WO2022246085A2

WO2022246085A2 - Layilin antibodies and ligand

Info

Publication number: WO2022246085A2
Application number: PCT/US2022/030069
Authority: WO
Inventors: Jeff E. Glasgow; James A. Wells
Original assignee: The Regents Of The University Of California
Priority date: 2021-05-20
Filing date: 2022-05-19
Publication date: 2022-11-24
Also published as: WO2022246085A3

Abstract

The disclosure describes anti-layilin antibodies that comprising a layilinbindingdomain that interferes with layilin binding to Type IV and/or Type V collagen, methods of generating such antibodies, and method of using such antibodies to inhibit layilin binding to Type IV and/or Type V collagen.

Description

LAYILIN ANTIBODIES AND LIGAND

BACKGROUND OF THE INVENTION

[0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No. 63/191,302, filed May 20, 2021, which is incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

[0002] C-type lectins (CLECs) comprise a large family of transmembrane and extracellular proteins that play numerous roles in cell signaling, adhesion, tissue structure, pathogen sensing, and protein turnover, among other functions (Brown et al., 2018). C-type lectin domains (CTLDs) are characterized by a conserved fold including anywhere from one to four Ca²⁺ ions that stabilize the structure and enable ligand binding (Valverde et al., 2020). Structurally related C-type lectin-like domains lacking Ca²⁺ are also widespread among cell surface receptors. Proteins in the CLEC family bind incredibly diverse ligand types, but many CLECs are able to recognize specific glycan motifs by forming Ca²⁺-coordination bonds with vicinal diols in sugar molecules. Calcium ion coordination is mediated by highly conserved motifs in the CLEC sequence, with ‘WND’ sites aiding in Ca²⁺ binding alongside either ΈRN’ or ‘QPD’ sites shaping the binding site for sugar specificity (Keller and Rademacher, 2020). These interactions typically have modest affinity and are often strengthened by multimeric or tandem CTLDs. In the immune system, numerous CLECs and CLEC-like proteins mediate cell-cell contacts, endocytosis of extracellular material, and detection of pathogen-associated molecular patterns.

[0003] The class of CLECs are widely distributed on tissues and therapeutic antibodies targeting of these receptors are beginning to emerge. Monalizumab, targets the inhibitory C- type-lectin-like receptor KLRC1/NKG2A (CD159a) on tumor-infiltrating T- and Natural Killer (NK) cells, and has shown promising in early clinical data (Andre et al., 2018). Ontuxizumab targets the collagen-binding CLEC tumor endothelial marker- 1 (TEM- l)/endosialin/CD248 although it had low efficacy in Phase 2 trials in melanoma patients (D’Angelo et al., 2018). As CLEC-family receptors play prominent roles in inflammation, antibody-mediated modulation of CLECs will likely become more prevalent in both immuno- oncology and anti-inflammatory drugs.

[0004] Layilin is a small Type I transmembrane protein with a single extracellular CTLD. Very little is known about the structure of layilin or how it interacts with other receptors, cells, or matrix. Layilin was first discovered through a yeast-two-hybrid screen for proteins that interact with the band 4.1, ezrin, radixin, moesin (FERM) domain of talin (Borowsky and Hynes, 1998). Layilin’ s intracellular domain competed with binding of b-integrin tails to the talin FERM domain and co-immunoprecipitated radixin and merlin (Bono et ak, 2005; Wegener et ak, 2008). Layilin’s CTLD was shown to bind hyaluronic acid and localize to membrane ruffles (Bono et ak, 2001). Collectively, these data point to layilin mediating interactions between the extracellular matrix and the cytoskeleton; however, its exact role in cell adhesion, interplay with integrins, and breadth of binding partners has not been clearly delineated.

[0005] Type IV collagen is primarily found in basement membranes; however, this molecule and its enzymatic post-translational modification machinery components, PLOD1 and PLOD3, are overexpressed in cancers such as hepatocellular carcinoma (HCC) (Yang et ak, 2020). Notably, layilin is highly enriched in HCC-infiltrating T cells, which may point to its role in T cell immunity in this cancer type (Zheng et ak, 2017). A close homologue of layilin, called chondrolectin, was recently found to interact with Type XIX collagen (Opri§oreanu et ak, 2019), which we also observed in our AP/MS. A unique splice variant of chondrolectin was found to be up-regulated upon T cell maturation and localized to the late endoplasmic reticulum (Weng et ah, 2003). The relationship between layilin, chondrolectin, and collagen glycosylation in T cell interactions with the ECM has not been fully characterized.

[0006] Early studies showed broad tissue distribution of layilin expression ((Borowsky and Hynes, 1998); however, in more recent years ayilin has repeatedly been found as one of the highest upregulated proteins in regulatory T cells (Tregs) (Bhairavabhotla et ah, 2016;

De Simone et ah, 2016), exhausted tumor infiltrating CD8+ T cells (Guo et ah, 2018; Zheng et ah, 2017), and highly activated IL-17-producing CD8+ T cells in psoriatic skin (Liu et ah, 2020). Knockdown studies in Tregs found that decreased layilin expression led to increased suppressive capacity in in vitro co-culture assays (Bhairavabhotla et ah, 2016). It was recently found that CD8+ T cell-specific knockout of layilin in mice led to decreased control of tumor growth in a melanoma model, demonstrating that layilin does not act as a traditional checkpoint but is important for continued cytotoxic activity as CD8+ cells become exhausted (Mahuron et al., 2020). Despite this strong association with T cell function in disease, the molecular basis for layilin’s role in these cells is largely unknown.

BRIEF SUMMARY OF THE INVENTION

[0007] In one aspect, the disclosure provides an antibody comprising a layilin-binding domain A (LBD-A), wherein the LBD-A specifically binds to a layilin extracellular domain (ECD) comprising SEQ ID NO: 11 and interferes with binding between the layilin ECD with one or both of collagen IV or collagen V. In some embodiments, the antibody comprises: (a) a heavy chain variable (VH) region comprising a heavy chain complementarity determining region (HCDR) 1 comprising SGFNFYSSYIH (SEQ ID NO:l), an HCDR2 comprising SISSYYGSTSYADSVKG (SEQ ID NO:2), and an HCDR3 comprising FSQYSWYTFSGLDY (SEQ ID NO:3); and (b) a light chain variable (VL) region comprising a light chain complementarity determining region (LCDR) 1 comprising R(A/T)SQSVSSAVA (SEQ ID NO:4), an LCDR2 comprising SASSLYS (SEQ ID NO:5), and an LCDR3 comprising QQASTYPIT (SEQ ID NO:6). In some embodiments, the VH region comprises:

EVQLVESGGGLVQPGGSLRLSCAASGFNFYSSYIHWVRQAPGKGLEWVASISSYYGS TSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFSQYSWYTFSGLDYW GQGTLVTVSS (SEQ ID NO:7); and the VL region comprises:

DIQMTQSPSSLSASVGDRVTITCR(A/T)SQSVSSAVAWYQQKPGKAPKLLIYSASSLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQASTYPITFGQGTKVEIK (SEQ ID NO: 8). In some embodiments, the heavy chain comprises

EVQLVESGGGLVQPGGSLRLSCAASGFNFYSSYIHWVRQAPGKGLEWVASISSYYGS TSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFSQYSWYTFSGLDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THT CPP CP APE AAGGP S VFLFPPKPKDTLMIS RTPEVTC V V VD V SHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKALGAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:9), and the light chain comprises:

DIQMTQSPSSLSASVGDRVTITCR(A/T)SQSVSSAVAWYQQKPGKAPKLLIYSASSLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQASTYPITFGQGTKVEIKRTVAAPSV FIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10). In some embodiment, the antibody comprises at least a second layilin binding domain. In some embodiments, the second layilin-binding domain comprises a VH region comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprises SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3; and a VL region comprising an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6. In some embodiments, the VH region of the second layilin-binding domain comprises SEQ ID NO;7 and the VL region of the second layilin-binding domain comprises SEQ ID NO: 8. In some embodiments, the second layilin-binding domain comprises the LBD-A. In other embodiments, the second layilin-binding domain comprises a layilin-binding domain B (LBD-B), wherein the LBD-B specifically binds to the layilin ECD. In some embodiments, the LBD-A and the LBD-B each bind a distinct layilin epitope of the layilin ECD. In some embodiments, the LBD-B does not interfere with binding between the layilin ECD with one or both of collagen IV or collagen V. In some embodiments, the LBD-B binds the layilin ECD with greater affinity than the LBD-A. In some embodiments, In some embodiments, the antibody comprises at least three layilin-binding domains. In some embodiments, each of the layilin-binding domains are independently selected from the LBD-A and the LBD-B. In some embodiments, the LBD-A and/or the LBD-B comprise an antagonistic layilin-binding domain. In some embodiments, the LBD-A and/or the LBD-B do not result in detectable levels of integrin signaling, e.g., as assessed by an M24-cell based signaling assay, upon binding to the layilin ECD when the layilin ECD is operably linked to a layilin intracellular domain. In some embodiments, the LBD-A and/or the LBD-B bind the layilin ECD in a calcium-dependent manner. In some embodiments, the LBD-A and/or the LBD-B do not bind the layilin ECD when Ca²⁺ is absent. In some embodiments, the antibody comprises at least two heavy chain variable regions and at least two light chain variable regions, the heavy chain variable regions each comprising aHCDRl, HCDR2, and HCDR3, and the light chain variable regions each comprising a LCDR1, LCDR2, and LCDR3. In some embodiments, each of the two heavy chain variable regions are identical and/or each of the two light chain variable regions are identical. In some embodiments, the LBD-B is operably linked to the heavy chain variable region or the light chain variable region of LBD-A. In some embodiments, the antibody further comprises one or more constant domains selected from the group consisting of: a CHI, a CH2, a CH3, and a CL constant domains; or comprises an Fc domain, such as an IgG constant domain. In some embodiments, the antibody comprises two of each of the CHI, the CH2, the CH3, and the CL constant domains. In some embodiments, each of the two respective constant domains are identical. In some embodiments, one or more of the constant domains comprise an engineered mutation with reference to an endogenous constant domain. In some embodiments, the antibody comprises an scFv fragment, a single domain antibody, a Fab fragment, an Fv fragment, a F(ab’)2 fragment, a Fab’ fragment, and/or an scFv-Fc fragment, or antigen binding fragment thereof. In some embodiments, the layilin ECD is a human, murine, or cynomolgus layilin ECD. In some embodiments, the collagen IV and/or collagen V is glycosylated. In some embodiments, the antibody interferes with binding between the layilin ECD with collagen VI.

[0008] In a further aspect, the disclosure provides a chimeric receptor comprising an antibody comprising a layilin-binding domain as described herein, e.g., in the preceding paragraphs, optionally comprising a transmembrane domain, and optionally comprising an intracellular signaling domain.

[0009] In an additional aspect, the disclosure provides a polynucleotide or set of polynucleotides encoding the antibody or the chimeric receptor comprising a layilin-binding domain as described herein or an antigen-binding portion thereof. In some embodiments, the disclosure provides a vector or set of vectors encoding the antibody or chimeric receptor and/or the polynucleotide or set of polynucleotides, optionally wherein the vector comprises a promoter operably linked to the polynucleotide encoding the antibody.

[0010] In another aspect, the disclosure provides a cell expressing an antibody or chimeric receptor comprising a layilin-binding domain as described herein and/or comprising a polynucleotide or set of polynucleotides; or comprising a vector or set of vectors encoding such an antibody or chimeric receptor.

[0011] The disclosure further provides a pharmaceutical composition comprising an antibody or chimeric receptor comprising a layilin-binding domain as described herein; a polynucleotide or set of polynucleotides encoding the antibody or chimeric receptor or a vector or set of vectors encoding the antibody or chimeric receptor; and/or a cell that expresses the antibody or chimeric receptor.

[0012] In another aspect, the disclosure provides a method of producing an anti-layilin antibody or chimeric receptor, wherein the method comprises expressing or having expressed the antibody or chimeric receptor comprising a layilin-binding domain as described herein in a cell and isolating or having isolated the anti-layilin antibody or chimeric receptor.

[0013] In an additional aspect, the disclosure provides a method of manufacturing a polynucleotide encoding an anti-layilin antibody or chimeric receptor as described herein, wherein the method comprises obtaining or having obtained a polynucleotide or set of polynucleotides encoding the antibody or chimeric receptor, and/or a vector or set of vectors encoding the antibody or chimeric receptor; amplifying or having amplified the polynucleotide or vector, and isolating the amplified polynucleotide or vector, optionally wherein the amplification comprises (1) transfecting or having transfected the polynucleotide or vector in a host cell under conditions sufficient for replication of the polynucleotide or vector in the host cell, and/or (2) a polymerase chain reaction.

[0014] The disclosure further provides a method of reducing binding of one or both of collagen IV and collagen V to layilin on a cell surface in the presence of the collagen IV and/or collagen V, the method comprising contacting, or having contacted, a binding molecule that specifically binds to the layilin and interferes with binding between the layilin and the collagen IV and/or collagen V. In some embodiments, the binding molecule comprises a binding peptide or an antibody. In some embodiments, the antibody comprises an comprising a layilin-binding domain as described herein. In some embodiments, the cell is a human CD8+ T-cell or a regulatory T-cell (Treg). In some embodiments, the cell is in a human and the binding molecule is administered to the human to a disease. In some embodiments, the human has cancer. In some embodiments, the human has an autoimmune disease.

[0015] In another aspect, the disclosure provides a method of reducing binding of human layilin on a cell surface of human CD8+ T-cells or regulatory T-cells (Tregs) to one or both of collagen IV or collagen V in a human subject having a disease, the method comprising: contacting the human CD8+ T-cells or Tregs with a binding molecule that binds human layilin on the cell surface and that interferes with binding between the human layilin and one or both of collagen IV or collagen V, in an amount effective to reduce binding of the human layilin to one or both of collagen IV and collagen V. In some embodiments, the binding molecule comprises a binding peptide or an antibody, such as an antibody comprising a layilin-binding domain as described herein. In some embodiments, the method further comprises detecting or having detected (a) one or both of the collagen IV or collagen V, and/or (b) a cell known or suspected of expressing one or both of the collagen IV or collagen V.

[0016] In an additional aspect, the disclosure provides a method of identifying a binding molecule that interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V; the method comprising: having contacted or contacting a polypeptide comprising the layilin ECD with one or more binding molecules in the presence of the collagen IV, collagen V, and/or a layilin-binding fragment thereof, having determined or determining that one or more binding molecules interferes with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof, thereby identifying the binding molecule that interferes with binding between the layilin ECD and collagen IV and/or collagen V.

[0017] The disclosure also provides a method of determining that a binding molecule interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V; the method comprising: having contacted or contacting the binding molecule with a composition comprising a polypeptide comprising the layilin ECD and one or more of collagen IV, collagen V, or a layilin-binding fragment thereof, optionally wherein the polypeptide comprising the layilin ECD and/or the collagen is expressed on the surface of a cell, having measured or measuring binding between the binding molecule and the polypeptide comprising the layilin ECD and/or collagen, and having determined or determining that the binding molecule interferes with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof.

[0018] In another aspect, the disclosure provides a method of determining that a binding molecule interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V, the method comprising: having contacted or contacting a polypeptide comprising the layilin ECD with at least one of collagen IV, collagen V, or a layilin-binding fragment thereof, optionally wherein the polypeptide comprising the layilin ECD and/or the collagen is expressed on the surface of a cell, under a first condition in which a binding molecule is present and under a second condition in which the binding molecule is absent, having measured or measuring binding between the polypeptide comprising the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof, and having determined or determining that the binding molecule interferes with binding between the polypeptide comprising the layilin ECD and the collagen IV, collagen V, and/or layilin- binding fragment thereof when the binding between the polypeptide and collagen is lower in the first condition relative to the second condition.

[0019] Additionally, the disclosure provides a method of determining that a binding molecule interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V; the method comprising: having contacted or contacting the binding molecule with a composition comprising a polypeptide comprising either (a) the layilin ECD or (b) the collagen IV, collagen V, and/or layilin-binding fragment thereof, optionally wherein the polypeptide comprising the layilin ECD and/or the collagen is expressed on the surface of a cell, subsequently having contacted or contacting the composition with (a) the collagen IV, collagen V, and/or layilin-binding fragment thereof when the composition comprises or comprised the layilin ECD, or (b) the layilin ECD when the composition comprises or comprised the collagen IV, collagen V, and/or layilin-binding fragment thereof, optionally wherein the subsequent contact is in the continued presence of the binding molecule, and having measured or measuring binding between the polypeptide comprising the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof, and having determined or determining the binding molecule interferes with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof when binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof is reduced or eliminated.

[0020] In some embodiments of the preceding methods of identifying a binding molecule and/or determining whether a binding molecule interferes with layilin ECD binding to collagen IV and/or collagen V, the binding molecule specifically binds layilin. In some embodiments, the binding molecule comprises an antibody, for example, an antibody comprising a layilin-binding domain as described herein. In some embodiments, the antibody specifically binds an epitope distinct from that bound by anti-layilin antibody 3F7D7E2 and/or 4C11. In some embodiments, the determining step further comprises comparing to the binding determined between the layilin and collagen under conditions wherein the binding molecule is an antibody comprising a layilin-binding domain as described herein. In some embodiments, the binding molecule is determined to interfere with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof when the binding between the layilin ECD and collagen is equal to or lower than under conditions wherein the binding molecule is an antibody comprising a layilin-binding domain as described herein . In some embodiments, the determining step further comprises comparing to the binding determined between the layilin and collagen under conditions wherein the binding molecule is anti-layilin antibody 3F7D7E2, 4C11, and/or a binding fragment thereof. In some embodiments, the binding molecule is determined to interfere with binding between the layilin ECD and the collagen IV, collagen V, and/or layibn-binding fragment thereof when the binding between the polypeptide and collagen is lower than under conditions wherein the binding molecule is anti-layilin antibody 3F7D7E2, 4C11, and/or a binding fragment thereof. In some embodiments, In some embodiments, the polypeptide comprises a multimer of the human layilin ECD. In some embodiments, the multimer comprises a dimer. In some embodiments, the multimer comprises a trimer. In some embodiments, the multimer comprises a pentamer. In some embodiments, the contacting is performed in the presence of Ca²⁺, optionally wherein prior to the contacting in the presence of Ca²⁺, the one or more binding molecules have been pre-contacted with the polypeptide comprising the layilin ECD or the collagen IV, collagen V, and/or layilin-binding fragment thereof in the absence of Ca2⁺ and one or more binding molecules that bind to the layilin ECD or collagen in a calcium- independent manner have been removed prior to the contacting. In some embodiments, the selecting step comprises eluting with a calcium-chelating agent, optionally wherein the calcium-chelating agent is EDTA or EGTA. In some embodiments, the collagen IV and/or collagen V is glycosylated. In some embodiments, the layilin ECD is a human, murine, or cynomolgus layilin ECD. In some embodiments, the layilin ECD is a human layilin ECD. In some embodiments, the binding molecule interferes with binding to a C-type lectin domain of the layilin ECD. In some embodiments, the binding molecule interferes with binding between the layilin ECD with collagen VI.

BRIEF DESCRIPTION OF THE DRAWINGS [0021] FIG. 1 A-E: Ligand discovery for layilin on A375 melanoma cells. A) Constructs used to characterize layilin binding include Fc fusions and small coiled-coil multivalent assemblies. B) Multivalency is a factor for efficient binding of soluble layilin to A375 cells as assessed in this experiment. C) Calcium-dependent binding of hLAYNl-COMP to A375 cells was observed. D) SILAC-AP/MS strategy to characterize calcium-dependent ligands for layilin. E) Quantitative AP/MS shows collagen and collagen-associated proteins are highly enriched in the presence of calcium.

[0022] FIG. 2A-H: Highly glycosylated collagens bind immobilized hLAYNl-CCDi. A) Type IV collagen, B)Type II collagen, C) Type V collagen, and D) Type VI collagen bind to immobilized layilin only in the presence of CaCh. E) Type I and F) Type III collagens do not bind. G) Oxidation of sugars on Type IV collagen ablates binding to immobilized layilin. H) Mutation of the conserved Ca²⁺-binding EPS motif to APS ablates binding of Type IV collagen to hLAYN2-Fc.

[0023] FIG. 3A-E: Differential phage display to isolate layilin-binding antibodies. A) Two strategies were used to isolate layilin binders in Strategy i, standard binders were selected by clearing the library of Fc-binders, selecting in PBS, followed by elution with TEV protease and infection of E. coli. In Strategy ii, Ca²⁺-sensitive binders were selected by clearing the library with EGTA-treated layilin, selecting in TBS with CaCh. and eluting with EGTA. B,

C, and D) Titration of soluble B) ML3D12 (strategy i), C) HL3A9 (strategy i), and D) HL2E8 (strategy ii) IgGs binding to immobilized hLAYNl-CCDi. E) Binning of IgGs shows that HL2E8 blocks Type IV collagen binding to immobilized hLAYNl-CCDi and is dependent on CaCh for full affinity.

[0024] FIG. 4A-E: Effects of Binding anti-Layilin antibodies to cells. A) HL2E8 binding is decreased by EGTA treatment, while B) HL3A9, C) ML3D12, and D) Sino 3F7D7E2 are unaffected. E) Binding of soluble hLAYNl-CCDi to A375 melanoma cells is affected by antibody binding. HL2E8 effectively blocks layilin from binding while alternate epitope binding mAbs HL3A9 and ML3D12 allow binding. Sino 3F7D7E2 also blocks binding.

[0025] FIG. 5A-B: Testing Layilin’s Binding to Hyaluronic Acid. A) Biotinylated layilin isoforms fused to either human IgGl Fc or COMP were tested for binding to plate-bound hyaluronic acid in TBST with 1 mM CaCh for 1 hour at room temperature. Biotinylated hyaluronic acid binding protein from bovine nasal cartilage (HABP) was used as a positive control. B) Hyaluronic acid in solution (0.03 mg/ml) showed no apparent binding to sensor- immobilized, dimerized human layilin isoform 1.

[0026] FIG. 6A-B: Collagenase treatment decreases binding of layilin fusions to A375 melanoma cells. A375 cells were lifted with EDTA and digested with collagenase for 1 hour at 37 °C. Cells were then washed and stained with biotinylated hLAYN2-Fc (A) or hLAYN2- COMP (B) followed by Streptavidin-PE and analyzed by flow cytometry.

[0027] FIG. 7A-F: Binding of different collagens to C-type lectins. A) Native Type IV collagen from Abeam binds immobilized hLAYNl-CCDi. B) Human Type I collagen (Sigma) is unable to bind. C) Type V collagen from Abeam binding is Ca²⁺-dependent D) Binding of Type IV collagen from Sigma is not affected by co-incubation with hyaluronic acid. Type IV collagen is also able to bind E) cynomolgus layilin and F) murine layilin fused to human IgGl Fc.

[0028] FIG. 8:Binning of commercially available anti-layilin Sino 3F7D7E2 against Type IV collagen shows the antibody is able to bind immobilized hLAYNl-CCDi while it is bound to collagen, but collagen binding is mostly blocked by the antibody.

[0029] FIG. 9A-E: Cross-reactivity and binning of HL2E8. A) HL2E8 is able to bind to immobilized hLAYNl-CCDi in the presence of Type IV collagen, but blocks collagen binding. B) Titrations of HL2E8 on immobilized B) cynomolgus and C) murine layilin show cross-reactivity with some increase in off-rate against murine protein. D) and E) Binding of HL2E8 is dependent on calcium against both D) cyno and E) murine protein.

[0030] FIG. 10. Octet binding analysis of HL2E8r binding to various collagen types.

[0031] FIG. 11: analysis of integrin LFA-1 activation.

DETAILED DESCRIPTION OF THE INVENTION

[0032] As used in herein, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules, and the like.

[0033] As used herein, the term “layilin” refers a human protein encoded by the LAYN gene localized to chromosome region 1 lq23. Layilin can refer to any isoform of layilin including. Illustrative layilin polypeptide sequence are available under UniProt entry Q6UX15.

Multiple have been identified, including three isoforms having the sequences designated as Q6UX15-1, Q6UX15-2, and Q6UX15-3 in the UniProt entry, which designated isoform-1 as the canonical sequence. The layilin amino acid sequences of isoform 1-3 are provides as SEQ ID NOS: 12-14, respectively. SEQ ID NO: 11 shows the sequence of the extracellular domain of isoform 1, corresponding to positions 22-235 of SEQ ID NO: 12. Other isoforms include, but are not limited to, UniProt Accession numbers E9PMI0, E9PQU7, A0A0D9SFG0, E9PK64, E9PR90, E9PQY8.

[0034] As used herein, a “layilin-binding domain” or “LBD” refers to the region of an antibody that specifically binds to an antigenic epitope of the extracellular domain (ECD) of layilin, e.g., the ECD of layilin isoform 1. An LBD comprises at least the portion of the VH region that comprises the three heavy chain CDRs. In typical embodiments, the layilin- binding domain comprises a VH region that comprises three heavy chain CDRs (HCDRs) and a VL region comprising three light chain CDRs (LCDRs).

[0035] As used herein, the term "antibody" means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and includes, but is not limited to, a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, single domain antibodies, such as nanobodies, diabodies, camelid- derived antibodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including, but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.

[0036] "Antibody fragments" comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific or mutlivalent antibodies formed from antibody fragments. A "Fab" fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. A F(ab')2 fragment has a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known. An "Fv" is a minimal antibody fragment that contains a complete antigen- recognition and binding site and is a dimer of one heavy- and one light-chain variable region domain.

[0037] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG4.

[0038] As used herein, “V -region” refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4.

[0039] As used herein, "complementarity-determining region (CDR)" refers to the three hypervariable regions that interrupt the four "framework" regions of s variable domain. The CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of each heavy or light chain are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. .

[0040] The amino acid sequences of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Rabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et ak, supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et ak, 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et ak, 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et ak, J.Mol.Biol 1997, 273(4)).

Definitions of CDRs are also described in the following: Ruiz et ak, IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan l;29(l):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymoh, 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996). Reference to CDRs as determined by Rabat numbering are based, for example, on Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0041] “Epitope" or "antigenic determinant" as used in the present disclosure in the context of antibody binding refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids and/or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Voh 66, Glenn E. Morris, Ed (1996). Binding of an antibody to an epitope can be influenced by other environmental factors, such as s the presence of calcium ions. [0042] The term "valency" as used herein refers to the number of different binding sites of an antibody for an antigen. A monovalent antibody comprises one binding site for an antigen. A multivalent antibody comprises multiple binding sites.

[0043] The term “monovalent antibody” as used herein, refers to an antibody that binds to a single epitope on a target molecule.

[0044] The term “bivalent antibody” as used herein, refers to an antibody that has two antigen binding sites.

[0045] The term “multivalent antibody” refers to a single binding molecule with more than one valency, where “valency” is described as the number of antigen-binding moieties present per molecule of an antibody construct. As such, the single binding molecule can bind to more than one binding site on a target molecule. Examples of multivalent antibodies include, but are not limited to, bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, and the like, as well as bispecific antibodies.

[0046] The term “bispecific antibody” as used herein, refers to an antibody that binds to two or more different epitopes. In some embodiments, a bispecific antibody binds to epitopes for two different target antigens. In some embodiments, a bispecific antibody binds to two different epitopes for the same target antigen.

[0047] The phrases “monoclonal antibody” or “monoclonal antibody composition” as used herein refers to polypeptides, including antibodies, bispecific antibodies, etc., that have substantially identical amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0048] As used herein, the term “specifically binds” to a target, e.g., layilin, when referring to a layibn-binding protein as described herein, refers to a binding reaction whereby the layibn-binding protein binds to layilin with greater affinity, greater avidity, and/or greater duration than it binds to a different target. In some embodiments, a layibn-binding protein has at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25- fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for layilin compared to an unrelated target when assayed under the same binding affinity assay conditions. The term “specific binding,” “specifically binds to," or “is specific for" a particular target (e.g., layilin), as used herein, can be exhibited, for example, by a molecule ( e.g ., a layilin-binding protein) having an equilibrium dissociation constant KD for layilin of, e.g., 10² M or smaller, e.g., 10³ M, 10⁴ M, lO ⁵ M, lO ⁶ M, 10⁷ M, 10⁸ M, 10⁹M, 10 ⁰ M, 10⁴¹ M, or 10⁴² M. In some embodiments, a laying binding protein has a Kx> of less than 100 nM or less than 10 nM. For purposes of this application, KD values can be determined by biolayer interferometry, e.g., using ForteBio Octet intstrumentation and methodology, using antibodies in an IgG format (see, the Examples section of the application).

[0049] The term “binding specificity” as used herein refers to the ability of an individual antibody to interact with one antigenic determinant and not with a different antigenic determinant.

[0050] The term “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down an undesired physiological change or disorder. For purpose of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0051] As used herein, the term “subject” refers to a mammal, e.g., preferably a human. Mammals include, but are not limited to, humans and domestic and farm animals, such as monkeys (e.g., a cynomolgus monkey), mice, dogs, cats, horses, and cows, etc.

[0052] As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present invention, the pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to the active ingredient. The nature of the carrier differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.

[0053] As used herein, the term “treat” refers to a therapeutic treatment of a disease in a subject, as well as prophylactic or preventative measures towards the disease. A therapeutic treatment slows the progression of the disease, ameliorates disease symptoms, improves the subject’s outcome ( e.g survival), eliminates the disease, and/or reduces or eliminates the symptoms of the disease. Beneficial or desired clinical results include, but are not limited to, alleviation of disease symptoms, diminishment of the extent of the disease, stabilization (i.e., not worsening) of the disease, delay or slowing of the disease progression, amelioration or palliation of the disease state, remission (whether partial or total, whether detectable or undetectable) and prevention of relapse or recurrence of the disease. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already having the disease, condition, or disorder, as well as those at high risk of having the disease, condition, or disorder, and those in whom the disease, condition, or disorder is to be prevented.

[0054] The terms “identical” or percent “identity,” in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotide or amino acid residues that are the same (e.g., at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region. Alignment for purposes of determining percent y can be performed in various methods, including those using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 11, an expect threshold of 0.05, M=2, N=-3, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 6, an expect threshold of 0.05, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). Thus, for purposes of this disclosure, BLASTP can be used with the default parameters to determine percent sequence identity.

[0055] The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a variable domain polypeptide “corresponds to” an amino acid in the variable domain polypeptide of SEQ ID NO: 1 when the residue aligns with the amino acid in SEQ ID NO: 1 when optimally aligned to SEQ ID NO: 1. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.

[0056] A “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, hydrophobicity, and/or size of the side group chain is maintained. Illustrative sets of amino acids that may be substituted for one another include (i) positively- charged amino acids Lys, Arg and His; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) large aliphatic nonpolar amino acids Val, Leu and lie; (vi) slightly polar amino acids Met and Cys; (vii) small-side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and Pro; (viii) aliphatic amino acids Val, Leu, lie, Met and Cys; and (ix) small hydroxyl amino acids Ser and Thr. Reference to the charge of an amino acid in this paragraph refers to the charge at physiological pH.

[0057] The terms “nucleic acid” and “polynucleotide” are used interchangeably and as used herein refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide, and combinations thereof. The terms also include, but is not limited to, single- and double- stranded forms of DNA. In addition, a polynucleotide, e.g., a cDNA or mRNA, may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. The nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analogue, intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The above term is also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed, triplex, hairpinned, circular and padlocked conformations. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term also includes codon- optimized nucleic acids that encode the same polypeptide sequence.

[0058] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. A “vector” as used here refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

DETAILED DESCRIPTION

[0059] Provided herein are anti-layilin antibodies that comprise a layilin-binding domain that specifically binds to the extracellular domain of layilin and interferes with binding between the layilin ECD and Type IV and/or Type V collagen. In som emodiments, the layilin binding domain interferes with binding between the layilin ECD and Type IV and Type II collagen. As used herein, the term “interferes with binding” means that the level of binding of layilin ECD is reduced in the presence of the layilin-binding domain in a binding reaction as further detailed below. A layilin-binding domain is considered to interfere with binding if it reduces binding of the layilin ECD Type IV and/or Type V collagen in a binding interaction. In some embodiments, binding is reduced when the layilin-binding domain is incubated with collagen in a binding reaction before ECD is introduced into the reaction. In some embodiments, binding is reduced when the layilin-binding domain and ECD are provided at the same time in the binding reaction. In some embodiments, binding is reduced when the layilin-binding domain is introduced into the binding reaction after ECD is incubated with the collagen target. In some embodiments, binding of the layilin-binding domain is Ca²⁺-dependent, i.e., calcium ions are provided in the binding reaction in order to achieve higher affinity binding compared to an identical binding reaction without calcium. In some embodiments, a layilin binding domain of the present disclosure does not substantially activate integrin LFA-1, e.g., activation of less than 1% when analyzed as described in the EXAMPLES section.

[0060] In some embodiments, a layilin binding domain that specifically binds a layilin ECD in a Ca²⁺-dependent manner has at least one, at least two, or three CDRs of a VH region sequence of SEQ ID NO:7 and/or at least one, at least two, or three CDRs of a VL region sequence of SEQ ID NO: 8.

[0061] In some embodiments, a layilin-binding domain comprises an HCDR3 of a VH region of SEQ ID NO:7 in which 1, 2, or 3 amino acids is substituted relative to SEQ ID NO:7. In some embodiments, a layilin-binding domain comprises an HCDR3 of SEQ ID NO:3 in which 1, 2, or 3 amino acids are substituted. In some embodiments, a layilin-binding domain further comprises a CDR1 of the VH region of SEQ ID NO:7 in which 1, 2, or 3 amino acid are substituted and/or a CDR2 of the VH region of SEQ ID NO:7 in which 1, 2, or 3 amino acids are substituted. The HCDRs of SEQ ID NO:7 can be defined by Chothia, IMGT, AbM, or Rabat, or a combination. In some embodiments, the CDRs are defined by Rabat. In some embodiments, a layilin-binding domain comprises an HCDR1 of SEQ ID NO:l in which 1, 2, or 3 amino acids are substituted and/or an HCDR2 of SEQ ID NO:2 in which 1, 2, or 3 amino acids are substituted. In some embodiments, a layilin-binding domain comprises an HCDR1 of SEQ ID NO: 1, or a variant thereof in which 1 or 2 amino acids are substituted; an HCDR2 of SEQ ID NO:2; or a variant thereof in which 1 or 2 amino acids are substituted; and an HCDR3 of SEQ ID NO:3, or a variant thereof in which 1 or 2 amino acids are substituted.

[0062] In some embodiments, a layilin-binding domain comprises an LCDR3 of a VL region of SEQ ID NO: 8 in which 1, 2, or 3 amino acids is substituted relative to SEQ ID NO: 8. In some embodiments, a layilin-binding domain comprises an LCDR3 of SEQ ID NO:6in which 1, 2, or 3 amino acids are substituted. In some embodiments, a layilin-binding domain comprises a CDR1 of the VL region of SEQ ID NO:8 in which 1, 2, or 3 amino acid are substituted and/or a CDR2 of the VL region of SEQ ID NO: 8 in which 1, 2, or 3 amino acids are substituted. The LCDRs of SEQ ID NO: 8 can be defined by Chothia, IMGT, AbM, or Rabat, or a combination. In some embodiments, the CDRs are defined by Rabat. In some embodiments, a layilin-binding domain comprises an LCDR1 of SEQ ID NO:4 in which 1, 2, or 3 amino acids are substituted and/or an LCDR2 of SEQ ID NO:5 in which 1, 2, or 3 amino acids are substituted. In some embodiments, a layilin-binding domain comprises: an LCDR1 of SEQ ID NO:4, or a variant thereof in which 1 or 2 amino acids are substituted; an LCDR2 of SEQ ID NO:5; or a variant thereof in which 1 or 2 amino acids are substituted; and an CDR3 of SEQ ID NO:6, or a variant thereof in which 1 or 2 amino acids are substituted.

[0063] In some embodiments, a layilin-binding domain comprises a VH region and a VL region, wherein: (a) the VH region comprises a CDR1 of the VH region of SEQ ID NO: 7, or a variant thereof in which 1 or 2 amino acid are substituted, a CDR2 of the VH region of SEQ ID NO: 7 or a variant thereof in which 1 or 2 amino acids are substituted; and an HCDR3 of a VH region of SEQ ID NO:7, or a variant thereof in which 1 or 2 amino acids are substituted; and (b) the VL region comprises a CDR1 of the VL region of SEQ ID NO: 8, or a variant thereof in which 1 or 2 amino acid are substituted, a CDR2 of the VL region of SEQ ID NO: 8 or a variant thereof in which 1 or 2 amino acids are substituted; and an HCDR3 of a VL region of SEQ ID NO: 8, or a variant thereof in which 1 or 2 amino acids are substituted. The CDRs can be defined by Chothia, IMGT, AbM, or Rabat, or a combination, e.g., of Rabat and Chothia. In some embodiments, the VH region comprises a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO:2, and a CDR3 of SEQ ID NO:3; and the VL region comprises a CDR1 of SEQ ID NO:4, a CDR2 of SEQ ID NO:5, and a CDR3 of SEQ ID NO:6.

[0064] In some embodiments, a layilin-binding domain of the present disclosure comprises a VH region having at least 80%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the VH region comprises substitutions, insertions, or deletions in the framework of the VH region of SEQ ID NO:7. In some embodiments, binding domain further comprises a VL region having at least 80%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VL region comprises substitutions, insertions, or deletions in the framework of the VL region of SEQ ID NO: 8.

[0065] In some embodiments, a variable region of SEQ ID NO:7 or SEQ ID NO:8 comprises an insertion or deletion, e.g., a deletion of 1, 2, 3, 4, 5, 6, or more amino acids; or an insertion of 1, 2, 3, 4, 5, or 6 or more amino acids. In some embodiments a CDR of any one of SEQ ID NOS: 1 to 6 comprises a 1 or 2 amino acid deletion; or a 1 or 2 amino acid insertion.

[0066] In some embodiments, a mutation, e.g., a substitution, is introduced into a CDR or FR to alter an N-linked glycosylation motif; to alter charge and/or hydrophobicity, or to remove, e.g., by substitution, one or more amino acid that may undergo chemical modification during storage, such as (1) oxidation (methionine, cysteine, histidine, tyrosine, tryptophan, and phenylalanine), (2) intra- and inter-residue cyclization (aspartic and glutamic acid, asparagine, glutamine, N-terminal dipeptidyl motifs), and (3) b-elimination (serine, threonine, cysteine, cystine) reactions.

Methods of identifying layilin-binding domains that bins to collagen type IV and/or Collagen Type V.

[0067] Additional layilin-binding domains that interfere with binding between layilin ECD and one or both of collagen Type IV or collagen Type V can be identified by evaluating the ability of a candidate layilin-binding domain to bind to collagen Type IV and/or Type V, or a layilin-binding fragment of collagen IV or collagen V, and disrupt ECD-collagen binding interactions. In some embodiments, the layilin ECD employed to assess binding is a multimer, e.g., atrimeric, tetrameric, or pentameric form. In some embodiments, binding to layilin ECD is assessed in as assay in which the ECD is a soluble ECD in the form of an ECD-Fc fusion protein. In some embodiments, the binding reaction comprises Ca²⁺. In some embodiments, the layilin ECD is human or cynomolgus. In some embodiments, the collagen IV and/or collagen V is glycosylated.

[0068] In the present disclosure, “interferes with binding” between layilin ECD and one or both of collagen Types IV and Type V, or a layilin -binding fragment of the collagen molecule, refers to the ability of a binding agent to prevent or inhibit binding of layilin ECD to collagen Type IV, collagen Type V, or both. In some emobdiments, a layilin binding domain of the present disclosure prevents or inhibits binding between layilin ECD and collagen Types IV and Type II. In some embodiments, a layilin binding domain of the present disclosure prevents or inhibits binding to Type IV collagen, Type II collagen, Type V collagen, and Type VI collagen. In some embodiments, a binding agent, such as a candidate antibody, inhibits binding by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, or greater. In some embodiments, the binding agent is a bivalent, bispecific, multivalent, or multispecific antibody in which at least one of the antigen binding domains comprises an antibody comprising the CDRs set forth in SEQ ID NO: 1-6.

[0069] Binding can be assessed using any method. Illustrative embodiments are further described below and are provided in the EXAMPLES section. In some embodiments, the candidate binding domain is provided in a binding reaction that comprises the layilin ECD for incubation with the collagen Type IV and/or collagen Type V. In other embodiments, a candidate layilin-binding domain is introduced into the binding reaction before the layilin ECD. In some embodiments, the candidate layilin-binding domain and ECD are introduced into the binding reaction at the same time. In some embodiments, the candidate layilin- binding domain is introduced into the binding reaction after the layilin EC. In some embodiments, the binding assay comprises a cell comprising collagen Type IV and/or collagen Type V on the surface to evaluate the ability of the candidate laylin binding domain to interfere with layin binding to collagen Type IV and/or Type V. In some embodiments, the ability of a candidate layilin-binding domain to interfere with binding between layilin ECD and one or both of collagen Types IV and/or V, can be assessed in a competitive binding assay employing an antibody that comprises the CDR sequences set forth in SEQ ID NOS: 1-6. In some embodiments, a candidate layilin-binding domain is further evaluated in a competitive binding assays employing 3F7D7E2 (Sino Biologicals) or 4C11 (Novus Biologicals). In some embodiments, the candidate binding agent is tested in the same antibody format, e.g., as an IgG, as an antibody comprising the CDR sequences set forth in SEQ ID NOS: 1-6. In some embodiments, the candidate binding agent is a variant of an antibody that comprises the CDRs of SEQ ID NOS: 1-6 in which at least one of the CDRs comprises one or two mutations, e.g., one or two substitutions.

[0070] In some embodiments, the disclosure provides a method of identifying a binding molecule that interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V; the method comprising contacting a polypeptide comprising the layilin ECD with one or more binding molecules in the presence of the collagen IV, collagen V, and/or a layilin-binding fragment thereof, and determining that one or more binding molecules interferes with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof, thereby identifying the binding molecule that interferes with binding between the layilin ECD and collagen IV and/or collagen V. In some embodiments, the candidate binding agent is an antibody. In some embodiments, contacting is performed in the presence of Ca²⁺, optionally wherein prior to the contacting in the presence of Ca²⁺, the one or more binding molecules have been pre contacted with the polypeptide comprising the layilin ECD or the collagen IV, collagen V, and/or layilin-binding fragment thereof in the absence of Ca²⁺ and one or more binding molecules that bind to the layilin ECD or collagen in a calcium-independent manner have been removed prior to the contacting. In some embodiments, the method further comprises a selecting step comprising eluting with a calcium-chelating agent, optionally wherein the calcium-chelating agent is EDTA or EGTA. In some embodiments, the collagen IV and/or collagen V is glycosylated. In some embodiments, the binding molecule interferes with binding to a C-type lectin domain of the layilin ECD. In some embodiments, the binding molecule interferes with binding between the layilin ECD with collagen VI.

[0071] In additional embodiments, the disclosure provides a method of determining that a binding molecule interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V; the method comprising contacting the binding molecule with a composition comprising a polypeptide comprising the layilin ECD and one or more of collagen IV, collagen V, or a layilin-binding fragment thereof, optionally wherein the polypeptide comprising the layilin ECD and/or the collagen is expressed on the surface of a cell, measuring binding between the binding molecule and the polypeptide comprising the layilin ECD and/or collagen, and determining that the binding molecule interferes with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin binding fragment thereof. In some embodiments, the candidate binding agent is an antibody. In some embodiments, contacting is performed in the presence of Ca²⁺, optionally wherein prior to the contacting in the presence of Ca²⁺, the one or more binding molecules have been pre contacted with the polypeptide comprising the layilin ECD or the collagen IV, collagen V, and/or layilin-binding fragment thereof in the absence of Ca²⁺ and one or more binding molecules that bind to the layilin ECD or collagen in a calcium-independent manner have been removed prior to the contacting. In some embodiments, the method further comprises a selecting step comprising eluting with a calcium-chelating agent, optionally wherein the calcium-chelating agent is EDTA or EGTA. In some embodiments, the collagen IV and/or collagen V is glycosylated. In some embodiments, the binding molecule interferes with binding to a C-type lectin domain of the layilin ECD. In some embodiments, the binding molecule interferes with binding between the layilin ECD with collagen VI.

[0072] In other embodiments, the disclosure provides a method of determining that a binding molecule interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V, the method comprising contacting a polypeptide comprising the layilin ECD with at least one of collagen IV, collagen V, or a layilin-binding fragment thereof, optionally wherein the polypeptide comprising the layilin ECD and/or the collagen is expressed on the surface of a cell, under a first condition in which a binding molecule is present and under a second condition in which the binding molecule is absent, measuring binding between the polypeptide comprising the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof, and determining that the binding molecule interferes with binding between the polypeptide comprising the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof when the binding between the polypeptide and collagen is lower in the first condition relative to the second condition.

In some embodiments, the candidate binding agent is an antibody. In some embodiments, contacting is performed in the presence of Ca²⁺, optionally wherein prior to the contacting in the presence of Ca²⁺, the one or more binding molecules have been pre-contacted with the polypeptide comprising the layilin ECD or the collagen IV, collagen V, and/or layilin-binding fragment thereof in the absence of Ca²⁺ and one or more binding molecules that bind to the layilin ECD or collagen in a calcium-independent manner have been removed prior to the contacting. In some embodiments, the method further comprises a selecting step comprising eluting with a calcium-chelating agent, optionally wherein the calcium-chelating agent is EDTA or EGTA. In some embodiments, the collagen IV and/or collagen V is glycosylated.

In some embodiments, the binding molecule interferes with binding to a C-type lectin domain of the layilin ECD. In some embodiments, the binding molecule interferes with binding between the layilin ECD with collagen VI.

[0073] In further embodiments, the disclosure provides a method of determining that a binding molecule interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V; the method comprising contacting the binding molecule with a composition comprising a polypeptide comprising either (a) the layilin ECD or (b) the collagen IV, collagen V, and/or layilin-binding fragment thereof, optionally wherein the polypeptide comprising the layilin ECD and/or the collagen is expressed on the surface of a cell, subsequently contacting the composition with (a) the collagen IV, collagen V, and/or layilin-binding fragment thereof when the composition comprises the layilin ECD, or (b) the layilin ECD when the composition comprises the collagen IV, collagen V, and/or layilin-binding fragment thereof, optionally wherein the subsequent contact is in the continued presence of the binding molecule; measuring binding between the polypeptide comprising the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof, and determining the binding molecule interferes with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof when binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof is reduced or eliminated. In some embodiments, the candidate binding agent is an antibody. In some embodiments, contacting is performed in the presence of Ca²⁺, optionally wherein prior to the contacting in the presence of Ca²⁺, the one or more binding molecules have been pre-contacted with the polypeptide comprising the layilin ECD or the collagen IV, collagen V, and/or layilin-binding fragment thereof in the absence of Ca²⁺ and one or more binding molecules that bind to the layilin ECD or collagen in a calcium-independent manner have been removed prior to the contacting. In some embodiments, the method further comprises a selecting step comprising eluting with a calcium-chelating agent, optionally wherein the calcium-chelating agent is EDTA or EGTA. In some embodiments, the collagen IV and/or collagen V is glycosylated. In some embodiments, the binding molecule interferes with binding to a C-type lectin domain of the layilin ECD. In some embodiments, the binding molecule interferes with binding between the layilin ECD with collagen VI.

Antibody formats

[0074] As previously explained, a layilin-binding domain, e.g., a VH region and/or a VL region of an anti-layilin antibody as described herein, may be incorporated into a bivalent antibody or a multivalent antibody that binds to the same, or a different, antigen. In some embodiments, a layilin-binding domain may be incorporated into a bispecific antibody or multispecific antibody that binds to the an antigen at different epitopes, or that binds to different antigens. Illustrative antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212).

[0075] In some embodiments, such an antibody comprising a layilin-binding domain as described herein further comprises an Fc region. The term “Fc region” as used herein refers to a polypeptide comprising the CH3, CH2 and at least a portion of the hinge region of a constant domain of an antibody. In some embodiments, an Fc region can include a CH4 domain, present in some antibody classes. In some embodiments, an Fc region, can comprise the entire hinge region of a constant domain of an antibody. In one embodiment, an antibody comprises an Fc region and a CHI region. In one embodiment, the antibody comprises an Fc region, a CHI region and a Ckappa/lambda region. In one embodiment, an antibody comprises a constant region, e.g., a heavy chain constant region. In some embodiments, such a constant region is modified compared to a wild-type constant region i.e., a constant region may comprise alterations or modifications to one or more of the CHI, CH2 or CH3 domain and/or to the CL domain. Example modifications include additions, deletions or substitutions of one or more amino acids in one or more domains. Illustrative mutations are known, e.g., mutations that modulate effector function and/or serum half-life.

[0076] In some embodiments, a layilin-binding domain employed in a binding interaction comprises an antibody fragment, e.g., aFab, a F(ab’)2, an Fv, an scFv antibody, a VH, or a VHH. In some embodiments, a layilin-binding domain of the present disclosure is linked to a second layilin-binding domain as described herein. In some embodiments, a layilin-binding domain is provided in an scFV antibody as part of a bispecific antibody. Thus, for example, in some aspects, an anti-layibn-binding domain of the present invention may be incorporated into a bispecific antibody having a second binding domain that targets a different antigen on an immune effector cell, such as a T cell.

[0077] In some embodiments, a layilin-binding domain as described herein may be a chimeric antibody, an affinity -mature, humanized, or human antibody. In some embodiments, a layilin-binding domain may be present as an antigen binding domain of a larger molecule, e.g., present as an antigen binding domain of a chimeric receptor, such as an antigen receptor or synthetic Notch receptor, as further described below below. In further embodiments, a bispecific antibody, multispecific antibody, chimeric antigen receptor, synthetic Notch receptor, or other layilin-binding domain-containing construct, may comprise more than one layilin-binding domain that differs in sequence and/or binding specificity. In some embodiments, two, three, or four layilin-binding domains may be present in an antibody, chimeric antigen receptor or synthetic Notch receptor.

[0078] Genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Optionally, phage or yeast display technology can be used to identify antibodies and Fab fragments that specifically bind to layilin and/or other selected antigen of a bispecific antibody. Techniques for the production of single chain antibodies or recombinant antibodies can also be adapted to produce antibodies. [0079] Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression, such as a hybridoma, or a CHO cell expression system.

Many such systems are widely available from commercial suppliers. In embodiments in which an antibody comprises both a VH and VL region, the VH and VL regions may be expressed using a single vector, e.g., in a di-cistronic expression unit, or under the control of different promoters. In other embodiments, the VH and VL region may be expressed using separate vectors. A VH or VL region as described herein may optionally comprise a methionine at the N-terminus. Methods of generating and screening hybridoma cell lines, including the selection and immunization of suitable animals, the isolation and fusion of appropriate cells to create the hybridomas, the screening of hybridomas for the secretion of desired antibodies, and characterization of the antibodies are known to one of ordinary skill in the art.

[0080] In some embodiments, the antibody is a chimeric antibody. Methods for making chimeric antibodies are known in the art. For example, chimeric antibodies can be made in which the antigen-binding region (heavy chain variable region and light chain variable region) from one species, such as a mouse, is fused to the effector region (constant domain) of another species, such as a human. As another example, “class switched” chimeric antibodies can be made in which the effector region of an antibody is substituted with an effector region of a different immunoglobulin class or subclass.

[0081] In some embodiments, the antibody is a humanized antibody. Generally, a non human antibody is humanized in order to reduce its immunogenicity. Humanized antibodies typically comprise one or more variable regions (e.g., CDRs) or portions thereof that are non human (e.g., derived from a mouse variable region sequence), and possibly some framework regions or portions thereof that are non-human, and further comprise one or more constant regions that are derived from human antibody sequences. Methods for humanizing non human antibodies are known in the art. Transgenic mice, or other organisms such as other mammals, can be used to express humanized or human antibodies. Other methods of humanizing antibodies include, for example, variable region resurfacing, CDR grafting, grafting specificity-determining residues (SDR), guided selection, and framework shuffling. CAR constructs comprising a layilin-binding domain

[0082] Chimeric antigen receptors (CARs) are recombinant receptor constructs comprising an extracellular antigen-binding domain (e.g., a layilin-binding domain as described herein) joined to a transmembrane domain, and further linked to an intracellular signaling domain (e.g., an intracellular T cell signaling domain of a T cell receptor) that transduces a signal to elicit a function. In certain embodiments, immune cells (e.g., T cells or natural killer (NK) cells) are genetically modified to express CARs that comprise one or more layilin-binding domains of the present disclosure and have the functionality of effector cells (e.g., T cell cytotoxic fucntions).

[0083] In a standard CAR, the components include an extracellular targeting domain, a transmembrane domain and intracellular signaling/activation domain, which are typically linearly constructed as a single fusion protein. In the present invention, the extracellular region comprises a layilin-binding domain as described herein. The "transmembrane domain" is the portion of the CAR that links the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the host cell that is modified to express the CAR, e.g., the plasma membrane of an immune effector cell. The intracellular region may contain a signaling domain of TCR complex, and/or one or more costimulatory signaling domains, such as those from CD28, 4- IBB (CD 137) and OX-40 (CD134). For example, a "first-generation CAR" generally has a CD3-zeta signaling domain. Additional costimulatory intracellular domains may also be introduced (e.g., second and third generation CARS) and further domains including homing and suicide domains may be included in CAR constructs. CAR components are further described below.

[0084] In some embodiments, the extracellular domain may comprise two or more layilin- binding domains that bind to Type IV and/or Type V collagen, e.g., in a calcium-dependent manner, as described herein. For example, the extracellular domain may comprise two, three or four layilin-binding domains. In some embodiments, the extracellular domain may comprises multiple copies of the same layilin-binding domain. In some embodiments, the extracellular domain may comprise a layilin-binding domain that comprises the six CDRs set forth in SEQ ID NOS: 1-6 and a layilin-binding domain that binds to a different layilin ECD epitope.

[0085] A CAR construct encoding a CAR may also comprise a sequence that encodes a signal peptide to target the extracellular domain to the cell surface. Hinge domain

[0086] In some embodiments, the CAR may contain one or more hinge domains that link the antigen binding domain comprising the anti-layilin-binding domain and the transmembrane domain for positioning the antigen binding domain. Such a hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region, e.g., a naturally occurring human immunglobuline hinge region, or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1 , CD 152, and CD7, which may be wild-type hinge regions from these molecules or may be altered.

Transmembrane domain

[0087] Any transmembrane suitable for use in a CAR construct may be employed. Such transmembrane domains, include, but are not limited to, all or part of the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4- IBB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB 1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, or NKG2C.

[0088] A transmembrane domain incorporated into a CAR construct may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.

Intracellullar signaling domain

[0089] A CAR construct of the present disclosure includes one or more intracellular signaling domains, also referred to herein as co-stimulatory domains, or cytoplasmic domains that activate or otherwise modulate an immune cell, (e.g., a T lymphocyte). The intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. In one embodiment, a co-stimulatory domain is used that increases CAR immune T cell cytokine production. In another embodiment, a co-stimulatory domain is used that facilitates immune cell (e.g., T cell) replication. In still another embodiment, a co-stimulatory domain is used that prevents CAR immune cell (e.g., T cell) exhaustion. In another embodiment, a co-stimulatory domain is used that increases immune cell (e.g., T cell) antitumor activity. In still a further embodiment, a co-stimulatory domain is used that enhances survival of CAR immune cells (e.g., T cells) (e.g., post-infusion into patients).

[0090] Examples of intracellular signaling domains for use in a CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0091] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.

[0092] Examples of IT AM containing primary intracellular signaling domains include those of CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3- zeta.

[0093] An intracellular signaling domain of a CAR can comprise a primary intracellular signaling domain only, or may comprise additional desired intracellular signaling domain(s) useful in the context of a CAR of the invention. For example, the intracellular signaling domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds to CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB 1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, and CD 19a.

[0094] In some embodiments, a CAR may be designed as an inducible CAR, or may otherwise comprise a mechanisms for reversibly expressing the CAR, or controlling CAR activity to largely restrict it to a desired environment. Thus, for example, in some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in more detail in publications WO2014/055442 and WO2014/055657.

[0095] In some embodiments, a host cell, e.g., a T cell, can be engineered such that a synthetic Notch receptor comprising an extracellular domain comprising a layilin-binding domain as described herein induces the expression of a CAR that targets a second antigen. Such systems are described, e.g., in U.S.. Patent Application Publication No. 20190134093; see also, synNotch polypeptides as described in US20160264665, each incorporated herein by reference. In some embodiments, a synNotch comprises a one or more layilin-binding domains as described herein. In some embodiments, one or more layilin-binding domains is incorporated into a CAR, the expression of which is activated by a synNotch expressed by the host cell.

[0096] In some embodiments, a cell expressing a CAR comprising one or more layilin- binding domains as described herein also expresses a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., that binds to the same target or a different target. [0097] In some embodiments, a host cell, e.g., a host T cell is modified to express a layilin- binding domain as described herein, or a chimeric molecules, such as a chimeric receptor comprising such a domain, using a gene editing system, such as a Cas/CRISPR system, a Transcription activator-like effector nuclease (TALEN) system, a homing endonuclease (HE) system, or a zinc-finger nuclease (ZFN) system.

[0098] Many methods for introducing nucleic acids and viral vectors (e.g., viral particles) into a target cell (e.g., a CD8⁺ T cell) are available. Non-limiting examples of suitable methods include electroporation (e.g., nucleofection), viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, microparticle- or nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, a viral vector may be used, such as an adenovirus, adeno- associated virus (AAv), lentivirus vector, a vaccinia virus vector, or any of a number of different vectors. In some

Activation and Expansion of Immune Effector Cells (e.g., T Cells)

[0099] The invention is not limited by the type of immune cells genetically modified to express a CAR, or synthetic Notch receptor. Illustrative immune cells include, but are not limited to, T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, macrophages, and myeloid-derived phagocytes. In some embodiments, the T cells are CD8+ T cells Treg cells. In some embodiments, the immune cells, e.g., T cells, are autologous cells from the patient to undergo immunotherapy. In some embodiments, the immune cells are allogeneic.

[0100] Immune effector cells such as T cells may be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 2006/0121005. Examples of immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0101] Methods of making CAR-expressing cells are described, e.g., in US2016/0185861 and US2019/0000880. Compositions and Methods of Interfering with layilin-collagen Type IV and or Type V binding interactions

[0102] A layilin-binding domain as described herein can be provided to disrupt binding of layiling to Type IV and/or Type V collagen. In some embodiments, the layilin-binding domain is administered to a subject as a pharmaceutical composition to treat a disease. In some embodiments, the layilin-binding domain is administered to treat cancer or an autoimmune disorder in the subject. In some embodiments, a genetically modified cells, such as a T cell, e.g., a CD8+ T cell or Treg that expresses the layilin-binding domain as a chimeric receptor is administered to treat cancer or an autoimmune disease.

[0103] Pharmaceutical compositions comprising a layilin-binding domain contain one or more pharmaceutically acceptable carriers. Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers, antioxidants, preservatives, polymers, amino acids, and carbohydrates. Pharmaceutical compositions may be administered parenterally in the form of an injectable formulation. Pharmaceutical compositions for injection (i.e., intravenous injection) can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), a-Modified Eagles Medium (a- MEM), F-12 medium). Formulation methods are known in the art, see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Francis Group, CRC Press (2006).

[0104] The pharmaceutical composition may be formed in a unit dose form as needed. The amount of active component, e.g., a layilin-binding protein (e.g., an anti-layilin antibody), included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided (e.g., a dose within the range of 0.01-500 mg/kg of body weight).

[0105] Pharmaceutical compositions described herein may be formulated for subcutaneous administration, intramuscular administration, intravenous administration, parenteral administration, intra-arterial administration, intrathecal administration, or intraperitoneal administration. The pharmaceutical composition may also be formulated for, or administered via, oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, various effective pharmaceutical carriers are known in the art. In some embodiments, pharmaceutical compositions may administered locally or systemically (e.g., locally). In particular embodiments, pharmaceutical compositions may be administered locally at the affected area, such as skin or cancerous tissue.

[0106] The dosage of the pharmaceutical compositions depends on factors including the route of administration, the disease to be treated, and physical characteristics, e.g., age, weight, general health, of the subject. In some embodiments, the amount of active ingredient (e.g., a layilin-binding domain or genetically modified cells, e.g., T cells comprising a chimeric receptor comprising a layilin-binding domain (e.g., modified CD8⁺ T cells) contained within a single dose are administered in an amount that effectively prevents, delays, or treats the disease without inducing significant toxicity. The dosage may be adapted by the physician in accordance with conventional factors such as the extent of the disease and different parameters of the subject.

[0107] The pharmaceutical compositions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The pharmaceutical compositions may be administered in a variety of dosage forms, e.g., subcutaneous dosage forms, intravenous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug release capsules). Pharmaceutical compositions containing the active ingredient (e.g., a layilin-binding protein (e.g., an anti-layilin antibody) or modified T cells (e.g., modified CD8⁺ T cells)) may be administered to a subject in need thereof, for example, one or more times (e.g., 1-10 times or more) daily, weekly, monthly, biannually, annually, or as medically necessary. Dosages may be provided in either a single or multiple dosage regimens. The timing between administrations may decrease as the medical condition improves or increase as the health of the patient declines.

[0108] In some embodiments, a layilin-binding domain or genetically modified cells as described herein comprising a layilin-binding domain are administered to a subject that has cancer. In some embodiments, the layilin-bidning domains is administered to a cancer, e.g., a cancer such as a skin cancer, e.g., melanoma, or any other cancer that contains Type IV and/or Type V collagen on the surface to which layilin binds. In some embodiments, the cancer is a hematological cancer. Examples of different types of cancer involving solid tumros include, but are not limited to, breast cancer, lung cancer (e.g., non-small cell lung cancer); digestive and gastrointestinal cancers such as colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and stomach (gastric) cancer; esophageal cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; ovarian cancer; prostate cancer, renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system; skin cancer; choriocarcinomas; head and neck cancers; osteogenic sarcomas; and hematological cancers, e.g., leukemias or lymphomas of any lineage, e.g., T cell lineage.

[0109] In some embodiments, a layilin-binding domain or genetically modified immune effector cells as described herein comprising a layilin-binding domain are administered to a subject having cancer in conjunction with other cancer therapeutics, e.g., chemotherapeutic agents such as alkylating agents, including thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Nicolaou et al. Angew. Chem Inti. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); combretastatin; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, 5-azacytidine, carmofur, cytarabine, dideoxy uridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®, Bristol- Myers Squibb Oncology, Princeton, N.J.), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®, Rhome-Poulene Rorer,

Antony, France); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP- 16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) (e.g., erlotinib (TarcevaTM)); and VEGF-A that reduce cell proliferation; vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g, ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifamib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); famesyltransferase inhibitors such as lonafamib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin.

[0110] In some embodiments, a layilin-binding domain or immune effector cells that express a chimeric receptor comprising the layilin-binding domain are administered in conjunction with an agent that targets an immune checkpoint antigen. In one aspect, the agent is a biologic therapeutic or a small molecule. In another aspect, the agent is a monoclonal antibody, a humanized antibody, a human antibody, a fusion protein or a combination thereof. In certain embodiments, the agents inhibit, e.g., by blocking ligand binding to receptor, a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDOl, ID02, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, GITR, HAVCR2, LAG3, KIR, LAIR1, LIGHT, MARCO, OX-40, SLAM, , 2B4, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137 (4-1BB), CD160, CD39, VISTA, TIGIT, a SIGLEC, CGEN-15049, 2B4, CHK 1 , CHK2, A2aR, B-7 family ligands or a combination thereof. In some embodiments, the agent targets PD-1, e.g., an antibody that blocks PD-L1 binding to PD-1 or otherwise inhibits PD-1. In some embodiments, agent targets CTLA-4. In some embodiments, the targets LAG3. In some embodiments, the agents targets TIM3. In some embodiments, the agents target ICOS.

[0111] In some embodiments, a layilin-binding domain or genetically modified immune effector cells as described herein comprising a layilin-binding domain are administered to a subject that has an autoimmune disorder. In some embodiments, a layilin-binding domain or genetically modified cell comprising a chimeric receptor comprising a layilin bidning domain is administered to a subject in conjunction with another immunosuppressive therapeutic agent. Examples o include, but are not limited to, corticosteroids ( e.g ., prednisone, budesonide, and prednisolone), kinase inhibitors (e.g., tofacitinib), calcineurin inhibitors (e.g., cyclosporine and tacrolimus), mTOR inhibitors (e.g., sirolimus and everolimus), IMDH inhibitors (e.g., azathioprine, leflunomide, and mycophenolate), and other biologies (e.g., abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, and daclizumab).

[0112] The following examples illustrate certain aspects of the claimed invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

EXAMPLES

[0113] To further elucidate layilin’s role we identified its ligand in a model antigen presenting line and engineered antibodies to antagonize this newly discovered interaction. Using coiled-coil fusions to enhance avidity, we found a strong association of layilin’s ECD with highly glycosylated collagen molecules. We then used a differential enrichment strategy to direct phage display antibody selections toward blocking epitopes to antagonize this interaction. Our results show a new mechanism by which layilin on T cells interacts with the extracellular matrix, and an antibody engineering strategy by which metal ion binding can be exploited to generate CTLD-blocking antibodies.

Example 1. Characterization of Layilin Binding

Layilin binds collagen on melanoma cells [0114] Layilin was originally described as a hyaluronic acid receptor (Bono et al., 2001). We were unable to recapitulate binding to multiple forms of hyaluronic acid by both ELISA and biolayer interferometry (BLI) (FIG. 5A-B), and so set out to discover other ligands that may be involved in T cell-mediated melanoma response. CTLD-ligand interactions are often weak and in often require multivalence for measurable binding; therefore, we expressed the layilin ECD fused to several multimerizing peptides (FIG. 1 A). C-terminally 8xHis- and Avi- tagged layilin ECD was monomeric by size exclusion chromatography, so we used coiled- coil peptides to make parallel dimers (CCDi), trimers (CCTri) (Fletcher et al., 2012), and pentamers ( Rattus norvegicus cartilage oligomeric matrix protein N-terminal domain,

COMP) (Kerr and Wright, 2012). Based on our previous experiments indicating that layilin expression on T cells affected response to the A375 melanoma line, we tested binding of these fusions to suspended A375 cells by flow cytometry (FIG. IB). Monomeric layilin ECD showed relatively weak binding, while dimeric, trimeric, and pentameric fusions bound more strongly. Binding in this assay was dependent on calcium and was similar between layilin isoforms 1 and 2 (FIG. 1C).

[0115] Using the pentameric isoform 1 ECD, we performed affinity purification-mass spectrometry to find new layilin ligands (FIG. ID). For specific, high-confidence detection of binding partners, we used stable isotope labeling with amino acids in cell culture (SILAC) to incorporate light or heavy arginine and lysine over several passages of the A375 cells (Paul et al., 2011). After gentle lysis and soni cation, the clarified lysate from heavy isotope or light isotope-labeled cells was incubated with streptavidin agarose bead-immobilized pentameric layilin ECD with in buffer containing CaCh or EDTA. We also collected data for samples where the heavy and light samples were reversed to control for any isotope effects. Heavy and light samples were mixed and washed extensively with cold buffer. Bound proteins were then acid-eluted, digested with trypsin, and analyzed by LC/MS/MS. As shown in FIG. IE, CaCh-containing samples were highly enriched in collagen chains al(IV) and a2(IV), the subunits in the most common form of type IV collagen, and a5(IV). Several other proteins associated with collagen processing were enriched: SERPINH1, which codes for HSP47, is a chaperone associated with collagen processing in the endoplasmic reticulum (Ito and Nagata, 2017). PLOD1, PLOD3, and COLGALT1 ( GLT25D1 ) are responsible for a collagen-specific glycosylation pathway wherein PLOD1 and PLOD3 oxidize lysine to d-hydroxylysine (Hyl), followed by addition of a galactosyl moiety to the resulting hydroxyl group by COLGALT1 to form Gai i-Hyl (Hennet, 2019). PLOD 3 then adds a glucose residue to form Glcal-2Gai i- Hyl (Scietti et al., 2018). We re-analyzed the data to include Hyl, Gai i-Hyl, Glcal-2Gai i- Hyl, and hydroxyproline (Basak et al., 2016), and observed other enriched proteins. Type IV collagen a3(IV), Type VI collagen al(VI), Type XVIII collagen al (XVIII), Type XIX collagen al(XIX), and Type XIII collagen (a non-fibrillar membrane-bound member of the family), were also detected but their enrichment did not reach statistical significance. We validated collagen’s role in layilin binding on A375 cells by showing that incubation of the cells with collagenase diminished binding of both Fc- and COMP-fused ECD (FIG. 6). These data strongly suggest that glycosylated collagens are ligands for layilin.

Collagen Glycosylation as Layilin Ligand

[0116] The 28 different human collagens come in a wide range of tertiary and quaternary structures, with varying levels of specialized crosslinks and post-translational modifications. We tested several commercially available solubilized collagens for binding to the layilin ECD by BLI. As our AP/MS data suggested, collagen IV from multiple vendors bound layilin reproducibly in a Ca²⁺-dependent manner (FIG. 2A and FIG. 7A). Collagen IV is the primary network-forming collagen component of basement membranes, and is heavily glycosylated (Basak et al., 2016). Type II collagen, also heavily glycosylated found in cartilage (Song and Mechref, 2013), bound slightly in EGTA but bound significantly better in CaCh (FIG. 2B). Type V collagen, a glycosylated fibrillar dermal collagen (Ishikawa et al., 2021), bound immobilized layilin but was variable between preparations (FIG. 2C and FIG. 7B), and Type VI bound significantly more in CaCh (FIG. 2D). Types I and III (FIG. 2E and 2F and FIG. 7C), the most common fibrillar types in the family, showed little to no detectable binding. While Type I collagen is glycosylated, it typically has fewer glycans than Type IV (Taga et al., 2013). Additionally, hyaluronic acid did not interfere with collagen IV (FIG. 7D). The binding of collagens to immobilized layilin by BLI displayed a very slow off-rate, likely due to avid binding of large collagen molecules to multiple immobilized layilin ECDs. Collagen types II, IV, V and VI are extensively glycosylated on Hyl residues heterogeneously throughout their triple-helical regions with either Gai i-Hyl or Glcal-2Gai i-Hyl, whereas types I and III have very little of these modifications. The binding of types II, IV, V and VI, but not I and III, to the layilin ECD suggest the importance one or both of these glycan species for layilin molecular recognition. Further supporting this, periodate oxidation of collagen IV inhibited binding by BLI, whereas periodate prequenched with glycerol had no effect (FIG. 2G). Binding to solubilized collagen was dependent on the presence of calcium ions in solution, but coordination of Ca²⁺ by layilin itself was also important, as E to A mutation in the ‘EPS’ motif completely ablated binding (FIG. 2H). We also expressed murine and cynomolgus homologues of layilin as Fc fusions and confirmed Ca²⁺-dependent binding of solubilized type IV collagen (FIG. 7E-F). Collectively, these data demonstrate a highly specific interaction between layilin’s CTLD and Hyl glycosylation in collagens.

[0117] An Octet assay was also used to evaluated binding to various collagen types (FIG. 10). The extracellular domain of human layilin isoform 1 fused to the human IgGl Fc domain and biotinylated via a C-terminal AviTag was diluted to 20 nM in 100 mM HEPES pH 7.5, 50 mM NaCl, 0.2% BSA w/v, and 0.05% v/v Tween20. Collagen molecules were diluted to 0.02 mg/ml and HL2E8r IgG or Fab domain were diluted to 100 nM in the same buffer plus 20 mM biotin. The layilin ECD was immobilized on streptavidin octet tips for 180 seconds. After a 180 second baseline step the sensors were dipped into the antibody solutions and allowed to associate for 600 seconds. The sensors were then dipped into a mixture of each collagen molecule with HL2E8r and allowed to associate for another 600 seconds. The sensors were then moved to buffer and allowed to dissociate for 900 seconds.

[0118] In the second association step, blocking of binding of each collagen type is observed for collagen types II, IV, V, and VI. Type V and VI show a small shift indicating some residual binding.

Example 2 Phage Display Strategies for Directing Binding Site Blockers

[0119] In light of our previous data showing the importance of layilin in T cell responses in mouse models of melanoma (Mahuron et ah, 2020), a species cross-reactive ligand-blocking antibody for this receptor would be a valuable resource for further elucidation of its function and downstream therapeutic application. Commercially available antibody 4C11 (Novus Biologicals) did not compete with collagen binding. We previously showed that clone 3F7D7E2 (Sino Biologicals) led to integral activation when bound to layilin overexpressing cells at high concentrations; this antibody blocked Type IV collagen when bound to immobilized layilin first but was not blocked when Type IV collagen was bound first, suggesting multiple conformations with differing binding abilities are present in the purified layilin protein (FIG. 8). In contrast to the Sino Biologicals clone 3F7D7E2, HL2E8 did not activate integrins in an integrin LFA-1 activation assay (FIG. 11) as follows: Jurkat cells stably expressing full-length human layilin isoform 1 were washed with HEPES -Buffered saline with 2% Fetal Bovine Serum and suspended to 10^L6 cells per ml. Anti-layilin antibodies were diluted to 0.1 mg/ml with a 1/100 dilution of m24-PE-Cy7 in the same buffer. MnC12 (5 mM final) was used as a positive control. 10^L5 cells were distributed to wells in a round-bottom 96-well plate, pelleted by centrifugation at 500 x g for 3 minutes, resuspended in each antibody solution, and incubated at 37 °C for 20 minutes. Cells were washed twice and analyzed by flow cytometry.

[0120] To obtain new classes of functional layilin-binding antibodies, we performed phage display to generate mAbs for both human and murine layilin. Selections utilized a dimeric layilin ECD construct, either mouse or human, followed by a TEV protease cleavage site fused to the IgG human Fc. This was immobilized on streptavidin magnetic beads via a C- terminal biotin introduced by an AviTag. A previously developed synthetic naive Fab-phage library (diversity ~3 x 10¹⁰) was precleared with bead-bound Fc domain to remove potential Fc binders, and the surviving free phage pool allowed to bind the layilin ECD-Fc antigen on beads. After extensive washing, high-affinity binders are eluted by treating with TEV protease following our standard catch-and-release protocol (FIG. 3A, Strategy i) (Hornsby et al., 2015) which leaves non-specific binders bound to the beads. Three to four rounds of selections were conducted, and individual phage were screened first by antigen competitive ELISA followed by direct affinity measurements using BLI. Epitope binning experiments showed that antibodies from Strategy i fell into two bins, neither of which competed with collagen, 3F7D7E2, or 4C11. One of these highly dominant epitopes is likely near the N- terminus of layilin as antibodies in this bin were specific to isoform 1 which has an eight amino acid N-terminal extension compared to isoform 2. Prototypic members of each of these bins, mouse-directed antibody ML3D12 and human-directed antibody HL3A9, were expressed as IgGs and showed high-affinity binding of 0.5 nam and 1.1 nM (FIG. 3B and 3C). However, none of these Fabs blocked binding to collagen IV.

[0121] We thus pursued a higher stringency differential selection strategy to generate collagen blocking antibodies. We reasoned that layilin’s calcium dependence could be leveraged to direct phage selections toward blocking epitopes. We devised a selection strategy to clear our phage libraries with EGTA-treated layilin, select on Ca²⁺-bound layilin, and specifically elute Ca²⁺-dependent antibodies with excess EGTA (FIG. 3A, Strategy ii). This strategy yielded one unique clone with that bound in a Ca²⁺-dependent manner, HL2E8. Despite low affinity as a Fab fragment, HL2E8 bound well (KD=0.6 nM) as an IgG (FIG.

3D). While ML3D12 and HL3A9 were unable to block collagen binding, HL2E8 potently inhibited this interaction in the presence of Ca2+ ions (FIG. 3E). Like Sino 3F7D7E2, HL2E8 was able to bind purified layilin in the presence of Type IV collagen (FIG. 9A). Conveniently, HL2E8 was cross-reactive with cyno and mouse layilin and retained its Ca2+- dependence (FIG. 9).

[0122] Each of these antibodies were then tested for binding to layilin on Jurkat cells transduced with full-length human layilin isoform 1 linked to GFP expression through a T2A site and compared to GFP-only controls (FIG. 4). Addition of EGTA modestly inhibited HL2E8 binding to cells (FIG. 4A), whereas ML3D12, HL3A9, and Sino 3F7D7E2 bound equally well with or without calcium in solution (FIG. 4B-4D). All four IgGs were then tested for blocking soluble layilin to A375 melanoma cells (FIG. 4E). HL2E8 and Sino 3F7D7E2 strongly inhibited soluble layilin binding, whereas ML3D12 and HL3A9 did not. This demonstrates the utility of the differential Ca²⁺-dependent selection method to find antibodies inhibiting layilin’ s interaction with collagen.

Summary of example findings

[0123] Here, we discovered a specific interaction between layilin and collagen glycans. This suggests that layilin maintains activated T cell contacts in collagen-rich tissues, possibly enhancing antigen-presenting cell interactions.

[0124] Further, these examples describe an effective strategy to isolate antibodies directed specifically to the Ca²⁺-binding conformation of layilin’ s CTLD, which is readily applicable to other CLECs and other Ca²⁺-dependent proteins. A Ca²⁺-dependent binder for the IL-6 receptor was selected by Iwaga and co-workers to promote endosomal dissociation of mAbs to the IL-6 receptor (Hironiwa et ak, 2016). Here we used the metal ion binding properties of the receptor itself to direct antibody function. Interestingly, Ca²⁺-dependent antibodies to macrophage galactose/N-acetylgalactosamine-specific calcium-type lectin (MGL, CleclOa), promoted, rather than inhibited, ligand binding (Hosoi et ak, 1998), demonstrating that Ca²⁺- dependent mAbs to CLECs can have complex functions. This phage display strategy will therefore be useful to enrich antibodies that modulate CLEC behavior in multiple ways. Species cross-reactive layilin-blocking antibodies generated by this strategy represent a promising tool to characterizing this receptor’s function in disease models and potentially as therapeutics to modulate T cell behavior.

Materials and Methods

Materials.

Chemicals were obtained from Millipore-Sigma unless otherwise noted. Restriction enzymes were obtained from New England Biolabs. Bovine Serum Albumin, Fraction V was obtained from Gemini Bio-products (#700-101P) Phosphate-buffered saline (PBS, Sigma P4417-100TAB) was diluted to lx (10 mM phosphate, 2.7 mM KC1, 137 mM NaCl, pH 7.4) and autoclaved. When necessary, Tween20 was added to 0.05% v/v and BSA was added to 0.2% w/v.

Tris-buffered saline (TBS) was made by diluting Tris (pH adjusted to 7.2 with HC1) to 25 mM and NaCl to 150 mM and sterilized by filtration using Nalgene Filter Systems, PES Membrane, Sterile, 1000 mL, 0.2 pm, Thermo Scientific 567-0020). When necessary, Tween20 was added to 0.05% v/v and BSA was added to 0.2% w/v, and CaCh. Ethylenediaminetetraacetic acid (EDTA), or Ethyleneglycol bis(2-aminoethyl ether)- /V,/V,/V',/V'-tetraacetic acid (EGTA, GoldBio E-217-25) was added to specified concentrations. HEPES Buffered Saline (HBS) was made by diluting 4-(2-Hydroxyethyl)piperazine-l- ethanesulfonic acid (pH adjusted to 7.4 with NaOH) to 25 mM and NaCl to 150 mM). When necessary, Benchmark Fetal Bovine Serum (FBS Gemini Bio-Products 100-106) was added to 2% v/v.

D-Biotin (B-950-25), DNase I from Bovine Pancreas (D-300-100), and DL-Dithiothreitol (DTT50) were obtained from GoldBio.

Streptavidin MagneSphere Paramagnetic Particles were obtained from Promega (Z5482)

High Capacity Neutravidin Agarose Resin was obtained from Thermo Fisher Scientific (29204)

Complete Mini, EDTA-free Protease Inhibitor Cocktail (Sigma 11836170001) was diluted to lx during AP/MS experiments.

Several collagens were used as indicated: purified collagen type I (Sigma C5483-1MG), Type IV collagen from human placenta (Sigma C5533-5MG), highly purified Type IV collagen (Sigma CC076), Native human collagen IV (AbCam ab7536). Type III collagen from human placenta (Sigma C4407-1MG), and Type V collagen from human placenta (Sigma C3657- 1MG)

Heavy amino acids L-Arg HCl (¹³C6; ¹⁵N4, CNLM-539-H-PK) and L-Lys 2HC1(¹³C6; ¹⁵N2, CNLM-291-H-1) were obtained from Cambridge Isotope Laboratory, Inc.

Layilin antibodies were obtained from Sino Biological (clone 3F7D7E2, #10208-MM02-200) andNovus Biologicals (clone OTI4C11, NBP2-01879)

Neutravidin was obtained from Thermo Fisher Scientific (PI31000)

Cell lines and tissue culture

[0125] Mammalian cells were grown at 37 °C with 5% CCh. A375 melanoma cells were obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose with 4 mM L-glutamine (VWR 16777-129) supplemented with 10% v/v FBS and lx penicillin/streptomycin (Gemini Bio-Products #400-109). For SILAC experiments, cells were cultured in Thermo Scientific DMEM for SILAC (#PI88364) supplemented with 0.8 mM Lysine 0.38 mM Arginine and 10% dialyzed FBS (Gemini Bio-Products #100-108). Jurkat E6 cells and derivatives were cultured in RPMI-1640 with 2.05 mM L-Glutamine (VWR 16777-145) supplemented with 10% FBS v/v and lx penicillin/streptomycin. Lenti-X 293T cells (Takara Bio) were cultured in DMEM-high glucose with 10% v/v FBS and lx penicillin/streptomycin. A cell line derived from Expi293 stably expressing an endoplasmic reticulum-localized biotin ligase (birA) used for protein expression was cultured in Expi293 medium (Thermo Fisher Scientific).

Cloning

[0126] PCRs were performed using Phusion polymerase (NEB). Plasmids were propogated in XL10-Gold E. coli cells. Layilin constructs and IgGs for mammalian protein production were cloned in a pFUSE-derived (Invivogen) vector pAB1200 containing sequences for an optimized IL-2 secretion signal, Spel restriction sites, a Tobacco Etch Virus (TEV) protease site, flexible (Ser-Gly3)2 linker, human IgGl Fc, a BamHI site, and a C-terminal AviTag. Coding sequences for layilin were purchased as gBlocks (Integrated DNA Technologies). Fc- fusions with native layilin signal sequences were constructed by digesting pAB1200 with Kasl and Spel-HF and inserting the layilin ECD by Gibson cloning. Layilin fusions to multimerizing peptides CCDi, CCTri, and COMP, were constructed by overlap-extension PCR with the layilin ECD followed by a TEV protease site, flexible (Ser-Gly3)2 linker, multimerizing peptide, and a C-terminal AviTag. Mutants to the layilin ECD were made by amplifying the ECD sequence with mutagenic primers and re-inserting the gene by Gibson cloning. IgG heavy chain sequences were cloned into a similar pFUSE-derived vector encoding an optimized IL-2 secretion signal, the human IgGl Fc, and a C-terminal AviTag. VH-encoding regions were constructed by digesting this plasmid with Spel-HF and Bsmbl, amplifying phage-derived antibody sequences by PCR, and inserting the new VH sequence by Gibson cloning. Light chain plasmids included an optimized IL-2 signal sequence. VL- encoding regions were constructed by digesting this plasmid with Spel-HF and Kpnl, amplifying phage-derived antibody sequences by PCR, and inserting the new VL sequence by Gibson cloning.

[0127] Expression vectors for Fab sequences were constructed in a custom E. coli polycistronic periplasmic expression vector, pBL347 (Hornsby et ak, 2015) encoding the light chain followed by the heavy chain with a C-terminal AviTag. Fab expression was driven by consecutive pTAC promoters. Periplasmic localization of the light chain was directed by the pelB signal sequence, and the heavy chain was directed by the stil signal sequence. Phage-derived Fab sequences were amplified by PCR and inserted into the NcoI-HF and Agel-HF-digested vector by Gibson cloning.

[0128] Lentiviral transfer vectors for stable cell line production were produced from pCDH-EFla-MCS- IRES-Puro by removing the IRES-Puro sequence through digestion with Xbal and Sall-HF. To preserve layilin’s native sequence, the gene encoding full-length layilin was inserted after the fluorescent protein Clover (Lam et al., 2012) and a T2A sequence.

Protein expression and purification

[0129] Expression of biotinylated layilin fragments and IgGs was carried out in a BirA- expressing Expi293-derived line cells. Transfections using Expifectamine were carried out as described in the manual. After five days of expression, cells were pelleted by centrifugation and the supernatant was filtered through a 0.45 pm syringe filter (Coming #431220). lOx PBS was added to a final concentration of 2x. Fc-fused proteins were purified by chromatography through 1 ml HiTrap Protein A columns (GE Healthcare #17-0402-01), washed with 10 volumes of lx PBS, and eluted with 4 volumes of 0.1 M acetic acid directly into 0.4 volumes of ice-cold 1 M Tris base. His-tagged proteins were purified by adding 1 M Tris pH 7.2 to a final concentration of 25 mM and imidazole to a final concentration of 20 mM and incubating Expi supernatants with 0.4 ml of high-density nickel agarose (GoldBio #H-320-100) at 4 °C for 1 hour with rotation. The protein-bound resin was washed with SO SO volumes of cold TBS with 20 mM imidazole and 1 mM CaCk, and protein was eluted in TBS with 300 mM imidazole and 1 mM CaCh. Expression of Fab fragments was carried out by autoinduction in C43 cells harboring a pTUM4 plasmid encoding biotin ligase BirA (Schlapschy and Skerra, 2011). An overnight culture of cells grown in Terrific Broth (TB) containing 50 pg/ml carbenicillin and 12.5 pg/ml chloramphenicol was subcultured 1/500 into 100 ml autoinduction media composed of TB supplemented with 0.5% glycerol, 0.05% glucose, 0.5% lactose, 1 mM galactose, 2 mM MgSCk, 0.005% antifoam, 50 pg/ml carbenicillin, 12.5 pg/ml chloramphenicol, and 20 pM biotin in 250 ml baffled flasks.

Cultures were shaken at 300 RPM for 6 hours at 37 °C followed by 20 hours at 30 °C. Cells were pelleted and resuspended in 5 ml PBS with 0.05 mg/ml DNAse I and 50 pM MnCh. followed by the addition of 5 ml Bacterial Protein Extraction Reagent (Thermo Scientific #78243). The lysate was incubated at 60 °C for 20 minutes, cooled on ice, and centrifuged at 20,000 x g for 30 minutes. Supernatants were then applied to HiTrap Protein A columns as described above. Proteins were buffer exchanged by 3-4 rounds of spin concentration through Amicon Ultra 30 kDa MWCO filters (EMD Milbpore UFC803096). Antibodies were buffer exchanged into PBS, while layilin constructs were buffer exchanged into TBS + 1 mM CaCh.

Stable cell line generation.

[0130] Stable expression of layilin in Jurkat E6 cells was established by lentiviral transduction. Plasmids encoding Clover-T2A-layibn sequences (1.5 pg) were mixed with packaging vectors pMD2.g (0.165 pg) and pCMVdr8.91 (1.35 pg) in 300 pi serum-free DMEM along with 7.5 pi of Fugene HD (Promega #E2311). After incubation at room temperature for 5 minutes, the transfection mixture was added to 80% confluent Lenti-X cells in a 6 well plate in 3 ml complete DMEM. After 3 days of lentivirus production, supernatants were filtered through a 0.45 pm syringe filter and 1.5 ml was added directly to 1 million Jurkat E6 cells in complete 1.5 ml RPMI containing 16 pg/ml polybrene (EMD Millipore TR-1003-G) in 6-well plates. Plates were centrifuged at 1000 x g for 2 hours at 33 °C to promote transduction. The next day cells were washed with fresh media and grown for several days. High expressers were isolated using Fluorescence Activated Cell Sorting on a Sony SH800. Cells were washed in PBS with 0.02% BSA and 10,000 cells representing the top -10% of layilin-expressing clones or a gate corresponding to identical fluorescence levels for GFP-only cells were isolated and expanded.

Flow Cytometry

[0131] Flow cytometry was carried out on a Beckman Coulter Cytoflex. To analyze purified layilin constructs binding to A375 cells, cells were washed once with PBS containing 0.04% EDTA (UCSF Cell Culture Facility #CCFAL005) and released from the plate by incubation for 8 minutes at 37 °C. 10⁶ cells were pelleted at 500 x g for 3 minutes, washed once with 5 ml staining buffer (HBS + 2% FBS + 1 mM CaCh). resuspended to 10⁶/ml in a 1/50 dilution of Human TruStain FcX (Biolegend #422302), and incubated for 15 minutes. Fc-blocked cells were distributed into a 96-well plate (10⁵ cells/well), pelleted as above, and resuspended in staining buffer with the desired concentration of layilin ECD fusion. After staining for 1 hour at room temperature with shaking and occasional mixing by pipetting, cells were washed 3x with 200 pi ice-cold staining buffer and stained with 200 pi of 1/1000 streptavidin-PE (Biolegend #405203) for 20 minutes at 4 °C. Cells were washed twice and analyzed immediately. [0132] Jurkat cells expressing human layilin isoforms 1 or 2 or a GFP-only control were washed with HEPES-buffered saline (25 mM HEPES pH 7.5, 150 mM NaCl) with 2% v/v FBS, with either 5 mM CaCh or 1 mM EGTA. For antibody testing, 10⁵ cells were stained with 100 pi of 100 nM of the desired antibody for 30 minutes at room temperature. The cells were then washed three times in the same buffer at 4 °C and stained with 1/1000 anti-human Alexa Fluor 647 at 4 °C for 20 minutes. The cells were then washed two more times and resuspended in 200 mΐ and analyzed by flow cytometry.

Hyaluronic Acid Binding ELISA

[0133] Hyaluronic acid was diluted to 0.3 mg/ml in 50 mM bicarbonate buffer pH 9.5. Nunculon 384-well plates were coated with 20 mΐ of the HA solution for 5 hours at 37 °C. PBST with 1% BSA was added and plates were blocked overnight at 4 °C. Plates were washed with PBST in a BioTek EL405 plate washer. Biotinylated layilin constructs diluted in TBST with 1 mM CaC12 and 1% BSA (20 mΐ) were added and incubated at room temperature for 1 hour. Wells were washed three times with PBST. A 1/1000 dilution of HRP-conjugated Neutravidin (20 mΐ) was added and incubated on ice for 30 minutes. Wells were washed another three times with PBST and developed with 20 mΐ TMB substrate for several minutes at room temperature, quenched with 20 mΐ of 1 M phosphoric acid, and absorbance was read at 450 nM. Biotinylated human IgGl Fc was used as a negative control, and hyaluronic binding protein from bovine nasal cartilage was used as a positive control.

Affinity Purification/Mass Spectrometry

[0134] Heavy and light SILAC-labeled A375 cells were lifted from one T225 each plate as above, pelleted, and lysed for 30 minutes at 4 °C in lysis buffer (HBS containing 1 mM CaCh. 1 mM MgCh. 0.2% v/v NP-40, and EDTA-free protease inhibitor). The lysate was sonicated at 20% power for 1 minute on ice (Is on/ls off) to shear the genomic DNA and centrifuged at 16,000 x g for 10 minutes at 4 °C. Protein concentration of the lysate was estimated by making serial dilutions starting at 1:50 and incubating with Bio-Rad Protein Assay Reagent (#5000006) according to the manufacturer’s instructions. High capacity Neutravidin Agarose (400 mΐ bed volume) was transferred into a Pierce Spin Column (Life Technologies #69725) and washed with lysis buffer, incubated with 1 ml of 1 mM Layilin- COMP (isoform 1) at 4 °C for 30 minutes, washed 3x with lysis buffer, and distributed equally into four eppendorf tubes. A375 lysate (500 mΐ of 7 mg/ml) was added to each tube (two heavy, two light). EDTA (500 mM pH 8.0 stock) was added to one heavy and one light tube to a final concentration of 5 mM and samples were rotated at 4 °C for 1 hour. The agarose was pelleted once to remove unbound proteins and EDTA, resuspended in lysis buffer, and heavy and light samples were mixed with corresponding EDTA-treated control samples in spin columns. The mixtures were washed three times with 1 ml ice-cold lysis buffer and once with 1 mM HEPES pH 7.4 with 150 mM NaCl to lower the buffering capacity. Tubes were eluted three times with 100 pi 0.1 M acetic acid. The eluate was frozen on dry ice and lyophilized overnight. The next day the protein residue was resuspended in 100 mΐ of 50 mM ammonium bicarbonate with 6 M urea, reduced with 5 mΐ 200 mM DTT for 1 hour at RT, alkylated with 20 mΐ 200 mM iodoacetamide for 1 hour at RT in the dark, and quenched with another 20 mΐ 200 mM DTT for 1 hour in the dark. Samples were diluted to 1 ml with mass spec grade water and digested overnight at RT in the dark with 10 pg of trypsin (Promega #V5113). Digested peptides were acidified with trifluoroacetic acid (1% v/v final), isolated with SOLA HRP SPE columns (Life Technologies #60109-001), washed with 500 mΐ each of 1% TFA and 2% acetonitrile/0.1% formic acid, and eluted twice with 250 mΐ 40% acetonitrile/0.1% formic acid. The eluted peptides were concentrated by vacuum centrifugation and stored at -80 °C.

Liquid Chromatography/T imdem Mass Spectrometry

[0135] Mass spectrometry was performed largely as previously described (Leung et ak, 2020). Desalted, digested peptides were separated using an UltiMate 3000 UHPLC system (Thermo) with pre-packed 0.75mm x 150mm Acclaim Pepmap C18 reversed phase columns (2pm pore size, Thermo) and analyzed on a Q Exactive Plus (Thermo Fisher Scientific) mass spectrometer. For all samples, 1 pg of resuspended peptides was injected for separation with a linear gradient of 3-35% solvent B (solvent A: 0.1% formic acid, solvent B: 80% acetonitrile, 0.1% formic acid) over 230 mins at 300 pL/min. Data-dependent acquisition was performed using a top 20 method (dynamic exclusion 35 seconds; selection of peptides with a charge of 2, 3, or 4). Full spectra with a resolution of 140,000 (at 200 m/z) were gathered in MSI using an AGC target of 3e6, maximum injection time of 120 ms, and scan range of 400 - 1800 m/z. Centroided data from MS2 scans were collected at a resolution of 17,500 (at 200 m/z) with an AGC target of 5e4 and maximum injection time of 60 milliseconds. The normalized collision energy was set at 27 and an isolation window of 1.5 m/z with an isolation offset of 0.5 m/z was used. Raw output files were then carried forward for database search and SILAC quantification.

Mass Spectrometry Data Analysis [0136] All mass spectrometry data were analyzed using PEAKS Online X version 1.5. Raw output files were uploaded to the PEAKS Online server and quantified using PEAKS Q for SILAC data. Briefly, the precursor mass error tolerance was set to 15 ppm and the fragment mass error tolerance set to 0.02 daltons. Data were searched with carbamidomethylation as a fixed modification and with deamidation (NQ), acetylation (N-term), and the SILAC labels as variable modifications. For collagen post-translational modification searches, Hyl, Gai i- Hyl, Glcal-2Gai i-Hyl, and hydroxyproline were also included as variable modifications. Forward and reverse SILAC data were concatenated to generate the final enrichment data.

The PEAKS output was filtered for protein and peptide identifications with a false discovery rate of less than 1%, and intensities in all samples were normalized using the total ion current. Quantified data were exported from PEAKS for further analysis and visualization using Python. Proteins demonstrating >4-fold enrichment over EDTA-treated samples with a P- value <0.01 were considered significantly enriched.

Biolayer Interferometry (BLI)

[0137] BLI was performed on an OctetRED384 instrument (ForteBio, now Sartorius) in TBS with 0.05% Tween and 0.2% BSA unless otherwise specified. Biotinylated proteins were loaded onto streptavidin sensors at a concentration of 20 nM for 180 seconds followed by a combined blocking/baseline step in the same buffer with 20 mM biotin. Samples were associated for 600 seconds and dissociated in the baseline/blocking well for 900 seconds. For binning experiments, proteins were first associated alone followed by association in the presence of competitor at specified concentrations.

Standard Phage Display

[0138] Standard catch-and-release phage display was performed as previously described with previously developed Fab-phage Library E (Miller et ak, 2012) and UCSF in-house libraries. For selection Round 1, approximately 10¹³ phage were pelleted in 30 ml PBS supplemented with 4% PEG8k with 0.5 M NaCl on ice for 1 hour followed by centrifugation at 20,000 x g for 20 minutes. After carefully removing the supernatant the phage pellet was resuspended in 1 ml PBS with 0.05% Tween 20 and 0.2% w/v BSA (PBSTB). Streptavidin magnetic beads (100 pi) were washed with 1 ml PBSTB, and 500 mΐ of biotinylated Fc was immobilized for 30 minutes at RT, followed by three more washes. The resuspended phage library was cleared for 30 minutes at RT with rotation, and Fc-bound beads were carefully removed. Fc-fused layilin ECDs (100 mΐ of 1 mM) were immobilized on 100 mΐ fresh beads as above and washed three times with PBSTB. The cleared library was added to the layilin- bound beads and incubated for 1 hour at RT with rotation. After three washes at RT, bound phage were eluted by adding 20 pg/ml TEV protease in 500 pi PBSTB. The eluted phage were used to infect 5 ml log-phase XLl-Blue E. coli cells for 20 minutes at RT. The infected E. coli were cultured overnight at 37 °C in 30 ml 2xYT with 50 pg/ml carbenicillin and 10¹⁰ pfu/ml K07 helper phage. For round 2, the supernatant from round 1 was isolated by centrifugation at 4000 x g for 10 minutes, and phage were PEG-precipitated from 30 ml overnight culture supernatant as in round 1, resuspended in 1 ml PBSTB, and cleared again by centrifugation at 16,000 x g for 10 minutes at RT. Streptavidin magnetic beads (100 mΐ) were washed with PBSTB, and 250 pi of biotinylated Fc was immobilized for 30 minutes at RT, followed by three more washes. Nonspecific phage were cleared as above. Phage were selected with Fc-fused ECDs as above (100 pi of 50 nM immobilized on 20 mΐ beads), eluted with 100 mΐ 20 pg/ml TEV protease, and 50 mΐ of eluate was used to infect 100 mΐ log-phase XLIO-Gold for 20 minutes at RT. Infected cells were cultured in 3 ml 2xYT with 50 pg/ml carbenicillin and 10¹⁰ pfu/ml K07 helper phage. For rounds 3 and 4, supernatant from 3 ml overnight cultures was isolated by centrifugation at 4000 x g for 10 minutes. Fab-displaying phage were isolated from supernatants with either 20 mΐ of Protein A mangetic beads alone (Library E; Thermo Fisher Scientific #88845) or 20 mΐ each of Protein A and Protein L magnetic beads (UCSF Library; Thermo Fisher Scientific #88849) previously washed with PBSTB, incubated 30 minutes at RT with rotation, washed three times with PBSTB, eluted for 10 minutes at RT with 100 mΐ of 0.1 M acetic acid, and neutralized with 11 pi Tris base. Biotinylated Fc was used to clear nonspecific binders from the eluted phage as above (100 mΐ of 200 nM immobilized on 40 mΐ beads). Phage were selected on Fc-fused ECDs (100 pi of 10 nM immobilized on 20 mΐ beads), eluted in 100 mΐ 20 pg/ml TEV protease, and propagated as above. In rounds 3 and 4, selection efficiency was compared to mock selections on Fc- biotin by tittering 10-fold serial dilutions of TEV eluate.

Calcium-selective Phage Display

[0139] Calcium-dependent antibodies were selected using several modifications to the standard selection protocol above. To avoid precipitation, Tris buffers were used in place of phosphate throughout. In round 1, the resuspended library was cleared with layilin-Fc (isoform 2; 100 mΐ of 1 pM immobilized on 100 mΐ SA beads) in the presence of 100 mM EGTA. For selections, CaCh was added to 1 mM to quench EGTA. After 1 hour of binding, beads were washed twice with TBS + 0.05% v/v Tween20 + 0.2% w/v BSA (TBSTB) with 1 mM CaCh followed by one wash with TBSTB, and finally eluted in 500 mΐ TBSTB with 20 mg/ml TEV protease and propagated as above. In round 2, resuspended phage were similarly cleared with layilin-Fc (isoform 2, 100 pi of 1 mM immobilized on 100 mΐ SA beads) in the presence of 100 mM EGTA. Selections were carried out with 1 mM CaCh added, with 20 mΐ of SA beads (100 mΐ 200 nM layilin isoform 2 immobilized). Beads were again washed twice with TBSTB + 1 mM CaCh followed by TBSTB and eluted in 100 mΐ TBSTB with 20 pg/ml TEV protease. For rounds 3 and 4, 50 mΐ of Protein A/L-eluted phage was mixed with 50 mΐ of TBSTB + 100 mM EGTA and cleared with 40 mΐ SA beads (100 mΐ of 1 mM layilin-Fc isoform 2 immobilized). Selections were again carried out in TBSTB with 1 mM CaCh and washed as above, but bound phage were instead eluted with 100 mΐ of 5 mM EGTA at RT for 10 minutes. Eluted phage (50 mΐ) were used to infect XLl-Blue cells (100 mΐ) as above and propagated in 3 ml 2xYT + 50 pg/ml carbenicillin and 10¹⁰ pfu/ml K07 helper phage. In rounds 3 and 4, selections were monitored by comparing to mock selections on layilin-Fc without CaCh added.

Phage Competition ELISA

[0140] Fab-phage from selection rounds 3 or 4 were used to infect XLl-Blue and dilutions of infected cultures ( 10⁵ or 10⁶) were plated on LB agar with 50 pg/ml carbenicillin. Single colonies were used to start 95 cultures and one negative control in deep well 96-well blocks in 500 mΐ 2xYT + 50 pg/ml carbenicillin and 10¹⁰ pfu/ml K07 helper phage and shaken overnight at 900 RPM at 37 °C. The cultures were centrifuged at 4000 x g for 20 minutes. Nunc Maxisorp 384 well plates (Thermo Fisher Scientific #464718) were coated overnight with 50 pi 0.5 pg/ml neutravidin in PBS and blocked with PBSTB. ELISA plates were washed using a BioTek ELx405 plate washer. After 3 washes the plates were coated with 20 mΐ antigen according to selection type. For standard selections, each phage clone was analyzed in four adjacent wells: upper left, direct antigen binding (coated in 20 nM biotinylated Fc-fused antigen); upper right, competition ELISA (coated in 20 nM biotinylated Fc-fused antigen, pre-bound to soluble Fc-fused antigen); lower left, non-specific binding (coated in biotinylated Fc); lower left, positive control (coated in 1/1000 CaptureSelect Biotin anti-IgG-CHl conjugate, Thermo Fisher Scientific #7103202100). For calcium-dependent selections, the positive control quadrant was replaced with 20 nM Fc fused layilin in EGTA. After immobilizing antigen for 30 minutes at RT, unbound neutravidin was quenched with 20 pi 20 pM biotin. Plates were washed with PBST and Fab-phage were diluted 1/5 in PBSTB or in PBSTB containing 25 nM Fc-fused antigen for competition quadrant. Diluted phage were added to the ELISA plate and incubated no longer than 15 minutes at RT followed by washing with PBST (Standard Selection) or TBST with 1 mM CaCh (calcium-dependent selection). 20 mΐ of HRP-conjugated secondary anti-M13 (Sino Biologicals 11973-MM05T- H), diluted 1/10,000, was added to each well and incubated for 30 minutes at RT. Plates were washed three more times and developed with 20 mΐ TMB substrate (VWR 50-76-03) for 3 minutes followed by quenching with 20 mΐ 1 M phosphoric acid. Absorbance was read at 450 nm.

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[0180] One or more features from any embodiments described herein or in the figures may be combined with one or more features of any other embodiment described herein in the figures without departing from the scope of the disclosure.

[0181] All publications, accession numbers, patents and patent applications cited in this specification are herein incorporated by reference for the purposes for which it is cited as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Table of Illustrative Sequences:

SEQ ID NO:l HL2E8 HCDR1 amino acid sequence as defined by Rabat

SGFNFYSSYIH

SEQ ID NO:2 HL2E8 HCDR2 amino acid sequence as defined by Rabat

SISSYYGSTSYADSVKG SEQ ID NO:3 HL2E8 HCDR3 amino acid sequence as defined by Rabat

FSQYSWYTFSGLDY

SEQ ID NO:4 HL2E8 LCDR1 amino acid sequence as defined by Rabat

R(A/T)SQSVSSAVA

SEQ ID NO:5 HL2E8 LCDR2 amino acid sequence as defined by Rabat

SASSLYS

SEQ ID NO:6 HL2E8 LCDR3 amino acid sequence as defined by Rabat

QQASTYPIT

SEQ ID NO:7 HL2E8 V_H amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGFNFYSSYIHWVRQAPGKGLEWVASISSYYGSTSYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFSQYSWYTFSGLDYWGQGTLVTVSS

SEQ ID NO:8: HL2E8 VL sequence amino acid sequence

DIQMTQSPSSLSASVGDRVTITCR(A/T)SQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGV PSRFSGSRSGTDFTLTISSLQPEDFATYYCQQASTYPITFGQGTKVEIK

SEQ ID NO:9: HL2E8 Heavy chain amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGFNFYSSYIHWVRQAPGKGLEWVASISSYYGSTSYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFSQYSWYTFSGLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW S VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

SEQ ID NO: 10 HL2E8 Light chain amino acid sequence

DIQMTQSPSSLSASVGDRVTITCR(A/T)SQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGV PSRFSGSRSGTDFTLTISSLQPEDFATYYCQQASTYPITFGQGTKVEIKRTVAAPSVFIFPP SDSQLKSGTASW CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:ll Layilin extracellular domain amino acid sequence corresponding to positions 22-235 of Isoform 1 (Q6UX15-1) amino acid sequence as defined in in the UniProt entry fRB-Q6UX15 TGRLLSAS DLDLRGGQPV CRGGTQRPCY KVIYFHDTSR RLNFEEAKEA CRRDGGQLVS IESEDEQKLI EKFIENLLPS DGDFWIGLRR REEKQSNSTA

CQDLYAWTDG SISQFRNWYV DEPSCGSEVC W MYHQPSAP AGIGGPYMFQ

WNDDRCNMKN NFICKYSDEK PAVPSREAEG EETELTTPVL PEETQEEDAK

KTFKESREAA LNLAY

SEQ ID NO: 12 layilin amino acid sequence, UniProt Q6UX15-1

MRPGTALQAV LLAVLLVGLR AATGRLLSAS DLDLRGGQPV CRGGTQRPCY

KVIYFHDTSR RLNFEEAKEA CRRDGGQLVS IESEDEQKLI EKFIENLLPS

DGDFWIGLRR REEKQSNSTA CQDLYAWTDG SISQFRNWYV DEPSCGSEVC

W MYHQPSAP AGIGGPYMFQ WNDDRCNMKN NFICKYSDEK PAVPSREAEG

EETELTTPVL PEETQEEDAK KTFKESREAA LNLAYILIPS IPLLLLLW T

TW CWVWICR KRKREQPDPS TKKQHTIWPS PHQGNSPDLE VYNVIRKQSE

ADLAETRPDL KNISFRVCSG EATPDDMSCD YDNMAVNPSE SGFVTLVSVE

SGFVTNDIYE FSPDQMGRSK ESGWVENEIY GY

SEQ ID NO: 13 layilin amino acid sequence, UniProt Q6UX15-2

MRPGTALQAV LLAVLLVGLR AATGRLLSGQ PVCRGGTQRP CYKVIYFHDT

SRRLNFEEAK EACRRDGGQL VSIESEDEQK LIEKFIENLL PSDGDFWIGL

RRREEKQSNS TACQDLYAWT DGSISQFRNW YVDEPSCGSE VCWMYHQPS

APAGIGGPYM FQWNDDRCNM KNNFICKYSD EKPAVPSREA EGEETELTTP

VLPEETQEED AKKTFKESRE AALNLAYILI PSIPLLLLLV VTTW CWVWI

CRKRKREQPD PSTKKQHTIW PSPHQGNSPD LEVYNVIRKQ SEADLAETRP

DLKNISFRVC SGEATPDDMS CDYDNMAVNP SESGFVTLVS VESGFVTNDI

YEFSPDQMGR SKESGWVENE

SEQ ID NO: 14 layilin amino acid sequence UniProt Q6UX15-3

MVTSGLGSGG VRRNKAIAQP ARTFMLGLMA AYHNLEKPAV PSREAEGEET

ELTTPVLPEE TQEEDAKKTF KESREAALNL AYILIPSIPL LLLLW TTW

CWVWICRKRK REQPDPSTKK QHTIWPSPHQ GNSPDLEVYN VIRKQSEADL

AETRPDLKNI SFRVCSGEAT PDDMSCDYDN MAVNPSESGF VTLVSVESGF

VTNDIYEFSP DQMGRSKESG WVENEIYGY

Claims

WHAT IS CLAIMED IS:

1. An antibody comprising a layilin-binding domain A (LBD-A), wherein the LBD-A specifically binds to a layilin extracellular domain (ECD) comprising SEQ ID NO: 11 and interferes with binding between the layilin ECD with one or both of collagen Type IV or collagen Type V.

2. The antibody of claim 1, wherein the antibody comprises: a heavy chain variable (VH) region comprising a heavy chain complementarity determining region (HCDR) 1 comprising SGFNFYSSYIH (SEQ ID NO:l), an HCDR2 comprising SISSYYGSTSYADSVKG (SEQ ID NO:2), and an HCDR3 comprising FSQYSWYTFSGLDY (SEQ ID NO:3); and a light chain variable (VL) region comprising a light chain complementarity determining region (LCDR) 1 comprising R(A/T)SQSVSSAVA (SEQ ID NO:4), an LCDR2 comprising SASSLYS (SEQ ID NO:5), and an LCDR3 comprising QQASTYPIT (SEQ ID NO:6).

3. The antibody of claim 2, wherein the VH region comprises: EVQLVESGGGLVQPGGSLRLSCAASGFNFYSSYIHWVRQAPGKGLEWVASISSYYGS TSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFSQYSWYTFSGLDYW GQGTLVTVSS (SEQ ID NO:7); and the VL region comprises:

DIQMTQSPSSLSASVGDRVTITCR(A/T)SQSVSSAVAWYQQKPGKAPKLLIYSASSLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQASTYPITFGQGTKVEIK (SEQ ID NO: 8).

4. The antibody of claim 2, comprising a heavy chain and a light chain, wherein the heavy chain comprises

EVQLVESGGGLVQPGGSLRLSCAASGFNFYSSYIHWVRQAPGKGLEWVASISSYYGS

TSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFSQYSWYTFSGLDYW

GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS

GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK

THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

DGVEVHNAKTKPREEQYNSTYRVV S VLTVLHQD WLN GKEYKCKV SNKALGAPIEK

TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:9), and the light chain comprises: DIQMTQSPSSLSASVGDRVTITCR(A/T)SQSVSSAVAWYQQKPGKAPKLLIYSASSLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQASTYPITFGQGTKVEIKRTVAAPSV FIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

5. The antibody of any one of claims 1-4, wherein the antibody comprises at least a second layilin-binding domain.

6. The antibody of claim 5, wherein the second layilin-binding domain comprises a VH region comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprises SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3; and a VL region comprising an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6.

7. The antibody of claim 6, wherein the VH region of the second layilin- binding domain comprises SEQ ID NO:7 and the VL region of the second layilin-binding domain comprises SEQ ID NO: 8.

8. The antibody of claim 5, wherein the second layilin-binding domain comprises the LBD-A.

9. The antibody of claim 5, wherein the second layilin-binding domain comprises a layilin-binding domain B (LBD-B), wherein the LBD-B specifically binds to the layilin ECD.

10. The antibody of any one of claims 5-9, wherein the antibody comprises at least three layilin-bindingdomains.

11. The antibody of claim 10, wherein each of the domains are independently selected from the LBD-A and the LBD-B.

12. The antibody of any one of claims 1-11, wherein the LBD-A and/or the LBD-B comprise an antagonistic layilin-binding domain.

13. The antibody of claim 12, wherein the LBD- A and/or the LBD-B do not result in detectable levels of integrin signaling upon binding to the layilin ECD when the layilin ECD is operably linked to a layilin intracellular domain.

14. The antibody of claim 13, wherein integrin signaling is assessed by an M24-cell based signaling assay.

15. The antibody of any one of claims 1-14, wherein the LBD- A and/or the LBD-B bind the layilin ECD in a calcium-dependent manner.

16. The antibody of claim 15, wherein the LBD- A and/or the LBD-B do not bind the layilin ECD when Ca²⁺ is absent.

17. The antibody of any one of claims 5 or 8-16, wherein the antibody comprises at least two heavy chain variable regions and at least two light chain variable regions, the heavy chain variable regions each comprising a HCDR1, HCDR2, and HCDR3, and the light chain variable regions each comprising a LCDR1, LCDR2, and LCDR3.

18. The antibody of claim 17, wherein each of the two heavy chain variable regions are identical and/or each of the two light chain variable regions are identical.

19. The antibody of any one of claims 9-18, wherein the LBD-B is operably linked to the heavy chain variable region or the light chain variable region of LBD- A.

20. The antibody of any one of claims 1-19, wherein the antibody further comprises one or more constant domains selected from the group consisting of: a CHI, a CH2, a CH3, and a CL constant domains.

21. The antibody of claim 20, wherein the antibody comprises an Fc domain.

22. The antibody of claim 20 or 21, wherein the Fc domain comprises an IgG constant domain.

23. The antibody of any one of claims 20-22, wherein the antibody comprises two of each of the CHI, the CH2, the CH3, and the CL constant domains.

24. The antibody of claim 23, wherein each of the two respective constant domains are identical.

25. The antibody of any one of claims 20-24, wherein one or more of the constant domains comprise an engineered mutation with reference to an endogenous wildtype constant domain.

26. The antibody of any one of claims 1-25, wherein the antibody comprises an scFv fragment, a single-domain antibody, a Fab fragment, an Fv fragment, a F(ab’)2 fragment, a Fab’ fragment, and/or an scFv-Fc fragment, or antigen binding fragment thereof.

27. The antibody of any one of claims 1-26, wherein the layilin ECD is a human, murine, or cynomolgus layilin ECD.

28. The antibody of any one of claims 1-27, wherein the collagen Type IV and/or collagen Type V is glycosylated.

29. The antibody of any one of claims 1-29, wherein the antibody interferes with binding between the layilin ECD with collagen Type VI.

30. A chimeric receptor comprising the antibody of any one of claims 1- 29, optionally comprising a transmembrane domain, and optionally comprising an intracellular signaling domain.

31. A polynucleotide or set of polynucleotides encoding the antibody or the chimeric receptor of any one of claims 1-30 or an antigen-binding portion thereof.

32. A vector or set of vectors encoding the antibody or chimeric receptor of any one of claims 1-30 and/or comprising the polynucleotide or set of polynucleotides of claim 31, optionally wherein the vector comprises a promoter operably linked to the polynucleotide encoding the antibody.

33. A cell expressing the antibody or chimeric receptor of any one of claims 1-30, comprising the polynucleotide or set of polynucleotides of claim 31, and/or comprising the vector or set of vectors of claim 32.

34. A pharmaceutical composition comprising the antibody or chimeric receptor of any one of claims 1-30, the polynucleotide or set of polynucleotides of claim 31, the vector or set of vectors of claim 32, and/or the cell of claim 33.

35. A method of producing an anti-layilin antibody or chimeric receptor, wherein the method comprises expressing or having expressed the antibody or chimeric receptor of any one of claims 1-30 in a cell and isolating or having isolated the anti-layilin antibody or chimeric receptor.

36. A method of manufacturing a polynucleotide encoding an anti-layilin antibody or chimeric receptor, wherein the method comprises obtaining or having obtained the polynucleotide or set of polynucleotides of claim 31 and/or the vector or set of vectors of claim 32, amplifying of having amplified the polynucleotide or vector, and isolating the amplified polynucleotide or vector, optionally wherein the amplification comprises (1) transfecting or having transfected the polynucleotide or vector in a host cell under conditions sufficient for replication of the polynucleotide or vector in the host cell, and/or (2) a polymerase chain reaction.

37. A method of reducing binding of one or both of collagen IV and collagen V to layilin on a cell surface in the presence of the collagen IV and/or collagen V, the method comprising contacting, or having contacted, a binding molecule that specifically binds to the layilin and interferes with binding between the layilin and the collagen IV and/or collagen V

38. The method of claim 37, wherein the binding molecule comprises a binding peptide or an antibody.

39. The method of claim 39, wherein the antibody comprises any one of the antibodies of any one of claims 1-29.

40. The method of any one of claims 37-39, wherein the cell is a human CD8 T-cell or a regulatory T-cell (Treg)

41. The method of claim 40, wherein the cell is in a human and the binding molecule is administered to the human.

42. The method of claim 41, wherein the human has cancer or an autoimmune disease.

43. A method of reducing binding of human layilin on a cell surface of human CD8⁺ T-cells or regulatory T-cells (Tregs) to one or both of collagen Type IV or collagen Type V in a human subject having a disease, the method comprising: contacting the human CD8⁺ T-cells or Tregs with a binding molecule that binds human layilin on the cell surface and that interferes with binding between the human layilin and one or both of collagen Type IV or collagen Type V, in an amount effective to reduce binding of the human layilin to one or both of collagen IV and collagen V.

44. The method of claim 43, wherein the binding molecule comprises a binding peptide or an antibody.

45. The method of claim 44, wherein the antibody comprises an antibody of any one of claims 1 to 29.

46. The method of any one of claims 37-45, wherein the method further comprises detecting or having detected (a) one or both of the collagen Type IV or collagen Type V, and/or (b) a cell known or suspected of expressing one or both of the collagen type IV or collagen Type V.

47. A method of identifying a binding molecule that interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V; the method comprising: having contacted or contacting a polypeptide comprising the layilin ECD with one or more binding molecules in the presence of the collagen IV, collagen V, and/or a layilin-binding fragment thereof, having determined or determining that one or more binding molecules interferes with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof, thereby identifying the binding molecule that interferes with binding between the layilin ECD and collagen IV and/or collagen V.

48. A method of determining that a binding molecule interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V; the method comprising: having contacted or contacting the binding molecule with a composition comprising a polypeptide comprising the layilin ECD and one or more of collagen IV, collagen V, or a layilin-binding fragment thereof, optionally wherein the polypeptide comprising the layilin ECD and/or the collagen is expressed on the surface of a cell, having measured or measuring binding between the binding molecule and the polypeptide comprising the layilin ECD and/or collagen, and having determined or determining that the binding molecule interferes with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof.

49. A method of determining that a binding molecule interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V, the method comprising: having contacted or contacting a polypeptide comprising the layilin ECD with at least one of collagen IV, collagen V, or a layilin-binding fragment thereof, optionally wherein the polypeptide comprising the layilin ECD and/or the collagen is expressed on the surface of a cell, under a first condition in which a binding molecule is present and under a second condition in which the binding molecule is absent, having measured or measuring binding between the polypeptide comprising the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof, and having determined or determining that the binding molecule interferes with binding between the polypeptide comprising the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof when the binding between the polypeptide and collagen is lower in the first condition relative to the second condition.

50. A method of determining that a binding molecule interferes with binding between a layilin extracellular domain (ECD) and one or both of collagen IV or collagen V; the method comprising: having contacted or contacting the binding molecule with a composition comprising a polypeptide comprising either (a) the layilin ECD or (b) the collagen IV, collagen V, and/or layilin-binding fragment thereof, optionally wherein the polypeptide comprising the layilin ECD and/or the collagen is expressed on the surface of a cell, subsequently having contacted or contacting the composition with (a) the collagen IV, collagen V, and/or layilin-binding fragment thereof when the composition comprises or comprised the layilin ECD, or (b) the layilin ECD when the composition comprises or comprised the collagen IV, collagen V, and/or layilin-binding fragment thereof, optionally wherein the subsequent contact is in the continued presence of the binding molecule, and having measured or measuring binding between the polypeptide comprising the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof, and having determined or determining the binding molecule interferes with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof when binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof is reduced or eliminated.

51. The method of any one of claim 47-50, wherein the binding molecule specifically binds layilin.

52. The method of claim 51, wherein the binding molecule comprises an antibody.

53. The method of claim 52, wherein the antibody comprises an antibody of any one of claims 1 to 29.

54. The method of claim 52-53, wherein the antibody specifically binds an epitope distinct from that bound by anti-layilin antibody 3F7D7E2 and/or 4C11.

55. The method of any one of claim 47-54, wherein the determining step further comprises comparing to the binding determined between the layilin and collagen under conditions wherein the binding molecule is an antibody of any one of claims 1-29.

56. The method of claim 55, wherein the binding molecule is determined to interfere with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof when the binding between the layilin ECD and collagen is equal to or lower than under conditions wherein the binding molecule is an antibody of any one of claims 1-29.

57. The method of any one of claim 47-56, wherein the determining step further comprises comparing to the binding determined between the layilin and collagen under conditions wherein the binding molecule is anti-lay ilin antibody 3F7D7E2, 4C11, and/or a binding fragment thereof.

58. The method of claim 57, wherein the binding molecule is determined to interfere with binding between the layilin ECD and the collagen IV, collagen V, and/or layilin-binding fragment thereof when the binding between the polypeptide and collagen is lower than under conditions wherein the binding molecule is anti-layilin antibody 3F7D7E2, 4C11, and/or a binding fragment thereof.

59. The method of any one of claims 47-50, wherein the polypeptide comprises a multimer of the human layilin ECD.

60. The method of claim 59, wherein the multimer comprises a dimer, a trimer, or a pentamer.

61. The method of claim 60, wherein the multimer comprises a pentamer.

62. The method of any one of claims 47-61, wherein the contacting is performed in the presence of Ca²⁺, optionally wherein prior to the contacting in the presence of Ca²⁺, the one or more binding molecules have been pre-contacted with the polypeptide comprising the layilin ECD or the collagen IV, collagen V, and/or layilin-binding fragment thereof in the absence of Ca²⁺ and one or more binding molecules that bind to the layilin ECD or collagen in a calcium-independent manner have been removed prior to the contacting.

63. The method of claim 62, wherein the selecting step comprises eluting with a calcium-chelating agent, optionally wherein the calcium-chelating agent is EDTA or EGTA.

64. The method of any one of claims 47-63, wherein the collagen IV and/or collagen V is glycosylated.

65. The method of any one of claims 47-64, wherein the layilin ECD is a human, murine, or cynomolgus layilin ECD.

66. The method of claim 65, wherein the layilin ECD is a human layilin ECD.

67. The method of any one of claims 47-66, wherein the binding molecule interferes with binding to a C-type lectin domain of the layilin ECD.

68. The method of any one of claims 47-67, wherein the binding molecule interferes with binding between the layilin ECD with collagen VI.