discussed herein, T cell activation is dependent on the activity of Kvl.3 to maintain the proper membrane necessary for Ca2+ influx. In the case of autoimmune diseases and conditions, the hyperactive immune cell may be an auto-reactive T cell. Autoreactive T cells are T cells that recognize and are activated by self-antigens, e.g., as opposed to a foreign antigen (e.g., a bacterial antigen). In some cases, the T cell is an auto-reactive effector memory T cell. Auto-reactive effector memory T cells have been implicated as playing key roles in a myriad of autoimmune and inflammatory conditions. In some cases, such effector memory T cells can exhibit a several-fold increase in the expression of Kvl.3. Such auto-reactive effector memory T cells are known in the art and described, for example, in Devarajan, Priyadharshini, and Zhibin Chen. " Autoimmune effector memory T cells: the bad and the good." Immunologic research 57 (2013): 12-22 and Cheng, Sixuan, et al. " Voltage-gated potassium channel 1.3: A promising molecular target in multiple disease therapy." Biomedicine & Pharmacotherapy 175 (2024): 116651, the disclosures of which are incorporated by reference herein.

[0409] In some embodiments, the methods of treating an inflammatory disease in a subject comprise modulating Kvl.3 activity in a T cell of the subject. In certain embodiments, the method of treatment comprises reducing activation of a T cell in a subject. For example, in some cases, the modulation ofAtty. Dkt: CRYS-029WO

[0410] Kvl.3 in a T cell will lead to a reduction in the activation of said T cell. In certain embodiments, the T cell may be an effector memory T cell or an auto-reactive T cell. In some cases, the T cell is an auto-reactive effector memory T cell.

[0411] In some embodiments, the subject may have an acute lung disease, e.g., acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) (which may have a variety of causes, including pneumonia or viral infection). In other embodiments the subject may have chronic obstructive pulmonary disease or interstitial lung disease (ILD), which describes a group of chronic disorders that involve inflammation and scarring in the lung.

[0412] In these embodiments, the interstitial lung disease may have been caused by (i) an autoimmune disease (lupus, rheumatoid arthritis, sarcoidosis, dermatomyositis, polymyositis, mixed connective tissue disease, Sjogren's syndrome, and scleroderma, etc.), (ii) exposure to a foreign substance (e.g., dust, fungus, mold, silica dust, asbestos fibers, grain dust, bird and animal droppings, coal dust, or cotton dust, for example), (iii) ingestion of a medicine (such as nitrofurantoin, sulfonamides, bleomycin, amiodarone, methotrexate, gold, infliximab, etanercept, and other chemotherapy medicines), (iv) radiation treatment to the chest; or (v) an infection (e.g., CO VID or influenza).

[0413] By treatment is meant at least an amelioration of the symptoms associated with the inflammatory condition afflicting the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the inflammatory condition being treated, such as an autoimmune disorder or condition. As such, treatment also includes situations where the inflammatory condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the subject no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.

[0414] In some embodiments, the treatment may comprise inhibiting Kvl.3 activity (e.g., inhibiting K+ permeability) of a target T cell by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, compared to the degree of Kvl.3 activity (e.g., K+ ion-permeability) in the target T cell in the absence of the subject polypeptide.

[0415] Methods of treating cancer

[0416] In certain embodiments, the present disclosure provides methods of treating cancer in a subject, the methods comprising administering an effective amount of a pharmaceutical composition (comprising an anti-Zip6 polypeptide of the present disclosure or a nucleic acid encoding the same) to a subject in need thereof. Cancers that can be treated with these methods include, without limitation, breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, melanoma, cervical cancer, uterine cancer,Atty. Dkt: CRYS-029WO

[0417] nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Ilippel- Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing’s sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi’s sarcoma, lung cancer, testicular cancer, Hodgkin’s disease, non-Hodgkin’s lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer. Moreover, cancers that can be treated with the aforementioned methods can include any cancer comprising cells comprising Zip6. In some embodiments, the method of treatment comprises killing or inhibiting the proliferation of a target cancer cell in the subject. In some cases, contacting the target cancer cell (e.g., a target cancer cell comprising Zip6) in a subject with the anti-Zip6 polypeptide may kill the cancer cell. By way of example, in cases where the anti-Zip6 polypeptide is a NK cell engager (as described herein), the anti-Zip6 polypeptide may bind to both the target cancer cell and an NK cell, which may cause activation of the NK cell and subsequent death of the cancer cell. As another example, in cases where the anti-Zip6 polypeptide is an ADC (as described herein) and conjugated to a cytotoxic payload, the polypeptide can deliver the cytotoxic payload to the target cancer cell, which may cause the subsequent death of the cancer cell. As yet another example, in cases where the subject anti-Zip6 polypeptide comprises, or is conjugated to, a radioactive agent, the subject polypeptide can deliver the radioactive agent to a target cell, causing the target cell to absorb radioactive emissions from the radioactive agent which may cause the subsequent death of the target cell. In some cases, contacting the target cancer cell (e.g., a target cancer cell comprising Zip6) in a subject with a anti-Zip6 polypeptide may inhibit proliferation of the cancer cell. By way of example, the polypeptide may bind to, and interfere with the function of, Zip6 on the target cancer cell, which may subsequently inhibit proliferation of the cancer cell. As another example, in cases where the anti-Zip6 polypeptide is an ADC (as described herein) and conjugated to a cytostatic payload, theAtty. Dkt: CRYS-029WO

[0418] polypeptide can deliver the cytostatic payload to a target cancer cell, which may subsequently inhibit proliferation of the target cell.

[0419] In certain embodiments, the provided methods comprise administering a subject anti-NKp46 polypeptide or a polynucleotide encoding the same (e.g., an RNA) (e.g., as comprised in a subject pharmaceutical composition) to a subject that has cancer, wherein the anti-NKp46 polypeptide is a multispecific binding molecule that comprises the anti-NKp46 knob domain and at least one other binding domain that binds to a cancer antigen on a cancer cell. The cancer antigen may be selected based on the cancer being treated. Suitable cancer antigen / cancers include: CD19, CD20 or BCMA (for B-cell malignancies, including acute myeloid leukemia (AML), multiple myeloma (MM), B-cell acute lymphoblastic leukemia (B-ALL), lymphoma, etc.), ALPP (for, e.g., ovarian and endometrial cancer), CS1 (SLAMF7) (for, e.g., R / R multiple myeloma), CLDN18.2 (for, e.g., pancreatic and gastric adenocarcinoma, gastric cancer), AXL (for, e.g., renal cell carcinoma), ROR2 (for, e.g., renal cell carcinoma), TM4SF1 (for, e.g., advanced solid tumors), ICAM-1 (for, e.g., anaplastic thyroid cancer), L1CAM (CD171) (for, e.g., neuroblastoma, ganglioneuroblastoma), CD4 (for, e.g., T-cell lymphoma, T-cell leukemia), CD5 (T-cell acute lymphoblastic lymphoma, non-Hodgkin T-cell lymphoma), CD7 (for, e.g., T-cell acute lymphoblastic lymphoma, T-cell acute lymphoblastic leukemia, non-Hodgkin T-cell lymphoma), CD10 (for, e.g., CD19-negative B-cell malignancies), CD38 (for, e.g., CD19-negative B-cell malignancies), CFA (for, e.g., lung, colorectal, liver, pancreatic, gastric, and breast cancer), FLT3 (for, e.g., R / R acute myeloid leukemia), CD70 (for, e.g., pancreatic, renal cell, ovarian, and breast cancer, melanoma), CD30 (for, e.g., Hodgkin lymphoma), CD37 (for leukemia and B-cell, T-cell, and Non-Hodgkin lymphoma), CD147 (for, e.g., glioblastoma). Subjects suitable for treatment with the aforementioned method include individuals who have cancer, including individuals who have been diagnosed as having cancer, individuals who have been treated for cancer but who failed to respond to the treatment, and individuals who have been treated for cancer and who initially responded but subsequently became refractory to the treatment and / or whose disease progressed while on the prior Irealmeni. Cancers that can be treated with the aforementioned method include any cancer that can be targeted with the present anti-NKp46 polypeptide. Cancers that can be treated with a method include carcinomas, sarcomas, melanoma, leukemias, lymphomas and multiple myeloma. Cancers that can be treated with the aforementioned method include solid tumors, and cancers that begin in blood-forming tissue, i.e., hematological cancers such as leukemias, lymphomas and multiple myeloma. Cancers that can be treated with a method include metastatic cancers. Carcinomas that can treated by the aforementioned method include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma,Atty. Dkt: CRYS-029WO

[0420] colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma. Sarcomas that can be treated by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Other solid tumors that can be treated by the aforementioned method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. Leukemias that can be amenable to therapy by the aforementioned method include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukemias (neoplastic transformation of a multipotential hematopoietic stem cell or a heinalopoieiic cell of restricted lineage potential; c) chronic lymphocytic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemias (characterized by accumulation of lymphoblasts). Lymphomas that can be heated using a subject method include, but are not limited to, B-cell lymphomas (e.g., Burkitt’s lymphoma); Hodgkin's lymphoma; non-Hodgkin's lymphoma, and the like. Other cancers that can be treated according to the aforementioned methods include atypical meningioma, islet cell carcinoma, medullary carcinoma of the thyroid, mesenchymoma, hepatocellular carcinoma, hepatoblastoma, clear cell carcinoma of the kidney, and neurofibroma mediastinum.

[0421] In certain embodiments, the present disclosure provides methods of treating cancer in a subject, the methods comprising administering an effective amount of a pharmaceutical composition comprising an anti-EGFR polypeptide of the present disclosure to a subject in need thereof. Cancers that can be treated with the aforementioned methods include, without limitation, breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, melanoma, cervical cancer, uterine cancer, nasopharyngeal cancer, pharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, lung cancer, bowel cancer, gastrointestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma,Atty. Dkt: CRYS-029WO

[0422] prolactinoma, T-cell leukemia / lymphoma, hematological cancer, neuroma, von Hippel- Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer, lymphoid cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, head and neck cancer, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer. In some embodiments, the method of treatment comprises killing or inhibiting the proliferation of a target cancer cell in the subject. In some cases, contacting the target cancer cell (e.g., a target cancer cell comprising EGFR) in a subject with the anti-EGFR polypeptide may kill the cancer cell. By way of example, in cases where the anti-EGFR polypeptide is a NK cell engager (as described herein), the polypeptide may bind to both the target cancer cell and an NK cell, which may cause activation of the NK cell and subsequent death of the cancer cell. As another example, in cases where the anti-EGFR polypeptide is an ADC (as described herein) and conjugated to a cytotoxic payload, the polypeptide can deliver the cytotoxic payload to the target cancer cell, which may cause the subsequent death of the cancer cell. As yet another example, in cases where the subject anti-EGFR polypeptide comprises, or is conjugated to, a radioactive agent, the subject polypeptide can deliver the radioactive agent to a target cell comprising EGFR, causing the target cell to absorb radioactive emissions from the radioactive agent which may cause the subsequent death of the target cell. In some cases, contacting the target cancer cell (e.g., a target cancer cell comprising EGFR) in a subject with a anti-EGFR polypeptide may inhibit proliferation of the cancer cell. By way of example, the anti-EGFR polypeptide may bind to, and interfere with the function of, EGFR on the target cancer cell, which may subsequently inhibit proliferation of the cancer cell. As another example, in cases where the anti-EGFR polypeptide is an ADC (as described herein) and conjugated to a cytostatic payload, the polypeptide can deliver the cytostatic payload to a target cancer cell, which may subsequently inhibit proliferation of the target cell. In some embodiments of the methods, the subject polypeptide can bind to EGFR and block binding of EGFR to its native ligand, epidermal growth factor (EGF).Atty. Dkt: CRYS-029WO

[0423] Combination therapies

[0424] In some embodiments of the methods for treating a cancer in a subject, the polypeptide (e.g., anti-Zip6, anti-NKp42, or anti-EGFR polypeptide) or a polynucleotide encoding the same (e.g., an RNA) (comprised, e.g., in a subject pharmaceutical composition) may be administered along with at least one additional therapeutic agent or therapeutic treatment (together or sequentially). Suitable additional therapeutic agents include, but are not limited to, a small molecule cancer chemotherapeutic agent, and an immune checkpoint inhibitor. Suitable additional therapeutic treatments include, e.g., radiation, surgery (e.g., surgical resection of a tumor), and the like.

[0425] The treatment method can comprise co-administration of a polypeptide (e.g., anti-Zip6, anti-NKp42, or anti-EGFR polypeptide) or a polynucleotide encoding the same (e.g., an RNA) (comprised, e.g., in a subject pharmaceutical composition) and at least one additional therapeutic agent. By “coadministration” is meant that both a polypeptide and at least one additional therapeutic agent are administered to an individual, although not necessarily at the same time, in order to achieve a therapeutic effect that is the result of having administered both the polypeptide and the at least one additional therapeutic agent. The administration of the polypeptide and the at least one additional therapeutic agent can be substantially simultaneous, e.g., the polypeptide can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of administration of the at least one additional therapeutic agent. In some cases, a polypeptide of the present disclosure (e.g., anti-Zip6, anti-NKp46, or anti-EGFR polypeptide) is administered to an individual who is undergoing treatment with, or who has undergone treatment with, the at least one additional therapeutic agent. The administration of the polypeptide can occur at different times and / or at different frequencies.

[0426] In some cases, the subject is an individual undergoing treatment with an immune checkpoint inhibitor. In some cases, the subject is an individual who has undergone treatment with an immune checkpoint inhibitor, but whose disease has progressed despite having received such treatment. In some cases, the subject is an individual who is undergoing treatment with, or who has undergone treatment with, a cancer chemotherapeutic agent. In some cases, the subject is an individual who is preparing to undergo treatment with, is undergoing treatment with, or who has undergone treatment with, an immune checkpoint inhibitor. In some cases, the subject is an individual who is preparing to undergo treatment with, is undergoing treatment with, or who has undergone treatment with, a cancer chemotherapeutic agent, radiation treatment, surgery, and / or treatment with another therapeutic agent. In some cases, a pharmaceutical composition comprising the polypeptide (e.g., anti-Zip6, anti-NKp42, or anti-EGFR polypeptide) is administered in the adjuvant or neoadjuvant setting.Atty. Dkt: CRYS-029WO

[0427] Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, 0X40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3. TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, 0X40, GITR, CD 122 and CD 137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT and VISTA.

[0428] Co-therapies include for example, (a) anthracycline therapy (e.g., by administering daunomycin, doxorubicin, or mitoxantrone), (b) alkylating agent therapy (e.g., by administering mechlorethane, cyclophosphamide, ifosfamide, melphalan, cisplatin, carboplatin, nitrosourea, dacarbazine and procarbazine or busulfan), (c) topoisomerase II inhibitor therapy (e.g., by administering etoposide or teniposide), (d) bleomycin therapy, (e) anti-metabolite therapy (e.g., by administering methotrexate, 5-fluorouracil, cytarabine, 6-mercaptopurine or 6-thioguanine), (f) vinca alkyloid therapy (e.g., by administering vincristine or vinblastine), (g) steroid therapy (e.g., by administering prednisone or dexamethasone and (h) radiation treatment, etc. Alternative therapies include targeted therapies and nontargeted chemotherapies, where targeted therapy includes treatment with erlotinib (Tarceva), afatinib (Gilotrif), gefitinib (Iressa) or osimertinib (Tagrisso) which may be administered to patients having an activating mutation in EGFR, crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa) or brigatinib (Alunbrig) which may be administered to patients having an ALK fusion, crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), ropotrectinib (TPX-0005), DS-6051b, ceritinib, ensartinib or cabozantinib which may be administered to patients having an ROS1 fusion, or dabrafenib (Tafinlar) or trametinib (Mekinist) which may be administered to patients having an activating mutation in BRAF. Many other actionable mutations are known. If the patient is going to be switched to a non-targeted chemotherapy, the therapy may be, for example, a platinum-based doublet chemotherapy (in which the platinum-based doublet chemotherapy may comprise a platinum-based agent selected from cisplatin (CDDP), carboplatin (CBDCA), and nedaplatin (CDGP)) and one third-generation agent (selected from docetaxel (DTX), paclitaxel (PTX), vinorelbine (VNR), gemcitabine (GEM), irinotecan (CPT-11), pemetrexed (PEM), and tegafur gimeracil oteracil (SI)).

[0429] Methods of treating viral infections

[0430] The present disclosure provides methods for treating viral infections in a subject. In certain embodiments, the method of treatment may comprise administering an anti-NKp46 polypeptide or aAtty. Dkt: CRYS-029WO

[0431] polynucleotide encoding the same (e.g., an RNA) (comprised, e.g., in a subject pharmaceutical composition) to a subject that has a viral infection, wherein the polypeptide is a multispecific binding molecule that comprises the anti NKp46 knob domain and at least one other binding domain that binds to a viral antigen on a virally-infected cell. The viral antigen recognized by the at least one other binding domain may be selected based on the viral infection of the subject. Viral infections that may be treated with the aforementioned methods may include, e.g., viral infections caused by chicken pox (varicella), coronaviruses (e.g., COVID-19), cytomegalovirus (CMV), dengue virus, influenza, human immunodeficiency virus (HIV), human papillomavirus (HPV), measles, ebolavirus, chikungunya virus, mpox, norovirus, mumps vims, respiratory syncytial virus (RSV), rotavirus, and zika virus, among others. In some embodiments, the methods of treatment comprise contacting the virally-infected cell with an NK cell and a subject anti-NKp46 polypeptide. In these embodiments, the polypeptide may bind to both the NK cell and the virally-infected cell, which may cause activation of the NK cell and subsequent death of the virally-infected cell.

[0432] Regarding the methods of treatment described herein, in certain embodiments an “effective amount” of a polypeptide or a polynucleotide encoding the same (e.g., an RNA) (comprised, e.g., in a subject pharmaceutical composition) is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual. For example, in some cases, an “effective amount” of a polypeptide or a polynucleotide encoding the same (e.g., an RNA) is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the number of cancer cells in the individual before administration of the polypeptide, or in the absence of administration with the polypeptide. In some cases, an “effective amount” of a polypeptide or a polynucleotide encoding the same (e.g., an RNA) is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual to undetectable levels. In some cases, an “effective amount” of a polypeptide or a polynucleotide (e.g., an RNA) encoding the same ) (comprised, e.g., in a subject pharmaceutical composition) is an amount that, when administered in one or more doses to an individual in need thereof, reduces the tumor mass / tumor volume in the individual. In some cases, an “effective amount” of a polypeptide is an amount that, when administered in one or more doses to an individual in need thereof, increases survival time of the individual. For example, in some cases, an “effective amount” of a polypeptide or a polynucleotide encoding the same (e.g., an RNA) is an amount that, when administered in one or more doses to an individual in need thereof, increases survival time of the individual by at least 1 month, at least 2 months, at least 3 months, from 3 months to 6 months, from 6 months to 1 year, from 1 year to 2 years, from 2Atty. Dkt: CRYS-029WO

[0433] years to 5 years, from 5 years to 10 years, or more than 10 years, compared to the expected survival time of the individual in the absence of administration with the polypeptide or a polynucleotide encoding the same (e.g., an RNA). in certain embodiments an “effective amount” of a polypeptide or a polynucleotide encoding the same (e.g., an RNA) (comprised, e.g., in a subject pharmaceutical composition) is an amount that, when administered in one or more doses to an individual in need thereof, reduces the level of inflammation and / or inflammatory markers in the individual. In some cases, an “effective amount” of a polypeptide or a polynucleotide encoding the same (e.g., an RNA) is an amount that, when administered in one or more doses to an individual in need thereof, reduces the level of C-reactive protein in the serum of the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%>, at least 80%, at least 90%, or at least 95%, compared to the level of CRP in the serum of the individual before administration of the polypeptide, or in the absence of administration with the polypeptide. In certain embodiments, an “effective amount” of a polypeptide or a polynucleotide encoding the same (e.g., an RNA) (comprised, e.g., in a subject pharmaceutical composition) is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of virally-infected cells in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the number of virally infected cells in the individual before administration of the polypeptide, or in the absence of administration with the polypeptide.

[0434] A suitable dosage for the treatments described herein can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently. A subject polypeptide described herein may be administered in amounts between 1 ng / kg body weight and 20 mg / kg body weight per dose, e.g. between 0.1 mg / kg body weight to 10 mg / kg body weight, e.g. between 0.5 mg / kg body weight to 5 mg / kg body weight; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it can also be in the range of 1 μg to 10 mg per kilogram of body weight per minute.

[0435] A subject pharmaceutical composition described herein may be administered to the subject via any conventional or pharmaceutically acceptable route. Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, intraventricular, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. As noted above, a pharmaceutical composition comprising a polypeptide or a polynucleotide encoding the same (e.g., anAtty. Dkt: CRYS-029WO

[0436] RNA) typically will be administered intravenously, but may also be administered by other routes that involve injection.

[0437] In any embodiment, the polypeptide may be administered to the subject by administering a polynucleotide (e.g., an RNA) encoding the subject polypeptide to a patient. See, e.g., van Hoecke et al (Journal of Translational Medicine 2019 17: 54, Deal et al (Vaccines 2021 9: 108), Tai et al (Nature Communications 2023 14: 8042) and Schlake et al (Molecular Therapy 201927: P773-784) for reviews of this category of therapeutic.

[0438] Methods of Imaging

[0439] The present disclosure provides methods of imaging cancer in a subject, the methods comprising administering an effective amount of a subject anti-EGFR polypeptide comprising, or conjugated to, a radioactive agent (or a pharmaceutical composition thereof) to a subject in need thereof. In these embodiments, the subject polypeptide can function as a radiotracer that localizes to a tumor (e.g., a tumor comprising cancer cells comprising EGFR) and enables imaging of the tumor via a variety of imaging techniques known in the art, such as PET or SPECT (see, e.g., Salsano and Treglia, Research and Reports in Nuclear Medicine. 2013: 3; 9-17). These methods may be used to image any suitable cancer or any cancer described herein including, e.g., brain cancer, breast cancer, esophageal cancer, gastrointestinal cancer, anal cancer, pancreatic cancer, cervical cancer, uterine cancer, ovarian cancer, lymphoid cancer, lung cancer, thyroid cancer, head and neck cancer, pharyngeal cancer, skin cancer, prostate cancer, kidney cancer, liver cancer, or hematological cancer.

[0440] Treatment Methods using anti-GIPR antibodies

[0441] The present disclosure provides a method of treating a metabolic disorder, such as a disorder of glucose metabolism (e.g. Type 2 diabetes, elevated glucose levels, elevated insulin levels, dyslipidemia, metabolic syndrome (Syndrome X or insulin resistance syndrome), glucosuria, metabolic acidosis, Type 1 diabetes, obesity and conditions exacerbated by obesity) by blocking or interfering with the biological activity of GIP. In one embodiment, a therapeutically effective amount of a subject antibody is administered to a subject in need thereof. Methods of administration and delivery are also provided A subject antibody can be used to treat, diagnose or ameliorate, a metabolic condition or disorder. In one embodiment, the metabolic disorder to be treated is diabetes, e.g., type 2 diabetes. In another embodiment, the metabolic condition or disorder is obesity. In other embodiments the metabolic condition or disorder is dyslipidemia, elevated glucose levels, elevated insulin levels or diabetic nephropathy. For example, a metabolic condition or disorder that can be treated or ameliorated using a subject antibody includes a state in which a human subject has a fasting blood glucose level of 125 mg / dLAtty. Dkt: CRYS-029WO

[0442] or greater, for example 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or greater than 200 mg / dL. Blood glucose levels can be determined in the fed or fasted state, or at random. The metabolic condition or disorder can also comprise a condition in which a subject is at increased risk of developing a metabolic condition. Tor a human subject, such conditions include a fasting blood glucose level of 100 mg / dL. Conditions that can be treated using a pharmaceutical composition comprising a subject antibody can also be found in the American Diabetes Association Standards of Medical Care in Diabetes Care-2011, American Diabetes Association, Diabetes Care Vol. 34, No. Supplement 1, S11-S61, 2010, incorporated herein by reference.

[0443] A metabolic disorder or condition, such as Type 2 diabetes, elevated glucose levels, elevated insulin levels, dyslipidemia, obesity or diabetic nephropathy, can be treated by administering a therapeutically effective dose of a subject antibody to a patient in need thereof. The administration can be performed as described herein, such as by IV injection, intraperitoneal (IP) injection, subcutaneous injection, intramuscular injection, or orally in the form of a tablet or liquid formation. In some situations, a therapeutically effective or preferred dose of a subject antibody can be determined by a clinician. A therapeutically effective dose of subject antibody will depend, inter alia, upon the administration schedule, the unit dose of agent administered, whether the subject antibody is administered in combination with other therapeutic agents, the immune status and the health of the recipient. The term "therapeutically effective dose," as used herein, means an amount of subject antibody that elicits a biological or medicinal response in a tissue system, animal, or human being sought by a researcher, medical doctor, or other clinician, which includes alleviation or amelioration of the symptoms of the disease or disorder being treated, i.e., an amount of a subject antibody that supports an observable level of one or more desired biological or medicinal response, for example lowering blood glucose, insulin, triglyceride, or cholesterol levels; reducing body weight; or improving glucose tolerance, energy expenditure, or insulin sensitivity.

[0444] It is noted that a therapeutically effective dose of a subject antibody can also vary with the desired result. Thus, for example, in situations in which a lower level of blood glucose is indicated a dose of subject antibody will be correspondingly higher than a dose in which a comparatively lower level of blood glucose is desired. Conversely, in situations in which a higher level of blood glucose is indicated a dose of subject antibody will be correspondingly lower than a dose in which a comparatively higher level of blood glucose is desired. In various embodiments, a subject is a human having a blood glucose level of 100 mg / dL or greater can be treated with a subject antibody.

[0445] In one embodiment, a method of the instant disclosure comprises first measuring a baseline level of one or more metabolically-relevant compounds such as glucose, insulin, cholesterol, lipid in a subject. A pharmaceutical composition comprising a subject antibody is then administered to the subject. After aAtty. Dkt: CRYS-029WO

[0446] desired period of time, the level of the one or more metabolically-relevant compounds (e.g., blood glucose, insulin, cholesterol, lipid) in the subject is again measured. The two levels can then be compared in order to determine the relative change in the metabolically-relevant compound in the subject.

[0447] Depending on the outcome of that comparison another dose of the pharmaceutical composition comprising a subject antibody can be administered to achieve a desired level of one or more metabolically-relevant compound.

[0448] It is noted that a pharmaceutical composition comprising a subject antibody can be coadministered with another compound. The identity and properties of compound co-administered with the GIPR binding protein will depend on the nature of the condition to be treated or ameliorated. A nonlimiting list of examples of compounds that can be administered in combination with a pharmaceutical composition comprising a GIPR binding protein include rosiglitizone, pioglitizone, repaglinide, nateglitinide, metformin, exenatide, sitagliptin, pramlintide, glipizide, glimepiride, acarbose, and miglitol.

[0449] Embodiments

[0450] For purposes of completeness, non-limiting aspects and embodiments of the present disclosure are further disclosed in the following sets of numbered clauses.

[0451] Clause Set A:

[0452] Clause 1. A monomeric or multimeric polypeptide comprising:

[0453] an antibody binding domain comprising:

[0454] (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a heavy chain variable domain selected from Table 1 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of a heavy chain variable domain selected from Table 1 except for up to 10 amino acid substitutions in the collective CDR regions; and

[0455] (ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 2 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 2 except for up to 10 amino acid substitutions in the collective CDR regions,

[0456] wherein the antibody binding domain binds to Kvl.3.

[0457] Clause 2. The polypeptide of clause 1, wherein:

[0458] the antibody binding domain comprises:Atty. Dkt: CRYS-029WO

[0459] (i) a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of the selected antibody; and

[0460] (ii) a light chain variable domain that is at least 90% identical to the light chain variable domain of the selected antibody.

[0461] Clause 3. The polypeptide of clause 1, wherein:

[0462] the antibody binding domain comprises:

[0463] (i) a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain of the selected antibody; and

[0464] (ii) a light chain variable domain that is at least 95% identical to the light chain variable domain of the selected antibody.

[0465] Clause 4. The polypeptide of any prior clause, wherein the heavy chain variable domain and the light chain variable domain are present in separate polypeptides.

[0466] Clause 5. The polypeptide of any one of clauses 1-3, wherein the heavy chain variable domain and the light chain variable domain are present in a single polypeptide.

[0467] Clause 6. The polypeptide of any one of clauses 1-4, wherein the polypeptide is an antibody.

[0468] Clause 7. The polypeptide of any one of clauses 1-6, wherein the antibody binding domain is a scFv or Fab.

[0469] Clause 8. The polypeptide of any prior clause, wherein the polypeptide is conjugated to a pharmacologically active agent.

[0470] Clause 9. The polypeptide of any prior clause, wherein the Kvl.3 is human Kvl.3.

[0471] Clause 10. The polypeptide of any prior clause, wherein the antibody binding domain binds human Kvl.3 with an affinity in the range of 107M-1to 1012M-1.

[0472] Clause 11. The polypeptide of any prior clause, wherein the polypeptide modulates Kvl. activity.

[0473] Clause 12. A pharmaceutical composition comprising:Atty. Dkt: CRYS-029WO

[0474] (a) the polypeptide of any prior clause; and

[0475] (b) a pharmaceutically acceptable carrier.

[0476] Clause 13. A method for modulating Kvl.3 activity, the method comprising contacting a cell comprising Kvl.3 with a polypeptide of any one of clauses 1-11.

[0477] Clause 14. The method of clause 13, wherein the cell is a T cell.

[0478] Clause 15. The method of clause 14, wherein the T cell is an effector memory T cell

[0479] Clause 16. The method of clause 14 or 15, wherein the T cell is an auto-reactive T cell.

[0480] Clause 17. The method of any one of clauses 13-16, wherein the cell is in vivo.

[0481] Clause 18. A method of treating an inflammatory disease in a subject, the method comprising administering an effective amount of the pharmaceutical composition of clause 12 to a subject in need thereof to treat the inflammatory disease in the subject.

[0482] Clause 19. The method of clause 18, wherein the treatment comprises modulating Kvl.3 activity in a T cell of the subject.

[0483] Clause 20. The method of clause 19, wherein the treatment comprises reducing activation of a T cell in the subject.

[0484] Clause 21. The method of any one of clauses 19-20, wherein the T cell is an effector memory T cell.

[0485] Clause 22. The method of any one of clauses 19-21, wherein the T cell is an auto-reactive T cell.

[0486] Clause 23. The method of any one of clauses 18-22, wherein the inflammatory disease is selected from: allergy, alopecia areata, Alzheimer's disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), contact-mediated dermatitis, delayed type hypersensitivity, fibrosis, glomerulonephritis, inflammatory bone resorption, inflammatory bowel disease, graft-versus host disease, hepatic fibrosis, multiple sclerosis, obesity, Parkinson’s disease, psoriasis, psoriatic arthritis, restenosis, rheumatoid arthritis,Atty. Dkt: CRYS-029WO

[0487] scleroderma, Sjogren’s syndrome, systemic lupus erythematosus, systemic sclerosis, type-1 diabetes mellitus, type -2 diabetes mellitus, transplant rejection, ulcerative colitis, and vasculitis.

[0488] Clause SetB:

[0489] Clause 1. A polypeptide comprising:

[0490] an antibody binding domain comprising:

[0491] i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 8 or are otherwise identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 8 except for up to 10 amino acid substitutions in the collective CDR regions; and

[0492] ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of the selected common light chain antibody or are otherwise identical to the light chain CDR1, CDR2, and CDR3 regions of the selected common light chain antibody except for up to 10 amino acid substitutions in the collective CDR regions,

[0493] wherein the antibody binding domain binds to Zip6.

[0494] Clause 2. The polypeptide of clause 1, wherein:

[0495] the antibody binding domain comprises:

[0496] i) a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of the selected common light chain antibody; and

[0497] ii) a light chain variable domain that is at least 90% identical to the light chain variable domain of the selected common light chain antibody.

[0498] Clause 3. The polypeptide of clause 1, wherein:

[0499] the antibody binding domain comprises:

[0500] i) a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain of the selected common light chain antibody; and

[0501] ii) a light chain variable domain that is at least 95% identical to the light chain variable domain of the selected common light chain antibody.

[0502] Clause 4. A polypeptide comprising:

[0503] an antibody binding domain comprising:Atty. Dkt: CRYS-029WO

[0504] i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 9 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of an antibody selected from Table 9 except for up to 10 amino acid substitutions; and

[0505] ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 10 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of an antibody selected from Table 10, wherein the antibody binding domain binds to Zip6.

[0506] Clause 5. The polypeptide of clause 4, wherein:

[0507] the antibody binding domain comprises:

[0508] i) a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of the selected antibody; and

[0509] ii) a light chain variable domain that is at least 90% identical to the light chain variable domain of the selected antibody.

[0510] Clause 6. The polypeptide of clause 4, wherein:

[0511] the antibody binding domain comprises:

[0512] i) a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain of the selected antibody; and

[0513] ii) a light chain variable domain that is at least 95% identical to the light chain variable domain of the selected antibody.

[0514] Clause 7. The polypeptide of any of the prior clauses, wherein the polypeptide is an antibody.

[0515] Clause 8. The polypeptide of any one of clauses 1-6, wherein the antibody binding domain is a scFv or Fab.

[0516] Clause 9. The polypeptide of any prior clause, wherein the polypeptide is conjugated to a pharmacologically active agent.

[0517] Clause 10. The polypeptide of any one of clauses 1-3, wherein the polypeptide is a bispecific binding molecule that comprises the selected antibody binding domain and at least one other binding domain.Atty. Dkt: CRYS-029WO

[0518] Clause 11. The polypeptide of clause 10, wherein the at least one other binding domain recognizes an immune cell antigen.

[0519] Clause 12. The polypeptide of clause 11, wherein the immune cell antigen is CD2 CD16, NKp30, NKp44, NKp46, NKG2C, NKG2D, DNAM1, SLAMF7, 2B4, KIR2DS, or KIR3DS.

[0520] Clause 13. A pharmaceutical composition comprising:

[0521] a) the polypeptide of any prior clause or a polynucleotide encoding the same; and

[0522] b) a pharmaceutically acceptable carrier.

[0523] Clause 14. A method of killing or inhibiting the proliferation of a target cell, the method comprising contacting a cell comprising Zip6 with a polypeptide of any one of clauses 1-13.

[0524] Clause 15. The method of clause 14, wherein the target cell is a cancer cell.

[0525] Clause 16. The method of clause 15, wherein the cancer cell is a cell of a solid tumor.

[0526] Clause 17. A method of treating a cancer in a subject, the method comprising administering an effective amount of the pharmaceutical composition of clause 13 to a subject in need thereof to treat the cancer in the subject.

[0527] Clause 18. The method of clause 17, wherein the method comprises killing or inhibiting the proliferation of a target cancer cell in the subject.

[0528] Clause 19. The method of clause 18, wherein the cancer cell is a cell of a solid tumor.

[0529] Clause 20. The method of any one of clauses 17-19, wherein the cancer is selected from breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, melanoma, cervical cancer, and uterine cancer.

[0530] Clause Set C:

[0531] Clause 1. An antibody that binds to the human glucose-dependent insulinotropic polypeptide (GIP) receptor, wherein the antibody comprises:Atty. Dkt: CRYS-029WO

[0532] (a) a variable domain comprising:

[0533] i. heavy chain CDR1, CDR2 and CDR3 regions that are identical to SEQ ID NOs. 3241, 3242 and 3243, respectively; and

[0534] ii. light chain CDR1, CDR2 and CDR3 regions that are identical to SEQ ID NOs. 3245, 3246 and 3247, respectively; or

[0535] (b) a variant of said variable domain of (a) that is otherwise identical to said antibody variable domain except for up to 10 amino acid substitutions in the collective CDR regions of the variable domain of (a).

[0536] Clause 2. The antibody of clause 1, wherein the antibody comprises:

[0537] a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to SEQ ID NO: 3240; and

[0538] a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to SEQ ID NO: 3244.

[0539] Clause 3. The antibody of any prior clause, wherein the antibody inhibits GIP receptor signaling.

[0540] Clause 4. The antibody of any prior clause, wherein the heavy chain variable domain and the light chain variable domain are present in separate polypeptides.

[0541] Clause 5. The antibody of any prior clause, wherein the heavy chain variable domain and the light chain variable domain are present in a single polypeptide.

[0542] Clause 6. The antibody of any prior clause, wherein the antibody binds the human GIP receptor with an affinity in the range of 10-7M-1to 10-12M-1.

[0543] Clause 7. The antibody of any prior clause, wherein the antibody comprises a covalently linked non-peptide synthetic polymer.

[0544] Clause 8. The antibody of clause 7, wherein the synthetic polymer is polyethylene glycol) polymer.

[0545] Clause 9. The antibody of any prior clause, wherein the antibody comprises a covalently linked lipid or fatty acid moiety.Atty. Dkt: CRYS-029WO

[0546] Clause 10. The antibody of any prior clause, wherein the antibody comprises a covalently linked polysaccharide or carbohydrate moiety.

[0547] Clause 11. The antibody of any prior clause, wherein the antibody is a single chain Fv (scFv) antibody.

[0548] Clause 12. The antibody of clause 10, wherein the scFv is multimerized.

[0549] Clause 13. A pharmaceutical composition comprising:

[0550] a) the antibody of any prior clause; and

[0551] b) a pharmaceutically acceptable carrier.

[0552] Clause 14. The pharmaceutical composition of clause 13, wherein the antibody is encapsulated in a liposome.

[0553] Clause 15. A method for inhibiting GIP receptor signaling, comprising contacting a cell comprising a GIP receptor with an antibody of any of any of clauses 1-12.

[0554] Clause 16. A method of inhibiting the GIP receptor in a subject, comprising administering to the subject an effective amount of the antibody of any of clauses 1-12.

[0555] Clause 17. A method of treating a metabolic disorder, comprising administering to a metabolic disorder or condition an effective amount of the antibody of any of any of clauses 1-14.

[0556] Clause Set D:

[0557] Clause 1. A polypeptide comprising:

[0558] a knob domain comprising an amino acid sequence that is the same as or comprises up to 10 amino acid substitutions relative to the knob domain of an ultralong CDR3 domain selected from Table 19, wherein the polypeptide binds to Nkp46.

[0559] Clause 2. The polypeptide of clause 1 wherein:

[0560] the knob domain comprises an amino acid sequence that is at least 90% identical to the sequence of an ultralong CDR3 domain selected from Table 19.Atty. Dkt: CRYS-029WO

[0561] Clause 3. The polypeptide of clause 1 or 2, wherein:

[0562] the knob domain comprises an amino acid sequence that is at least 95% identical to the sequence of an ultralong CDR3 domain selected from Table 19.

[0563] Clause 4. The polypeptide of any prior clause, wherein the polypeptide comprises an antibody binding domain, and wherein the antibody binding domain comprises a heavy chain variable domain that is at least 90% identical to the sequence of a heavy chain variable domain selected from Table 21, and a light chain variable domain that is at least 90% identical to the sequence of a light chain variable domain selected from Table 22.

[0564] Clause 5. The polypeptide of any prior clause, wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain.

[0565] Clause 6. The polypeptide of clause 5, wherein the at least one other binding domain recognizes a cancer antigen or a viral antigen.

[0566] Clause 7. The polypeptide of clause 6, wherein the cancer antigen is CD19, CD20, BCMA, ALPP, CS1 (SLAMF7), CLDN18.2, AXL, ROR2, TM4SF1, ICAM-1, L1CAM (CD171), CD4, CD5, CD7, CD10, CD38, CEA, FLT3, CD70, CD30, CD37, or CD 147.

[0567] Clause 8. The polypeptide of any prior clause, wherein the polypeptide is a bispecific binding molecule that comprises (i) the knob domain and (ii) a binding domain that recognizes a cancer antigen or a viral antigen.

[0568] Clause 9. The polypeptide of any prior clause, wherein the polypeptide is a bispecific binding molecule that comprises (i) the knob domain, (ii) a binding domain that recognizes a cancer antigen and (iii) either a binding domain that recognizes a co-stimulatory receptor on NK cells or a binding domain that recognizes a second cancer antigen or a second viral antigen.

[0569] Clause 10. The polypeptide of clause 9, wherein the co-stimulatory receptor is 2B4, DNAM1, or CD2.Atty. Dkt: CRYS-029WO

[0570] Clause 11. The polypeptide of any of clauses 6-10, wherein the cancer antigen is a blood cancer antigen.

[0571] Clause 12. The polypeptide of any one of clauses 6-10, wherein the cancer antigen is a solid tumor antigen.

[0572] Clause 13. The polypeptide of any prior clause, wherein the polypeptide is conjugated to a pharmacologically active agent.

[0573] Clause 14. A pharmaceutical composition comprising:

[0574] a) the polypeptide of any prior clause or a polynucleotide, e.g., an RNA, encoding the same; and b) a pharmaceutically acceptable carrier.

[0575] Clause 15. The pharmaceutical composition of clause 14, wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen.

[0576] Clause 16. A method of increasing binding between an NK cell and a cancer cell, comprising incubating the NK cell and a cancer cell with a polypeptide of any one of clauses 1-13, wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell.

[0577] Clause 17. A method of killing a cancer cell, comprising contacting the cancer cell with an NK cell and a polypeptide of any of clauses 1-13, wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell.

[0578] Clause 18. A method of treatment comprising administering a polypeptide of any of clauses clause 1-13 or a polynucleotide encoding the same (e.g., an RNA) to a subject that has cancer, wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell.

[0579] Clause 19. A method of increasing binding between an NK cell and a virally-infected cell,Atty. Dkt: CRYS-029WO

[0580] comprising incubating the NK cell and a virally-infected cell with a polypeptide of any of clauses 1-13, wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a viral antigen on the virally-infected cell.

[0581] Clause 20. A method of killing a virally-infected cell, comprising contacting the virally-infected cell with an NK cell and a polypeptide of any of clauses 1-13, wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a viral antigen on the virally-infected cell.

[0582] Clause 21. A method of treatment comprising administering a polypeptide of any of clauses 1-13 or a polynucleotide encoding the same (e.g., an RNA) to a subject that has a viral infection, wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a viral antigen on a virally-infected cell.

[0583] Clause Set E:

[0584] Clause 1. A polypeptide comprising:

[0585] a knob domain comprising an amino acid sequence that is the same as or comprises up to 10 amino acid substitutions relative to the knob domain of an ultralong CDR3 domain selected from Table 24 or Table 37.

[0586] Clause 2. A polypeptide comprising:

[0587] a VHH domain comprising:

[0588] (a) CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a VHH antibody selected from Table 25 or Table 38; or

[0589] (b) CDR1, CDR2, and CDR3 regions that are otherwise identical to the CDR1, CDR2, and CDR3 regions of a VHH antibody selected from Table 25 or Table 38 except for up to 10 amino acid substitutions in the collective CDR regions;

[0590] wherein the VHH domain binds to EGFR.

[0591] Clause 3. A polypeptide comprising:

[0592] an antibody binding domain comprising:

[0593] (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 36 or are otherwise identical to the heavy chainAtty. Dkt: CRYS-029WO

[0594] CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 36 except for up to 10 amino acid substitutions in the collective CDR regions; and

[0595] (ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody or are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody except for up to 10 amino acid substitutions in the collective CDR regions,

[0596] wherein the antibody binding domain binds to EGFR.

[0597] Clause 4. A polypeptide comprising:

[0598] an antibody binding domain comprising:

[0599] (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 39 or are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 39 except for up to 10 amino acid substitutions in the collective CDR regions; and

[0600] (ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 40 or are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 40 except for up to 10 amino acid substitutions in the collective CDR regions,

[0601] wherein the antibody binding domain binds to EGFR.

[0602] Clause 5. The polypeptide of any one of clauses 1 -4, wherein:

[0603] the knob domain comprises an amino acid sequence that is at least 90% identical to the sequence of the knob domain of an ultralong CDR3 domain selected from Table 24 or Table 37;

[0604] the amino acid sequence of the VHH domain is at least 90% identical to the variable domain of the selected VHH antibody;

[0605] the antibody binding domain comprises a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of the selected common light chain antibody and a light chain variable domain that is at l...

Claims

Atty. Dkt: CRYS-029WOCL IMSWhat is claimed is:

1. A polypeptide comprising:an antibody binding domain comprising:i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a heavy chain variable domain selected from Table 1 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of a heavy chain variable domain selected from Table 1 except for up to 10 amino acid substitutions in the collective CDR regions, andii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 2 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of a light chain variable domain selected from Table 2 except for up to 10 amino acid substitutions in the collective CDR regions,wherein the antibody binding domain binds to Kvl.3.

2. The polypeptide of claim 1, wherein the antibody binding domain comprises:a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain selected from 'fable 3 and a light chain variable domain that is at least 90% identical to the light chain variable domain selected from Table 4.

3. The polypeptide of claim 2, wherein the antibody binding domain comprises:a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain selected from Table 3 and a light chain variable domain that is at least 95% identical to the light chain variable domain selected from Table 4.

4. A polypeptide comprising:a) an antibody binding domain comprising:i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 9 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of an antibody selected from Table 9 except for up to 10 amino acid substitutions in the collective CDR regions, andii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 10 or are otherwise identical to the CDR1, CDR2, and CDR3 regions of an antibody selected from Table 10,Atty. Dkt: CRYS-029WOexcept for up to 10 amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to Zip6; orb) an antibody binding domain comprising:i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 8 or are otherwise identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 8 except for up to 10 amino acid substitutions in the collective CDR regions, andii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of the selected common light chain antibody or are otherwise identical to the light chain CDR1, CDR2, and CDR3 regions of the selected common light chain antibody except for up to 10 amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to Zip6.

5. The polypeptide of claim 4, wherein:a) the antibody binding domain comprises a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain selected from Table 12 and a light chain variable domain that is at least 90% identical to the light chain variable domain selected from fable 13; orb) the antibody binding domain comprises a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of a common light antibody selected from Table 11 and a light chain variable domain that is at least 90% identical to the light chain variable domain of the selected common light chain antibody.

6. The polypeptide of claim 5, wherein:a) the antibody binding domain comprises a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain selected from Table 12 and a light chain variable domain that is at least 95% identical to the light chain variable domain selected from Table 13; orb) the antibody binding domain comprises a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain of a common light antibody selected from Table 11 and a light chain variable domain that is at least 95% identical to the light chain variable domain of the selected common light chain antibody.

7. A polypeptide comprising:Atty. Dkt: CRYS-029WOa knob domain comprising an amino acid sequence that is the same as or comprises up to 10 amino acid substitutions relative to the knob domain of an ultralong CDR3 domain selected from Table 19, wherein the knob domain binds to NKp46.

8. The polypeptide of claim 7, wherein the knob domain comprises:an amino acid sequence that is at least 90% identical to the sequence of the knob domain of an ultralong CDR3 domain selected from Table 19.

9. The polypeptide of claim 8, wherein the knob domain comprises:an amino acid sequence that is at least 95% identical to the sequence of the knob domain of an ultralong CDR3 domain selected from Table 19.

10. The polypeptide of claim 8 or 9, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of an ultralong CDR3 selected from Table 19.

11. The polypeptide of any one of claims 7-10, wherein the polypeptide comprises an antibody binding domain, and wherein the antibody binding domain comprises a heavy chain variable domain that is at least 90% identical to the sequence of a heavy chain variable domain selected from Table 23, and a light chain variable domain that is at least 90% identical to the sequence of a light chain variable domain selected from Table 22.

12. A polypeptide comprising:a) a knob domain comprising an amino acid sequence that is the same as or comprises up to 10 amino acid substitutions relative to the knob domain of an ultralong CDR3 domain selected from Table 24, Table 37, or Table 62, wherein the knob domain binds to EGFR;b) a VHH domain comprising:i) CDR1, CDR2, and CDR3 regions that are identical to the CDR1, CDR2, and CDR3 regions of a VHH antibody selected from Table 25 or Table 38, orii) CDR1, CDR2, and CDR3 regions that are otherwise identical to the CDR1, CDR2, and CDR3 regions of a VHH antibody selected from Table 25 or Table 38 except for up to 10 amino acid substitution in the collective CDR regions,wherein the VHH domain binds to EGFR;c) an antibody binding domain comprising:Atty. Dkt: CRYS-029WOi) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 39 or are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 39 except for up to 10 amino acid substitutions in the collective CDR regions, andii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2, and CDR3 regions of an antibody selected from Table 40 or are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of an antibody selected from Table 40 except for up to 10 amino acid substitutions in the collective CDR regions,wherein the antibody binding domain binds to EGFR; ord) an antibody binding domain comprising:i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the heavy chain CDR1, CDR2, and CDR3 regions of a common light chain antibody selected from Table 36 or are otherwise identical to the heavy chain CDR1, CDR2 and CDR3 regions of a common light chain antibody selected from Table 36 except for up to 10 amino acid substitutions in the collective CDR regions, andii) a light chain variable domain comprising CDR1, CDR2, and CDR3 regions that are identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody or are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected common light chain antibody except for up to 10 amino acid substitutions in the collective CDR regions, wherein the antibody binding domain binds to EGFR.

13. The polypeptide of claim 12, wherein:a) the knob domain comprises an amino acid sequence that is at least 90% identical to the sequence of the knob domain of an ultralong CDR3 domain selected from Table 24, Table 37, or Table 62;b) the amino acid sequence of the VHH domain is at least 90% identical to a VHH sequence selected from Table 28 or Table 43;c) an antibody binding domain comprising a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain selected from Table 44 and a light chain variable domain that is at least 90% identical to the light chain variable domain selected from Table 45; ord) an antibody binding domain comprising a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of a common light antibody selected from Table 41 and a light chain variable domain that is at least 90% identical to the light chain variable domain of the selected common light chain antibody.Atty. Dkt: CRYS-029WO14. The polypeptide of claim 13, wherein:a) the knob domain comprises an amino acid sequence that is at least 95% identical to the sequence of the knob domain of an ultralong CDR3 domain selected from Table 24, Table 37, or Table 62;b) the amino acid sequence of the VHH domain is at least 95% identical to a VHH sequence selected from Table 28 or Table 43;c) an antibody binding domain comprising a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain selected from Table 44 and a light chain variable domain that is at least 95% identical to the light chain variable domain selected from Table 45; ord) an antibody binding domain comprising a heavy chain variable domain that is at least 95% identical to the heavy chain variable domain of a common light antibody selected from Table 41 and a light chain variable domain that is at least 95% identical to the light chain variable domain of the selected common light chain antibody.

15. The polypeptide of claim 13 or 14, wherein the knob domain comprises an amino acid sequence that is at least 90% identical to the sequence of an ultralong CDR3 selected from Table 24, 37, or 62.

16. The polypeptide of any one of claims 12-15, wherein the polypeptide comprising a VHH domain is humanized.

17. The polypeptide of any one of claims 1-6, and 12-14, wherein the antibody binding domain is humanized.

18. The polypeptide of any prior claim, wherein the polypeptide is an antibody.

19. The polypeptide of any one of claims 1-6, 12-14, and 17, wherein the antibody binding domain is a scFv or Fab.

20. The polypeptide of any prior claim, wherein the polypeptide is fused to an albumin or Fc domain.

21. The polypeptide of any prior claim, wherein the polypeptide is a multispecific binding molecule that comprises the knob domain, the VHH domain, or the antibody binding domain and at least one other binding domain.Atty. Dkt: CRYS-029WO22. The polypeptide of claim 21, wherein the at least one other binding domain recognizes a cancer antigen, an immune cell antigen, or a viral antigen.

23. The polypeptide of claim 22, wherein:a) the antibody binding domain binds to Zip6, and wherein the immune cell antigen is CD2 CD16, NKp30, NKp44, NKp46, NKG2C, NKG2D, DNAM1, SLAMF7, 2B4, KIR2DS, or KIR3DS; b) the knob domain binds to NKp46, and wherein the cancer antigen is CD19, CD20, BCMA, ALPP, CS1 (SLAMF7), CLDN18.2, AXL, R0R2, TM4SF1, ICAM-1, L1CAM (CD171), CD4, CD5, CD7, CD10, CD38, CEA, FLT3, CD70, CD30, CD37, or CD147;c) the knob domain, VHH domain, or antibody binding domain binds to EGFR, and wherein the cancer antigen is EGFR, HER2, VEGFR2, DLL4, ANG2, c-MET, PD-L1, LGR5, ZIP6, or MET; or d) the knob domain, VHH domain, or antibody binding domain binds to EGFR, and wherein the immune cell antigen is CD2 CD16, NKp30, NKp44, NKp46, NKG2C, NKG2D, DNAM1, SLAMF7, 2B4, KIR2DS, KIR3DS, CD3, or CD28.

24. The polypeptide of any one of claims 21-23, wherein the polypeptide is a bispecific binding molecule that comprises (i) the knob domain and (ii) a binding domain that recognizes a cancer antigen or a viral antigen, wherein the knob domain binds to NKp46.

25. The polypeptide of claim 24, wherein the polypeptide is a bispecific binding molecule that comprises (i) the knob domain, (ii) a binding domain that recognizes a cancer antigen or a viral antigen and (iii) either a binding domain that recognizes a co-stimulatory receptor on NK cells or a binding domain that recognizes a second cancer antigen or a second viral antigen.

26. The polypeptide of claim 25, wherein the co-stimulatory receptor is 2B4, DNAM1, or CD2.

27. The polypeptide of any one of claims 22-26, wherein the cancer antigen is a blood cancer antigen.

28. The polypeptide of any one of claims 22-26, wherein the cancer antigen is a solid tumor antigen.

29. The polypeptide of any prior claim, wherein the polypeptide is conjugated to a pharmacologically active agent.Atty. Dkt: CRYS-029WO30. The polypeptide of any one of claims 1-28, wherein the polypeptide comprises, or is conjugated to, a radioactive agent.

31. The polypeptide of any one of claims 1-28, wherein the polypeptide is in the extracellular domain of an engineered immune cell receptor.

32. A pharmaceutical composition comprising:a) the polypeptide of any prior claim or a polynucleotide, e.g., an RNA, encoding the same, and b) a pharmaceutically acceptable carrier.

33. The pharmaceutical composition of claim 32, wherein the polypeptide is a multispecific binding molecule that comprises the VHH domain, the antibody binding domain, or the knob domain and at least one other binding domain that binds to a cancer antigen, an immune cell antigen, or a viral antigen.

34. A method for modulating Kvl.3 activity, the method comprising contacting a cell comprising Kvl.3 with a polypeptide of any one of claims 1-3, 17-22, and 29-31, wherein the antibody binding domain of the polypeptide binds Kvl.3.

35. The method of claim 34, wherein the cell is a T cell.

36. The method of claim 35, wherein the T cell is an effector memory T cell.

37. The method of claim 35 or 36, wherein the T cell is an auto-reactive T cell.

38. The method of any one of claims 34-37, wherein the cell is in vivo.

39. A method of treating an inflammatory disease in a subject, the method comprising administering an effective amount of the pharmaceutical composition of claim 32 to a subject in need thereof to treat the inflammatory disease in the subject, wherein the antibody binding domain of the polypeptide binds to Kvl.3.

40. The method of claim 39, wherein the treatment comprises modulating Kvl.3 activity in a T cell of the subject.Atty. Dkt: CRYS-029WO41. The method of claim 40, wherein the treatment comprises reducing activation of a T cell in the subject.

42. The method of any one of claims 40-41, wherein the T cell is an effector memory T cell.

43. The method of any one of claims 40-42, wherein the T cell is an auto-reactive T cell.

44. The method of any one of claims 39-43, wherein the inflammatory disease is selected from: allergy, alopecia areata, Alzheimer’s disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), contact-mediated dermatitis, delayed type hypersensitivity, fibrosis, glomerulonephritis, inflammatory bone resorption, inflammatory bowel disease, graft-versus host disease, hepatic fibrosis, multiple sclerosis, obesity, Parkinson’s disease, psoriasis, psoriatic arthritis, restenosis, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, systemic lupus erythematosus, systemic sclerosis, type-1 diabetes mellitus, type-2 diabetes mellitus, transplant rejection, ulcerative colitis, and vasculitis.

45. A method of killing or inhibiting the proliferation of a target cell, the method comprising contacting a cell comprising Zip6 with a polypeptide of any one of claims 4-6, 17-23, and 29-31 wherein the antibody binding domain of the polypeptide binds to Zip6.

46. The method of claim 45, wherein the target cell is a cancer cell.

47. The method of claim 46, wherein the cancer cell is a cell of a solid tumor.

48. A method of treating a cancer in a subject, the method comprising administering an effective amount of the pharmaceutical composition of claim 32 to a subject in need thereof to treat the cancer in the subject, wherein the antibody binding domain of the polypeptide binds to Zip6.

49. The method of claim 48, wherein the method comprises killing or inhibiting the proliferation of a target cancer cell in the subject.

50. The method of claim 49, wherein the cancer cell is a cell of a solid tumor.Atty. Dkt: CRYS-029WO51. The method of any one of claims 48-50, wherein the cancer is selected from breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, melanoma, cervical cancer, and uterine cancer.

52. A method of increasing binding between an NK cell and a cancer cell, comprising incubating the NK cell and a cancer cell with a polypeptide of any one of claims 7-11 and 21-31, wherein the knob domain of the polypeptide binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell.

53. A method of killing a cancer cell, comprising contacting the cancer cell with an NK cell and a polypeptide of any one of claims 7- 11 and 21-31, wherein the knob domain of the polypeptide binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell.

54. A method of treatment comprising administering a polypeptide of any one of claims 7-11 and 21-31 or a polynucleotide encoding the same (e.g., an RNA) to a subject that has cancer, wherein the knob domain of the polypeptide binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a cancer antigen on the cancer cell.

55. A method of increasing binding between an NK cell and a virally-infected cell, comprising incubating the NK cell and a virally-infected cell with a polypeptide of any one of claims 7-11 and 21-31, wherein the knob domain of the polypeptide binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a viral antigen on the virally-infected cell.

56. A method of killing a virally-infected cell, comprising contacting the virally-infected cell with an NK cell and a polypeptide of any one of claims 7-11 and 21-31, wherein the knob domain of the polypeptide binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a viral antigen on the virally-infected cell.

57. A method of treatment comprising administering a polypeptide of any one of claims 7-11 and 21-31 or a polynucleotide encoding the same (e.g., an RNA) to a subject that has a viral infection, whereinAtty. Dkt: CRYS-029WOthe knob domain of the polypeptide binds to NKp46, and wherein the polypeptide is a multispecific binding molecule that comprises the knob domain and at least one other binding domain that binds to a viral antigen on a virally-infected cell.

58. A method of killing or inhibiting the proliferation of a target cell, the method comprising contacting a cell comprising EGFR with a polypeptide of any one of claims 12-23 and 29-31, wherein the knob domain, the VHH domain, or the antibody binding domain of the polypeptide binds to EGFR.

59. The method of claim 58, wherein the target cell is a cancer cell.

60. The method of claim 59, wherein the cancer cell is a cell of a solid tumor.

61. A method of treating and / or imaging cancer in a subject, the method comprising administering an effective amount of the pharmaceutical composition of claim 32 to a subject in need thereof to treat and / or image the cancer in the subject, wherein the knob domain, the VHH domain, or the antibody binding domain of the polypeptide binds to EGFR.

62. The method of claim 61, wherein the method comprises killing or inhibiting the proliferation of a target cancer cell in the subject.

63. The method of claim 61, wherein the method comprises imaging a target cancer cell in the subject.

64. The method of claim 62 or 63, wherein the target cancer cell comprises EGFR.

65. The method of any one of claims 62-64, wherein the cancer cell is a cell of a solid tumor.

66. The method of any one of claims 61-65, wherein the cancer is selected from brain cancer, breast cancer, esophageal cancer, gastrointestinal cancer, anal cancer, pancreatic cancer, cervical cancer, uterine cancer, ovarian cancer, lymphoid cancer, lung cancer, thyroid cancer, head and neck cancer, pharyngeal cancer, skin cancer, prostate cancer, kidney cancer, liver cancer, or hematological cancer.

67. An antibody that binds to the human glucose-dependent insulinotropic polypeptide (GIP) receptor, wherein the antibody comprises:Atty. Dkt: CRYS-029WO(a) a variable domain comprising:i. heavy chain CDR1, CDR2 and CDR3 regions that are identical to SEQ ID NOs. 3241, 3242 and 3243, respectively; andii. light chain CDR1, CDR2 and CDR3 regions that are identical to SEQ ID NOs. 3245, 3246 and 3247, respectively; or(b) a variant of said variable domain of (a) that is otherwise identical to said antibody variable domain except for up to 10 amino acid substitutions in the collective CDR regions of the variable domain of (a).

68. The antibody of claim 67, wherein the antibody comprises:a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to SEQ ID NO: 3240; anda light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 95%) identical to SEQ ID NO: 3244.

69. The antibody of any prior claim, wherein the antibody inhibits GIP receptor signaling.

70. The antibody of any prior claim, wherein the heavy chain variable domain and the light chain variable domain are present in separate polypeptides.71 The antibody of any prior claim, wherein the heavy chain variable domain and the light chain variable domain are present in a single polypeptide.

72. The antibody of any prior claim, wherein the antibody binds the human GIP receptor with an affinity in the range of 10-7M-1to 10-12M-1.

73. The antibody of any prior claim, wherein the antibody comprises a covalently linked non-peptide synthetic polymer.

74. The antibody of claim 73, wherein the synthetic polymer is polyethylene glycol) polymer.

75. The antibody of any prior claim, wherein the antibody comprises a covalently linked lipid or fatty acid moiety.Atty. Dkt: CRYS-029WO76. The antibody of any prior claim, wherein the antibody comprises a covalently linked polysaccharide or carbohydrate moiety.

77. The antibody of any prior claim, wherein the antibody is a single chain Fv (scFv) antibody.

78. The antibody of claim 77, wherein the scFv is multimerized.

79. A pharmaceutical composition comprising:a) the antibody of any prior claim; andb) a pharmaceutically acceptable carrier.

80. The pharmaceutical composition of claim 79, wherein the antibody is encapsulated in a liposome.

81. A method for inhibiting GIP receptor signaling, comprising contacting a cell comprising a GIP receptor with an antibody of any of any of claims 67-78.

82. A method of inhibiting the GIP receptor in a subject, comprising administering to the subject an effective amount of the antibody of any of claims 67-78.

83. A method of treating a metabolic disorder, comprising administering to a metabolic disorder or condition an effective amount of the antibody of any of claims 1-80.

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