Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1)-binding variable heavy (VH) domains

Abstract

The present disclosure provides VH domains that specifically bind to ENPP1. The VH domains and molecules comprising the VH domains that specifically bind to ENPP1can be used in methods, such as, methods for treating cancer or insulin resistance or diabetes (e.g., type 2 diabetes) in a subject.

Description

ECTONUCLEOTIDE PYROPHOSPHATASE PHOSPHODIESTERASE 1 (ENPPl)-BINDING VARIABLE HEAVY (VH) DOMAINSCROSS-REFERECE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 499,466, filed May 1, 2023, which application is incorporated herin by reference in its entirerty.INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

[0002] A Sequence listing is provided herwith as a Sequence Listing XML, “UCSF- 722WO_SEQ_LIST.xml” created on April 28, 2024 and having a size of 23,661 bytes. The contents of the Sequence listing XML are incorporated by reference in their entirety.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under P41 CA196276 awarded by the National Institutes of Health. The government has certain rights in the invention.INTRODUCTION

[0004] Ectodomain phosphatase / phosphodiesterase-1 (ENPP1) is an extracellular enzyme that is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing cyclic guanosine monophosphate adenosine monophosphate (cGA P). an immune-stimulant. There are small molecule ENPP1 inhibitors that are in early clinical phases, however biologies inhibiting ENPP1 activity have not yet been generated. A significant therapeutic advantage of biologies is that they can be recombinantly engineered into multi-functional formats for greater tissue selectivity and efficacy as immunotherapies .

[0005] The present disclosure addresses these as well as other needs.SUMMARY

[0006] The present disclosure provides VH domains and antibodies comprising VH domains disclosed herein that specifically bind to ENPP1. In certain aspects, the VH domains disclosed herein inhibit the activity of ENPP1 resulting in a significant reduction in hydrolysis of cGAMP. In certain aspects, the VH domains disclosed herein inhibit the activity of ENPP1 resulting in a significant reduction in hydrolysis of ATP.

[0007] In certain aspects, a VH domain as provided herein comprises a HCDR 1 comprising the sequence: DIYYSYYI (SEQ ID NO:1); a HCDR 2 comprising the sequence: RISPSYGSTS (SEQ ID NO:2); and a HCDR 3 comprising the sequence: FAY / APW / FYVADDAL (SEQ ID NOG).

[0008] In another aspect, a VH domain as provided herein comprises a heavy chain complementarity determining region 1 (HCDR 1) comprising the sequence: AIYYYDI(SEQ ID NO:4); a HCDR 2 comprising the sequence: RISPYSGSTY (SEQ ID NOG); and a HCDR 3 comprising the sequence: AHWDGYGYVDDWVAI (SEQ ID NOG).

[0009] In another aspects, a VH domain as provided herein comprises a HCDR 1 comprising the sequence: DIYYSEI (SEQ ID NO:7); a HCDR 2 comprising the sequence: RISPYYGSTS (SEQ ID NOG); and a HCDR 3 comprising the sequence: FDPYHYFSDYYGI (SEQ ID NO:9).

[0010] In another aspects, a VH domain as provided herein comprises a HCDR 1 comprising the sequence: AIYSYEI (SEQ ID NO: 10); a HCDR 2 comprising the sequence: RISPYSSSTY (SEQ ID NO: 11); and a HCDR 3 comprising the sequence: FWGPWFSSAL (SEQ ID NO: 12).

[0011] The VH chain may be complexed with, conjugated to or fused to another entity including but not limited to a peptide, polypeptide, labels, drugs, and the like. In certain aspects, the VH chain is complexed with a polypeptide such as a polypeptide that binds to an immune checkpint marker, e.g., POLE

[0012] Nucleic acids provided herein encode one or more VH domains that are described herein.Cells comprising such nucleic acids are also provided herein. Kits comprising one or more described herein are disclosed.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG 1. Phage-display generated high affinity VH domains recognizing native ENPP1 on PDX-derived osteosarcoma cells, and VH27 inhibited ATP and cGAMP hydrolysis. A.Structure of extracellular domain of ENPP1. B. Recombinant ENPP1 C-terminal Fc-fusion antigen for phage-display (ENPPl-Fc). ENPP1 ectodomain was modified by truncating SMB1 and SMB2 domains and mutating the catalytic threonine to alanine. Fc-fusion included an Avi tag for biotinylation and a TEV protease site. C,D. Schema of phage-display selection rounds. VH-phagemid library underwent four rounds of negative (Fc-biotin) and positive (ENPPl-Fc) selection with increasing stringency. To elute, bound phage were released by TEV protease treatment (C) or eluted using lOmM ATP substrate (D). E. Representative biolayer interferometry signals and fits for each VH-Fc binding ENPPl-Fc antigen or Fc- biotin control (no signal). F. Table of KD values measured by biolayer interferometry for VH-Fc panel (N=2). G. On-cell binding was tested using PDX-derived OS384 cell line engineered with ENPP1 knockout (KO) or safe-guide (SG). OS384 SG and ENPP1 KO cells were stained with 50nM of binderand median fluorescent signal was analyzed by flow cytometry. The bar graph reports the mean and SEM of the fold-change (SG / KO) in signal (N=3 or 4 independent replicates). Statistics were calculated using one-tailed Student’s t-test. H, I. Michaelis-Menten kinetics were determined for VH27-Fc inhibitor for ATP (H) or cGAMP (I) substrates (N=3, mean and standard deviation). J. VH27-Fc inhibited secreted ENPP1 in ex vivo plasma supplemented with ImM cGAMP over 90 min and 24 hr time courses. Fc isotype treatment and condition with no cGAMP added (No cGAMP) were included as controls (N=3 for Donor 1).

[0014] FIG. 2. Affinity maturation of VH27 improved affinity, stability, and inhibitory potency. A. Yeast display coupled with in cellulo continuous diversification was performed using the AHEAD hypermutation yeast strain. Yeast populations expressing mutated VH27 were stained with ENPPl-Fc and anti-HA antibody and FACS sorted for optimal antigen binding and stable expression. Five selection rounds of increasing antigen stringency were performed and the final population was amplicon sequenced by NGS. B. T75I and A89V framework mutations were ranked 2 and 3 respectively, with read frequencies over 1%. C. Affinity constants and microscopic rate constants for VH27-Fc, VH27 / T75I-Fc, VH27 / A89V-Fc, and VH27 / T75I / A89V-Fc binding to ENPPl-Fc antigen were determined by biolayer interferometry (N=2, mean and standard deviation). D,E. Michaelis-Menten enzyme kinetics for VH27 / T75I / A89V-Fc for ATP (D) and cGAMP (E) hydrolysis (N=3, mean and standard deviation). F. Human plasma supplemented with 1 mM cGAMP was treated with a titration of VH27 / T75I / A89V-Fc for 90 min and cGAMP degradation was measured. Fc isotype treatment and condition with no cGAMP added (No cGAMP) were included as controls (N=3 for each donor). G. Differential scanning fluorimetry was used to measure melting temperature of VH27 (WT) versus the double mutant (VH27 / T75I / A89V) as single-domain VHs in non-reducing and reducing (6.25% BME) conditions. Bar graph reports mean and SEM for N=4 or 5 biological replicates and statistics were calculated using two-tailed Student’s t-test. H. VH27-Fc and VH27 / T75I / A89V-Fc were analyzed by SEC and peak area ratios between ‘aggregate peak’ and ‘Fc peak’ were analyzed. Bar graph reports mean and SEM for N=2 or 3 biological replicates and statistics were calculated using two-tailed Student’s t-test.

[0015] FIG. 3. VH27.2 was ported into bi-paratopic and bispecific formats driving improved localization to tumor cells and stronger inhibition of ENPP1 on cell membranes. A.Structure of bi-paratopic tetravalent Fc inhibitor recognizing inhibitory VH27.2 epitope and a second nonoverlapping epitope on ENPPl-Fc. B,C. Biolayer interferometry was used to map the epitopes of the VH panel with respect to the VH27 epitope. VH24-Fc, VH31-Fc, and VH38-Fc could bind ENPPl-Fc after VH27-Fc was pre-loaded on the sensor (B), and VH27-Fc could bind ENPPl-Fc after VH24-Fc, VH31- Fc, or VH38-Fc were pre-loaded on the sensor (C). D. Representative biolayer interferometry signals and fits for bi-paratopic VH27.2 / VH31 inhibitor binding ENPPl-Fc antigen or Fc-biotin control (no signal).KD was measured to be sub-nanomolar (N=2, mean and standard deviation). E. Bi-paratopic molecule inhibited ENPP1 in human plasma (ImM cGAMP, 90 min). Fc isotype treatment and condition with no cGAMP added (No cGAMP) were included as controls (N=2 for each donor). F. Structure of bispecific tetravalent Fc inhibitor combining VH27.2 with the PD-L1 checkpoint inhibitor Envafolimab. G,H. Representative biolayer interferometry signals and fits for bispecific inhibitor binding ENPPl-Fc antigen (G) or PDLl-Fc antigen (H), with no signal observed for Fc-biotin controls. KD values were measured to be sub-nanomolar for both ENPPl-Fc and PD-Ll-Fc (N=2, mean and standard deviation). I. Bispecific molecule inhibited ENPP1 in human plasma (ImM cGAMP, 90 min). Fc isotype treatment and condition with no cGAMP added (No cGAMP) were included as controls (N=2 for each donor). J. OS384 SG and ENPP1 KO cells were stained with 50 nM of binder and median fluorescent signal was analyzed by flow cytometry. The bar graph reports the mean and SEM of the fold-change (SG / KO) in signal (at least N=5 biological replicates). Statistics were calculated using one-tailed Student’s t-test. K. MDA-MB-231 cells were stained with a titration of VH27.2-Fc, VH31-Fc, bi-paratopic molecule (VH27.2 / VH31), bispecific molecule (VH27.2 / Envafolimab), or Fc-Envafolimab and median fluorescence intensity values were used to fit the EC50 curve. Data represent mean and SEM for N=2 or 3 biological replicates. L. MDA-MB-231 cells were treated with 50 uM cGAMP and the indicated concentration of VH27.2-Fc, bi-paratopic inhibitor, or bispecific inhibitor. After 12 hr, cell media was harvested and remaining cGAMP was measured using a cGAMP ELISA. Data were normalized to the range between the cGAMP in media treated without cells and cells treated with PBS shown in M. Fc isotype treated at 3 uM was included as a control and data are reported in M. N. MDA-MB-231 cells were treated with 200 uM pNP-TMP and the indicated concentration of VH27.2-Fc, bi-paratopic inhibitor, or bispecific inhibitor. After 5 hr, absorbance at 405 mn was measured. Data were normalized to the PBS condition presented in O. Fc isotype treated at 3 uM was included as a control and data are reported in O. Data in L-O represent mean and SEM for N = 3-5 biological replicates.

[0016] FIG. 4. Recombinant engineering of VH inhibitor into immunotherapies and targeted protein degraders. A. Representative biolayer interferometry signals for 50 nM bivalent VH27.2-Fc, tetravalent bispecific Fc, and single domain VH27.2 (no Fc) binding to CD16-Fc antigen or Fc-biotin control (N=2). B. Structure of bispecific T cell engager (BiTE) combining VH27.2 with arm recognizing CD3 (OKT3 scFv) in a linked tandem format. C. Jurkat cells expressing NFAT-GFP reporter were incubated with beads coated with 0, 10, or lOOnM Fc-ENPPl or a no bead control, and were treated with 10 nM BiTE or PBS control. After 20 hr GFP expression driven by NF AT activation was measured by flow cytometry. Data were normalized to the no bead / no BiTE condition. Bar graph reports mean and SEM for N=3 biological replicates. Statistics were calculated using two-tailed Student’s t-test. D.Structure of ‘knob-into-hole’ bispecific AbTAC degrader combining anti-ENPPl VH with RNF43- recruiting IgG arm. E, F. MDA-MB-231 cells were treated with PBS or a dose titration (500, 100, 20, 4 nM) of VH27.2 AbTAC and ENPP1 levels were measured by immunoblot. Cells were also treated with 500 nM VH27.2-Fc and Fc isotype controls. ENPP1 densities were normalized to ACTIN loading control. The percent of ENPP1 remaining relative to the PBS treatment was calculated. E. A representative immunoblot for one experiment. F. Graph summarizes the mean and SEM for N=3-5 biological replicates. Statistics were calculated using two-tailed Student’s t-test. G. MDA-MB-231 cells were pretreated with 1 uM VH27.2 AbTAC, indicated control molecules, or PBS for 24 hr. Solutions containing molecules were removed, cells were washed, and cells were incubated with 200 uM pNP-TMP for 5 hr. Absorbance at 405 nm was measured. Data were normalized to PBS condition and graph represents mean and SEM for N= 3 biological replicates with 1 or 2 technical replicates. Statistics were calculated using two-tailed Student’ s t-test.

[0017] FIG. 5. Cryo-EM reveals VH binding ENPP1 proximal to the catalytic site. A. Cryo- EM 3D reconstruction of VH27.2 bound to ENPP1 ectodomain. B. View of the CDR Hl epitope. C. View of the CDR H2 epitope. D. View of the CDR H3 epitope. E. Table summarizing interactions between VH and ENPP1 residues. F. Biolayer interferometry comparing binding kinetics of VH27.2-Fc alanine mutants at 25 nM. Traces are representative of two experiments. G. Inhibitory potencies of VH27.2-Fc alanine mutants relative to WT VH27.2-Fc treated at 500 nM for ATP, pNP-TMP, and cGAMP substrates. Bar graph reports mean and SEM for two biological replicates. H. Biolayer interferometry comparing affinity of VH27.2-Fc to WT, K528A, F346A, and H38OA ENPPl-Fc. Traces are representative of two experiments. I. Biolayer interferometry comparing binding kinetics of VH27.2- Fc phenylalanine mutants at 25nM. Traces are representative of two experiments. J. Inhibitory potencies of VH27.2-Fc phenylalanine mutants relative to WT VH27.2-Fc treated at 500 nM for ATP, pNP-TMP, and cGAMP substrates. Bar graph reports mean and SEM for two biological replicates. K. ATP / cGAMP K; fold-change value for WT, Y102A, and W104F VH27.2 -Fc. L. Linear regression and R2values correlating KD and I<; values for ATP and cGAMP substrates for WT, Y102A, and W104F VH27.2-Fc mutants.

[0018] FIG. 6. Sequences, cellular binding, and stability of VH panel. A. CDR Hl-3 sequences for VH panel (first row, SEQ ID NOs:4-6; second row, SEQ ID NOs:l-2 and 13; third row, SEQ ID NOs:7-9; fourth row, SEQ ID NOs: 10-12). B. Representative flow cytometry histograms used to generate bar graph summarizing fold-changes for binding OS384 SG and KO in Figure 1G. C. SEC traces for VH24-Fc, VH27-Fc, VH31-Fc, and VH38-Fc.

[0019] FIG. 7. AHEAD yeast display campaign to affinity mature VH27. A. FACS gating used for sorting less than 1 % AHEAD yeast cell population based on expression (HA tag) and antigen binding.In round 4 the fluorophores were switched (SA-488 and anti-HA-647). B-D. Biolayer interferometry signals and fits to determine KD, kassociation, and kdissociation for VH27-Fc with scaffold mutations T75I (B), A89V (C), and T75I / A89V (D). Data are representative of two experiments. E,F. Mouse ENPP1 (mENPPl) was treated with 500 nM VH27 / T75I / A89V-Fc, Fc isotype, or PBS to evaluate crossreactivity for ATP (E) and cGAMP (F) substrates (N=3 biological replicates for each substrate). Bar graph reports mean and SEM and statistics were calculated using two-tailed Student’s t-test. G. Dose titration of VH27 / T75I / A89V-Fc or indicated controls were tested for ENPP1 inhibition in C57BL / 6J mouse plasma supplemented with 1 mM cGAMP for 90 min (N=3 mice, 1 experiment per mouse).

[0020] FIG. 8. Additional data for multivalent constructs. A, B. SEC traces for bi-paratopic inhibitor (A) and bispecific inhibitor (B). C. Representative flow cytometry histograms used to generate bar graph summarizing fold-changes for binding OS384 SG and KO in Figure 3J. D. Representative flow cytometry histograms for NFAT-GFP Jurkat activation assays when treated with indicated concentrations of ENPP1 -coupled beads or no beads and 10 nM BiTE or PBS. E. Unformatted immunoblot with visible ladder used for image in Figure 4F.

[0021] FIG. 9. Additional data for structural and mechanistic analyses. A-D. Validation of VH-ENPP1 complex. Complex was purified by SEC (A) and co-elution of VH and ENPPl-Fc was analyzed by SDS-PAGE gel (B). Mass photometry of antigen alone (C) and SEC-eluted complex peak (D) demonstrated mass shift of approximately 30 KDa suggesting 2:1 VH:antigen stoichiometry. E. Cryo- EM structure of VH-ENPP1 complex aligned to PDB 6wjf. F. Cryo-EM structure of VH-ENPP1 complex aligned to PDB 4gtw with sequence alignment of human and mouse ENPP1. Residues colored red were within 5A of VH CDR Hl, H2, or H3 (top SEQ ID NO:24; bottom SEQ ID NO:25). G. Close-up of clashing with pApG when VH-ENPP1 complex (cyan) is aligned to PDB 6aek (yellow). H.Representative biolayer interferometry signals and fits for VH27.2-Fc Y102A (N=2, mean and standard deviation). I, J. Michaelis-Menten kinetic analysis for VH27.2-Fc Y102A for ATP (I) and cGAMP (J) (N=2, mean and standard deviation). K. Representative biolayer interferometry signals and fits for VH27.2-Fc W104A (N=2, mean and standard deviation). L,M. Michaelis-Menten kinetic analysis for VH27.2-Fc W104A for ATP (L) and cGAMP (M) (N=2, mean and standard deviation).DEFINITIONS

[0022] “Derived from” in the context of an amino acid sequence or polynucleotide sequence is meant to indicate that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid, and is not meant to be limiting as to the source or method in which the protein or nucleic acid is made.

[0023] An “anti-ENPPl antibody” as used herein refers to an antibody that includes a VH domain provided in this disclosure and binds ENPP1. ENPP1 may be human ENPP1. ENPP1 may be mouse ENPP1. In certain aspects, the anti-ENPPl antibody reduces enzymatic activity of ENPP1. An anti-ENPPl antibody may reduce enzymatic activity of ENPP1 by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more. The enzymatic activity may be hydrolysis of cGAMP. The hydrolysis of cGAMP may be measured using an assay provided herein or another assay.

[0024] The terms "polypeptide", and "protein" are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acid residues are usually in the natural "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. In addition, the amino acids, in addition to the 20 "standard" amino acids, include modified and unusual amino acids, which include, but are not limited to those listed in 37 CFR (§ 1.822(b)(4)). Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates either a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group. However, the absence of a dash should not be taken to mean that such peptide bonds or covalent bond to a carboxyl or hydroxyl end group is not present, as it is conventional in representation of amino acid sequences to omit such. The term "peptide” also refers to a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues but is generally shorter than a protein or a polypeptide, c.g., less than 100 amino acids long, less than 80 amino acids long, less than 70 amino acids long, less than 60 amino acids long, or shorter, e.g., up to 5, 10, 15, 20, 30, 40 or 50 amino acids in length.

[0025] The term “antibody” (also used interchangeably with “immunoglobulin”) encompasses polyclonal and monoclonal antibody preparations where the antibody may be of any class of interest (e.g., IgM, IgG, and subclasses thereof), as well as preparations including hybrid antibodies, altered antibodies, F(ab')j fragments, F(ab) molecules, Fv fragments, scFv fragments, single chain antibodies, single domain antibodies, e.g., VH domain antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, multi-specififc antibodies, and functional fragments thereof which exhibit binding properties of the parent antibody molecule. The antibodies may be conjugated to other moieties, such as, labels, and / or may be bound to a support (e.g., a solid support), such as a polystyrene plate or bead, test strip, and the like.

[0026] Immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGi, IgG . IgG:, IgGr), delta, epsilon and mu heavy chains or equivalents in other species. Full- length immunoglobulin “light chains” (usually of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region atthe COOH-terminus. Full-length immunoglobulin “heavy chains” (of about 50 kDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).

[0027] An immunoglobulin light or heavy chain variable region is composed of a “framework” region (FR) interrupted by three hyper variable regions, also called “complementarity determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, (1991) and Lefranc et al. IMGT, the international ImMunoGeneTics information system®. Nucl. Acids Res., (2005) 33:D593-D597)). A detailed discussion of the IMGTS system, including how the IMGTS system was formulated and how it compares to other systems, is provided on the World Wide Web at imgt.cines.fr / textes / IMGTScientificChart / Numbering / IMGTnumberingsTable.html. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. All CDRs and framework provided by the present disclosure are defined according to Kabat et al, supra, unless otherwise indicated. The three light chain CDRs, as used herein, are also referred to as “LCDR1”, “LCDR2”, and “LCDR3”. The three heavy chain variable CDRs, as used herein, are also referred to as “HCDR1”, “HCDR2”, and “HCDR3”.

[0028] An "antibody" thus encompasses a protein having one or more polypeptides that can be genetically cncodablc, e.g., by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0029] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer.Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0030] Antibodies encompass intact immunoglobulins as well as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab') dimer into an Fab' monomer. TheFab’ monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies, including, but are not limited to, VH, Fab'2, IgG, IgM, IgA, scFv, dAb, nanobodies, unibodies, and diabodies.

[0031] Antibodies and fragments of the present disclosure encompass those that arc bispccific. Bispecific antibodies or fragments can be of several configurations. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). Bispecific antibodies may be produced by chemical techniques (Kranz et al. (1981) Proc. Natl. Acad. Sei., USA, 78: 5807), by "polydoma" techniques (see, e.g., U.S. Pat. No. 4,474,893), or by recombinant DNA techniques. Bispecific antibodies may have binding specificities for at least two different epitopes, at least one of which is an epitope of ENPP1. Bispecific antibodies include formats such as bispecific IgG (optionally wherein the IgG has a common light chain), DVD-Ig (see DiGiammarino et al., Meth. Mo. Biol., 2012, 889, 145-156), mAh- (see W02008 / 003103, the description of the mAb2format is incorporated herein by reference), FIT-Ig (see W02015 / 103072, the description of the FIT-Ig scaffold is incorporated herein by reference), mAb-dAb, dock and lock. Fab-arm exchange, SEEDbody, Triomab, orthogonal Fab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab- scFv, intrabody, BiTE, diabody, DART, scDiabody, scDiabody-CH3, Diabody-CH3, Triple body, Miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes with common light chain and charge pairs, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG- 2scFv, scFv4-Ig, etc. In another embodiment, the bispecific molecule comprises an antibody which is fused to another non-Ig format, for example a T-cell receptor binding domain; an immunoglobulin superfamily domain; a fibronectin domain; a heat shock protein (such as and epitope binding domain derived from GroEl and GroES); a transferrin domain (e.g., a trans-body); ankyrin repeat protein; C-type lectin domain; human y-cry stallin or human ubiquitin; a PDZ domain; scorpion toxin; and a kunitz type domain of a human protease inhibitor. In certain aspects, the bispecific antibody includes a VH domain as disclosed herein and a VH domain that binds to transmembrane E3 ligases ring finger 43 (RNF43). Such an antibody may be referred to as AbTAC.

[0032] An "antigen-binding site" or "binding portion" refers to the part of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-tcrminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as "hypervariable regions" which are interposed between more conserved flanking stretches known as "framework regions" or "FRs". Thus, the term "FR" refers to amino acid sequences that are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen binding "surface". This surface mediates recognition and binding of the target antigen. The three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs" and are characterized, for example by Kabat et al. Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, MD (1987).

[0033] As used herein, the term "binding" refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of binding interactions can be expressed in terms of the dissociation constant (KD) of the interaction, wherein a smaller KD represents a greater affinity. Binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (kon) and the "off rate constant" (koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of koff / konenables cancellation of all parameters not related to affinity and is thus equal to the equilibrium dissociation constant KD (see, generally, Davies el al. Ann. Rev. Biochem. 1990, 59: 439-15 473).

[0034] An “epitope" is a site on an antigen (e.g., ENPP1) to which an antibody binds.

[0035] “Isolated” refers to an entity of interest that is in an environment different from that in which the entity may naturally occur or is initially produced in. An “isolated” compound (e.g., an “isolated” antibody) is separated from all or some of the components that accompany it and may be substantially enriched, e.g., may be purified so that the compound is at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99%, or greater than 99% pure, or free of impurities, contaminants, and / or components other than the compound. “Isolated” also refers to the state of a compound separated from all or some of the componentsthat accompany it during manufacture (e.g., chemical synthesis, recombinant expression, culture medium, and the like).

[0036] A single chain Fv ("scFv") polypeptide is a covalently linked VH" VL hctcrodimcr which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker (Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883). A number of structures are available for converting the light and heavy polypeptide chains from an antibody V region into an scFv molecule which will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Patent Nos. 5, 091,513 and 5,132,405 and 4,956,778.

[0037] Recombinant design methods may be used to develop suitable chemical structures (linkers) for converting two heavy and light polypeptide chains from an antibody variable region into a scFv molecule which will fold into a three-dimensional structure that is substantially similar to native antibody structure.

[0038] Design criteria include determination of the appropriate length to span the distance between the C-terminal of one chain and the N-terminal of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures. Such methods have been described in the art. See, e.g.. U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al.-, and U.S. Patent No. 4,946,778 to Ladner et al.

[0039] In this regard, the first general step of linker design involves identification of plausible sites to be linked. Appropriate linkage sites on each of the VH and VL polypeptide domains include those which will result in the minimum loss of residues from the polypeptide domains, and which will necessitate a linker comprising a minimum number of residues consistent with the need for molecule stability. A pair of sites defines a "gap" to be linked. Linkers connecting the C-terminus of one domain to the N-terminus of the next generally comprise hydrophilic amino acids which assume an unstructured configuration in physiological solutions and may be free of residues having large side groups which might interfere with proper folding of the VH and VL chains. Thus, suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include glutamic acid and lysine residues inserted to enhance solubility. One particular linker has the amino acid sequence (Gly4Ser)3 (SEQ ID NO:21). Another example of a suitable linker is a linker that has the amino acid sequence comprising 2 or 3 repeats of | (ScrhGly | (SEQ ID NO:22), such as [(Ser^Glyh (SEQ ID NO:23), and the like. Nucleotide sequences encoding such linker moieties can be readily provided using various oligonucleotide synthesis techniques known in the art (see, e.g., Sambrook, supra.).

[0040] The phrase "specifically binds to" or "specifically immunoreactive with", when referring to an antibody that specifically binds to an antigen refers to a binding reaction which is determinative ofthe presence of the antigen in the presence of a heterogeneous population of other molecules, proteins, DNA, and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular antigen and do not bind in a significant amount to other molecules present in the sample. Specific binding to an antigen under such conditions may require an antibody that is selected for its specificity for the antigen. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase enzyme-linked immunosorbent assay (ELISA) immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an antigen. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0041] The term "conservative substitution" is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) of the molecule. Typically, conservative amino acid substitutions involve substituting one amino acid for another amino acid with similar chemical properties (e.g., charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0042] By “treatment” it is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration refers to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment includes situations where the condition, or at least symptoms associated therewith, are reduced or avoided. Treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful or otherwise undesired state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease.DETAILED DESCRIPTION

[0043] The present disclosure provides a VH domain that specifically bind to ENPP1. The present disclosure provides molecules that include the VH and specifically bind to ENPP1. Such molecules include VH domain that is modified to generate a ENPP1 -binder having a variety of antibody formats including but not limited to multispecific antibodies (e.g., bispecific antibody), BiTEs, scFvs, Tr- Fab, single chain diabodies and the like.

[0044] The VH domains provided herein fins use in a variety of method including treatment of cancers.Variable Heavy Chain Domain (VH Domain)

[0045] The present disclosure provides a VH domain that specifically bind to ENPP1. In certain aspects, the VH domain inhibits ENPP1 enzymatic activity upon binding to ENPP1. In certain aspects, the VH domain is an allosteric inhibitor of ENPP1 enzymatic activity, where it binds to a site different from the ENPPl’s active site. In certain aspects, the VH domain binds to a site adjacent to the ENPPl’s active site. In certain aspects, the VH domain is an allosteric inhibitor of ENPP1 enzymatic activity and binds to an epitope comprising H380 residue of ENPP1. In certain aspects, the VH domain is an allosteric inhibitor of ENPP1 enzymatic activity and binds to an epitope comprising H380 and K528 residues of ENPP1. In certain aspects, the VH domain is an allosteric inhibitor of ENPP1 enzymatic activity and binds to an epitope comprising G342 residue of ENPP1 . In certain aspects, the VH domain is an allosteric inhibitor of ENPP1 enzymatic activity and binds to an epitope comprising G342, S377, F346, and R395 residues of ENPP1. In certain aspects, the VH domain is an allosteric inhibitor of ENPP1 enzymatic activity and binds to an epitope comprising H380, K528 G342, S377, F346, and R395 residues of ENPP1. In certain aspects, the VH domain binds to ENPP1 and inhibits the ENPP1 -mediated hydrolysis of cGAMP. In certain aspects, the VH domain binds to ENPP1 and inhibits the ENPP1 -mediated hydrolysis of ATP. In certain aspects, the VH domain binds to ENPP1 and inhibits the ENPP1 -mediated hydrolysis of cGAMP and ATP.

[0046] A “VH domain” or “VH chain” refers to the variable region of a heavy chain polypeptide. As compared to the binding of an antigen by an antigen binding site formed by a VL and VH domain, the binding of the antigen by the VH domain alone may be weaker but as demonstrated herein is advantageous in identifying inhibitory molecules. In certain aspects, a VH domain may be used without a VL domain and / or without a constant region, e.g., an Fc region. Such a VH domain may also be referred to as a domain antibody (dAb). In certain aspects, a VH domain may be fused to a constant region of an antibody, e.g., Fc region of an IgG antibody. In certain aspects, a VH domain may be combined with an VH domain that has the same CDRs to form a bivalent antibody. In certain aspects, a VH domain may be combined with a VH domain that binds to a different epitope on the same antigen to form a biparatropic antibody. In certain aspects, a VH domain may be combined with a VH domain that binds to a different antigen to form a monovalent bispecific antibody. In certain aspects, a VH domain may be combined with a two or more VH domains that bind to different antigens to form a multispccific antibody. The VH domains provided herein may be modified in any manner as long as the modified VH domains retain the ability to bind ENPP1.

[0047] In certain aspects, a VH domain as provided herein comprises a HCDR 1 comprising the sequence: (SEQ ID NO:1); a HCDR 2 comprising the sequence: RISPSYGSTS (SEQ ID NO:2); and a HCDR 3 comprising the sequence: FAY / APW / FYVADDAL (SEQ ID NOG). In certain aspects, the VH domain may have an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence:

[0048] EVQLVESGGGLVQPGGSLRLSCAASGFDIYYSYYIGWVRRAPGKGEELVARISPS YGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFAYPWYVADDALDYWGQG TLVTVSS (VH27) (SEQ ID NO: 16)

[0049] or

[0050] EVQLVESGGGLVQPGGSLRLSCAASGFDIYYSYYIGWVRRAPGKGEELVARISPSYGSTSYADSVKGRFTISADISKNTAYLQMNSLRVEDTAVYYCARFAYPWYVADDALDYWGQGT LVTVSS (SEQ ID NO: 17) (VH27.2)

[0051] In another aspect, a VH domain as provided herein comprises a heavy chain complementarity determining region 1 (HCDR 1) comprising the sequence: AIYYYDI(SEQ ID NO:4); a HCDR 2 comprising the sequence: RISPYSGSTY (SEQ ID NO:5); and a HCDR 3 comprising the sequence: AHWDGYGYVDDWVAI (SEQ ID NO:6). In certain aspects, the VH domain may have an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence:

[0052] EVQLVESGGGLVQPGGSLRLSCAASGFAIYYYDIGWVRRAPGKGEELVARISPYS GSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARAHWDGYGYVDDWVAIDYWG QGTLVTVSS (VH24) (SEQ ID NO: 18).

[0053] In another aspect, a VH domain as provided herein comprises a HCDR 1 comprising the sequence: DIYYSEI (SEQ ID NO:7); a HCDR 2 comprising the sequence: RISPYYGSTS (SEQ ID NOG); and a HCDR 3 comprising the sequence: FDPYHYFSDYYGI (SEQ ID NO:9). In certain aspects, the VH domain may have an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence:

[0054] EVQLVESGGGLVQPGGSLRLSCAASGFDIYYSEIGWVRRAPGKGEELVARISPYY GSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFDPYHYFSDYYGIDYWGQGTL VTVSS (VH31, SEQ ID NO: 19).

[0055] In another aspect, a VH domain as provided herein comprises a HCDR 1 comprising the sequence: AIYSYEI (SEQ ID NO: 10); a HCDR 2 comprising the sequence: RISPYSSSTY (SEQ IDNO:11); and a HCDR 3 comprising the sequence: FWGPWFSSAL (SEQ ID NO:12). In certain aspects, the VH domain may have an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence:

[0056] EVQLVESGGGLVQPGGSLRLSCAASGFAIYSYEIGWVRRAPGKGEELVARISPYSS STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFWGPWFSSALDYWGQGTLVTV SS (VH38, SEQ ID NO:20).

[0057] In certain embodiments, the amino acid sequences of various VH domains that bind toENPP1, as well as each CDR and framework region, are shown below:

[0058] The framework regions in the sequences of VH chains are underlined and the CDRs are italicized.

[0059] It will be appreciated that the amino acid sequence of a CDR can also be defined using alternative systems, which will be readily apparent to and applied by the ordinarily skilled artisan (see, “Sequences of Proteins of Immunological Interest,” E. Kabat ct al., U.S. Department of Health and Human Services, (1991); and Lefranc et al. IMGT, the international ImMunoGeneTics information system. Nucl. Acids Res., (2005) 33:D593-D597)). A detailed discussion of the IMGTS system, including how the IMGTS system was formulated and how it compares to other systems, is provided on the World Wide Web at imgt.cines.fr / textes / IMGTScientificChart / Numbering / IMGTnumberingsTable.html.

[0060] Using the teachings and the sequence information provided herein, the variable light and variable heavy chains can be joined directly or through a linker e.g., (Gly4Ser)3, SEQ ID NO:) to form a single-chain Fv antibody. The various CDRs and / or framework regions can be used to form human antibodies, chimeric antibodies, antibody fragments, polyvalent antibodies, and the like.

[0061] In certain aspects, the VH domains provided herein are not associated with a variable light (VL) domain and binds ENPP1 in absence of a VL domain. In other words, the VH domain is sufficient for binding to ENPP1 in absence of a VL domain.

[0062] In certain aspects, the VH domains provided herein bind human ENPP1. In certain aspects, the VH domains provided herein bind mouse ENPP1. In certain aspects, the VH domains provided herein bind human ENPP1 with a KD of 0.1-10 nM, e.g., 0.5-5 nM, or 0.5-3 nM.

[0063] In certain aspects, the VH domains provided herein bind ENPP1 on a cell surface of a mammalian cell. The cell may be a human cell. The cell may be a cancer cell.

[0064] In certain aspects, the VH domains provided herein bind ENPP1 at a site different from the enzymatic active site. In certain aspects, the VH domains provided herein are allosteric inhibitors of ENPP1.

[0065] The VH domains provided herein may be fused to another peptide or polypeptide via a peptide bond or may be conjugated to another entity, e.g., a peptide or polypeptide or a chemical compound via a non-peptide bond, e.g., a disulphide bond. The VH domains may be delectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The VH domains may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. The VH domains may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like.

[0066] In certain aspects, the VH domain may be fused to an immunoglobulin heavy chain constant region, e.g., Fc region. In certain aspects, the VH domain may be fused to an immunoglobulin heavy chain constant region, e.g., constant heavy domain 1 (CHI), constant heavy domain 2 (CH2), and constant heavy domain 3 (CH3). In certain aspects, a VH domain of the present disclosure further comprises a heavy chain constant region and a light chain constant region of a human IgG antibody, e.g.,human IgG2, IgG4, or IgGl antibody. In certain embodiments, the VH domain comprises a human IgGl constant region having the amino acid sequence sequence set forth in UniProt: PO 1857-1, version 1.

[0067] In certain aspects, the VH domain may be a first VH domain that is combined with a second VH domain, where the fir st and second VH domains bind to different epitopes on ENPP1. In certain aspects, the first VH domain is fused to CH2 and CH3 and the second VH domain is fused to CH2 and CH3. In certain aspects, the first VH domain is fused to CHI, CH2 and CH3 and the second VH domain is fused to CHI, CH2 and CH3. In certain aspects, the CH2 and CH3 regions may include amino acids that facilitate knob-in-hole interactions or charged pair interactions to favour formation of heterodimers as opposed to homodimers.Preparation of VH domains and Molecules containing VH domains

[0068] Using the information provided herein, the VH domains of the present disclosure are prepared using standard techniques well known to those of skill in the art.

[0069] For example, the polypeptide sequences provided herein can be used to determine appropriate nucleic acid sequences encoding the VH domainand the nucleic acids sequences then used to express the VH domain. The nucleic acid sequence(s) can be optimized to reflect particular codon "preferences" for various expression systems according to standard methods well known to those of skill in the art.

[0070] Using the sequence information provided, the nucleic acids may be synthesized according to a number of standard methods known to those of skill in the art. Oligonucleotide synthesis can be earned out on commercially available solid phase oligonucleotide synthesis machines (Needham- VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168) or manually synthesized using, for example, the solid phase phosphoramidite triester method described by Beaucage et al. (1981) Tetrahedron Letts. 22(20): 1859-1862.

[0071] Once a nucleic acid encoding a VH domain of the present disclosure is synthesized it can be amplified and / or cloned according to standard methods. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are known to persons of skill. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds„ Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel).Methods of producing recombinant immunoglobulins are also known in the art. See, Cabilly, U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Nat’lAcad. Sci. USA 86: 10029-10033.

[0072] Examples of techniques sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Q[3-rcplicasc amplification and other RNA polymerase mediated techniques are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis etal. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomeli et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4. 560; and Barringer et al. (1990) Gene 89, 117. Improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.

[0073] Once the nucleic acid for a VH domain of the present disclosure is isolated and cloned, one can express the gene in a variety of recombinantly engineered cells known to those of skill in the art. Examples of such cells include bacteria, yeast, filamentous fungi, insect (especially employing baculoviral vectors), plant, and mammalian cells. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of antibodies.

[0074] In brief summary, the expression of natural or synthetic nucleic acids encoding VH domains of the present disclosure will typically be achieved by operably linking a nucleic acid encoding the antibody to a promoter (which is either constitutive or inducible), and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the VH domain. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, i.e., shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems. See Sambrook et al (1989) supra.

[0075] To obtain high levels of expression of a cloned nucleic acid it is common to construct expression plasmids which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription / translation terminator. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky (1984) J. Bacterial., 158:1018-1024, and the leftward promoter of phage lambda (PL) as described by Herskowitz and Hagen (1980) Ann. Rev. Genet., 14:399-445 and the L-arabinose (araBAD) operon (Better (1999) Gene Exp Systems pp95-107 Academic Press, Inc., San Diego, CA). The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. See Sambrook et al (1989) supra for details concerning selection markers, e.g., for use in E. coli.

[0076] Expression systems for expressing VH domains of the present disclosure are available using, for example, E. coli. Bacillus sp. (see,e-S-> Palva, et al. (1983) Gene 22:229-235; Mosbach et al. (1983) Nature, 302: 543-545), and Salmonella. E. coli systems may also be used.

[0077] The VH domains produced by prokaryotic cells may require exposure to chaotropic agents for proper folding. During purification from, e.g., E. coli, the expressed protein is optionally denatured and then renatured. This can be accomplished, e.g., by solubilizing the bacterially produced antibodies in a chaotropic agent such as guanidine HC1. The antibody is then renatured, either by slow dialysis or by gel filtration (see, e.g., U.S. Patent No. 4,511,503). Alternatively, nucleic acid encoding the VH domains of the present disclosure may be operably linked to a secretion signal sequence such as pelB so that the VH domains are secreted into the medium in correctly-folded form (Better et al (1988) Science 240: 1041-1043).

[0078] Methods of transfecting and expressing genes in mammalian cells are known in the art (see e.g., Birch and Racher Adv. Drug Deliv. Rev. 2006, 58: 671-685). Transducing cells with nucleic acids can involve, for example, incubating viral vectors containing the nucleic acids with cells within the host range of the vector (see, e.g., Goeddel (1990) Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego, CA or Krieger (1990) Gene Transfer and Expression — A Laboratory Manual, Stockton Press, New York, N.Y. and the references cited therein).

[0079] The culture of cells used in the present disclosure, including cell lines and cultured cells from tissue or blood samples is well known in the art (see, e.g., Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, N. Y. and the references cited therein).

[0080] The present disclosure also provides a cell, e.g., a genetically modified cell, that comprises a subject nucleic acid. A subject genetically modified cell can be a prokaryotic cell (e.g., a bacterial cell); or a eukaryotic cell (e.g., an insect cell; a mammalian cell, such as a mammalian cell line suitable for in vitro cell culture; a yeast cell; etc.), where the cell may produce the encoded VH domain.Modification of VH Domains

[0081] The VH domains provided herein can be modified into different formats which retain ability to bind ENPP1. Examples of these formats include bispecific, multispecific, scFv, BiTE, CAR, degrader, UniBodies (produced from IgG4 antibodies by eliminating the hinge region of the antibody,affibodies that are class of affinity proteins based on a 58-amino acid residue protein domain, derived from one of the IgG-binding domains of staphylococcal protein A.

[0082] A VH domain provided herein may be modified to generate a bispccific antibody, where the second arm of the antibody binds to an antigen of interest. The antigen of interest may be a tumor associated antigen (TAA), a B cell receptor, a T cell receptor, etc.

[0083] The term “tumor-associated antigen” or “TAA” is used herein to refer to tumor-specific antigens (TSA) that are exclusively expressed by tumor cells and to antigens that are overexpressed by tumor cells but are also found in normal cells. Tumor associated antigens (TAAs) that can be targeted by bispecific antibodies disclosed herein include, without limitation, EpCAM, ABCB1, CD47, HER2, EGFR, PD-L1, AXL, B7-H4, LIVE LY6E, LRRC15, Nectin4, TIM3, gpNMB, Fuc-GMl, cMET, ENPP3, CD19, CD20, CD27, CD30, CD33. D44, CD66e, CD70, CD73, CD79b, CD115, CD221, and CD228.

[0084] In certain aspects, a VH domain provided herein may be fused to a Fc region which may be associated with an Fc region fused to another VH domain that binds to an antigen other than ENPP1.

[0085] In certain aspects, a VH domain provided herein may be fused, e.g., via a linker, to another VH domain that binds to an antigen other than ENPP1.

[0086] In certain aspects, the other VH domain may be associated with a VL domain. In certain aspects, the VL domain may include a light chain constant region.

[0087] In certain aspects, a bispecific antibody may include a VH domain provided herein and a VH domain or VH and VL domains of an antibody selected from Magrolimab, Tratuzumab, Pertuzumab, Cetuximab, Necitumumab, atezolizumab, Avelumab, Durvalumab, YW327.6S2, HuMax-AXL-ADC, Enapotamab, AXL-107-MMAE, Ladiratuzumab SGN-LIV1A, ABBV-085 (ADC), Enfortumab (ADC) PADCEV, Cobolimab, Sabatolimab, Glembatumumab, Telisotuzumab, Amivantamab, LY2875358, Tafasitamab, Xmab5574, COLTUXIMAB, Loncastuximab, Inebilizumab, Blinatumomab, Ocrelizumab, Veltuzumab, Obinutuzumab, Ofatumumab, Ibritumomab, Rituximab, Tositumomab, Ublituximab, Gentuzumab, Lintuzumab, Labetuzumab, FPA150, XmAb2513, MDX-060 Iratumumab, 5f 11, R05429083, Bivatuzumab, BIWA4, Cusatuzumab, Vorsetuzumab, Oleclumab, Mupadolimab, CPI-006, Polatuzumab, Emactuzumab, AVE1642Enfortumab, Robatumumab, or Figitumumab.

[0088] In certain aspects, a bispecific antibody may be a bispecific T cell engager (BiTE) molecule. In certain aspects, the BiTE molecule may include a VH domain for binding to ENPP1 and an arm for recognizing CD3.

[0089] VH domains and antibodies comprising the VH domains can be labeled by any of a number of methods known to those of skill in the art. The label can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field ofimmunoassays and, in general, any label useful in such methods can be applied in the various embodiments of the present disclosure. Thus, a label is any entity detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include magnetic beads (e.g. DynabeadsTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, Alexa fluor dyes and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g., LacZ, CAT, horse radish peroxidase, luciferase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker gene products or in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads. For example, VH domains and antibodies comprising the VH domains can include a fluorescent label, a chemiluminescent label, a radiolabel, a chromogenic label, or other suitable label.

[0090] The label may be coupled directly or indirectly to the VH domains and antibodies comprising the VH domains. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, ease of conjugation of the compound, stability requirements, etc.

[0091] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule(e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands.

[0092] The VH domains and molecules comprising the VH domains can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems which may be used, see, U.S. Patent No. 4,391,904, which is incorporated herein by reference.

[0093] A modified VH domain may be assessed for retention of binding to ENPP1 by measuring its binding affinity.Measurement of antibody affinity

[0094] The KD of a modified VH domain and the kinetics of binding to antigen can determined in a BIAcore, a biosensor based on surface plasmon resonance. For this technique, antigen is coupled to aderivatized sensor chip capable of detecting changes in mass. When modified VH domain is passed over the sensor chip, VH domain binds to the antigen resulting in an increase in mass that is quantifiable.

[0095] Measurement of the rate of association as a function of VH domain concentration can be used to calculate the association rate constant (kon). After the association phase, buffer is passed over the chip and the rate of dissociation of modified VH domain (koff) determined. The equilibrium constant Kd is then calculated as koff / konand thus is typically measured in the range 107to 1012M. Affinities measured in this manner usually correlate well with affinities measured in solution by fluorescence quench titration.Compositions and Formulations

[0096] The present disclosure provides compositions, including pharmaceutical compositions, comprising a VH domain, e.g., a modified VH domain, such as antibodies comprising VH domains provided herein. The pharmaceutical compositions of the present disclosure are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ. The compositions for administration can comprise a solution of one or more VH domains in a pharmaceutically acceptable carrier, which may be an aqueous carrier. A variety of aqueous earners can be used, e.g., buffered saline and the like.

[0097] In general, a pharmaceutical formulation comprises an effective amount of the subject VH domain in combination with a pharmaceutically acceptable excipient. An “effective amount” means a dosage sufficient to produce a desired result, e.g., reduction in a cancer of a subject, reduction in the growth rate of a cancer in a subject, amelioration of a symptom of cancer, and the like. Generally, the desired result is at least a reduction in a symptom of a cancer, reduction in the growth of a cancer, reduction in the size of a cancer, etc., as compared to a control. The effective amount of a VH domain or a VH domain containing molecule present in the formulation is determined by considering the desired dose volumes and mode(s) of administration, for example.

[0098] The following formulations, excipients and methods are merely exemplary and are in no way limiting.

[0099] The pharmaceutical composition may, for example, be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. Thus, formulations of the subject a VH domain or a VH domain containing molecule may be in a solid form, which present advantages, such as improved stability and increased shelf-life as well as simpler storage and transportation. Such solid formulations may be prepared by a variety of drying technologies, including lyophilization and spray dry manufacturing processes. Solid formulations may be reconstituted prior to use. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent tothe volume removed during lyophilization), however, solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0100] Thus, pharmaceutical compositions of the subject VH domain might be in a variety of formulations, including lyophilized powders and liquid, typically aqueous, formulations, which may be concentrated solutions that require dilution prior to administration or ready-to-use solutions suitable for subcutaneous administration. Generally, the compositions contain the a VH domain or a VH domain containing molecule, an excipient to adjust tonicity or osmolality for solutions or a lyoprotectant for lyophilized powders, a buffer, and a surfactant. The ionic tonicity-adjusting excipient may, for example, be sodium chloride, and non-ionic osmolality-adjusting excipients include, for example, trehalose, sucrose, mannitol, maltose, and sorbitol. Typical lyoprotectants include trehalose and sucrose.

[0101] A tonicity agent may be included in the VH domain formulation to modulate the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 nM.

[0102] A surfactant may also be added to the VH domain formulation to reduce aggregation of the formulated a VH domain or a VH domain containing molecule and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-poly oxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan- fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactant may range from about 0.001% to about 1% w / v.

[0103] A lyoprotectant may also be added in order to protect the labile active ingredient (e.g. a protein) against destabilizing conditions during lyophilization, if any. For example, known lyoprotectants include sugars (including trehalose, glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Typical lyoprotectants are trehalose and sucrose. Lyoprotectants can be included in an amount of about 10 mM to 500 nM.

[0104] Pharmaceutical formulations of the subject VH domain include aqueous formulations comprising the a VH domain or a VH domain containing molecule in a pH-buffered solution. The buffer used in aqueous pharmaceutical formulations herein has a pH in the range from about 4.8 to about 8.0. In certain embodiments the pH is in the range from 5.5 to 7.0, from pH 5.5 to 6.5, in the range from pH 5.7 to 6.8, in the range from pH 5.8 to 6.5, in the range from pH 5.9 to 6.5, in the range from pH 6.0 to 6.5, or in the range from pH 6.2 to 6.5.

[0105] As another example, a subject parenteral formulation is a liquid or lyophilized formulation which may comprise, for example, about 1 mg / mL to about 200 mg / mL of a subject VH domain; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose or sucrose; and has a pH of 5.5.

[0106] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for a subject a VH domain or a VH domain containing molecule may depend on the particular a VH domain or a VH domain containing molecule employed and the effect to be achieved, and the pharmacodynamics associated with each a VH domain or a VH domain containing molecule in the host. In pharmaceutical dosage forms, a subject VH domain can be administered in conjunction with a pharmaceutically acceptable excipient, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[0107] In some embodiments, a subject VH domain is formulated in a controlled release formulation. Sustained-release preparations may be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the a VH domain or a VH domain containing molecule in which the matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers and poly-D-(-)-3-hydroxybutyric acid. Possible loss of biological activity and possible changes in immunogenicity of VH domain comprised in sustained-release preparations may be prevented by using appropriate additives, by controlling moisture content and by developing specific polymer matrix compositions.Methods

[0108] As summarized above, methods of the present disclosure include methods of contacting a cell with a VH domain (e.g., a modified VH domain such as a molecule that includes a VH domain of thepresent disclosure), methods of treating a subject according to a method that involves administering to the subject a VH domain of the present disclosure, methods of making elements described in the instant application, including e.g., multi-specific antibodies, AbTAC, compositions and formulations, nucleic acids, expression vectors, cells, and the like.

[0109] As summarized above, methods of the present disclosure include contacting a cancer cell with the VH domain of the present disclosure, e.g., to facilitate and / or enhance killing of the cancer cell. In some instances, killing of the cancer cell is mediated by an immune response or immune cell acting upon the cancer cell as a result of opsonization of the cancer cell by bispecific targeting when the two targets are co-expressed on the cancer cell. In some instances, killing of the cancer cell is mediated by an immune response or immune cell acting upon the cancer cell. Methods that involve contacting a cancer cell with a multi-specific antibody of the present disclosure may or may not include contacting the cancer cell with an additional therapy or active agent, including e.g., a chemotherapeutic, an immunotherapy, radiation therapy, or the like.

[0110] Contacting a cancer cell with a multi-specific antibody of the present disclosure will generally enhance the killing of the cancer cell, e.g., as compared to the level of killing of the cancer cell in the absence of the multi-specific antibody.

[0111] The present disclosure also provides a method for decreasing binding of ENPP1 to insulin receptor. For example, the VH domains of the present disclosure can be used to decrease the binding of ENPP1 to insulin receptor. The present disclosure also provides a method for treating insulin resistance and / or type 2 diabetes in a subject. In certain aspects, a subject suffering from insulin resistance may be pre-diabetic or may have type 1 diabetes.

[0112] In certain aspects, the subject is a human, non-human primate, cattle, equine, canine, feline, or rodent. In certain aspects, the subject is human.Treatment Methods

[0113] The present disclosure provides methods of treating a cancer, the methods generally involving administering to an individual in need thereof (e.g., an individual having a cancer) an effective amount of a subject VH domain, alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents.

[0114] The present disclosure also provides methods of tr eating insulin resistance and / or type 2 diabetes in a subject, the methods generally involving administering to an individual in need thereof (e.g., an individual having a prediabetes, type 1 diabetes, or type 2 diabetes) an effective amount of a subject VH domain, alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents.

[0115] Administration of a VH domain of the present disclosure may be performed by any convenient and appropriate route of delivery.

[0116] Aspects of the present disclosure include the VH domain according to the preceding section of the specification for use in a method of treating cancer in a subject, the method comprising administering the VH domain to the subject. The method comprises administering the VH domain in combination with at least one additional active agent, wherein the at least one additional active agent comprises a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapy agent, or a combination thereof.

[0117] Administration includes but is not limited to e.g., delivery of the VH domain by injection, delivery of a VH domain or a VH domain containing molecule by infusion, delivery of a nucleic acid or expression vector encoding the VH domain, delivery of the VH domain by administering to the subject a cell that expresses and secretes the VH domain, and the like.

[0118] In some embodiments, an effective amount of a subject VH domain is an amount that, when administered alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents, in one or more doses, is effective to reduce an adverse symptom of cancer or insulin resistance or type 2 diabetes by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the severity of the adverse symptom in the absence of treatment with the VH domain.

[0119] In some embodiments, an effective amount of a subject VH domain is an amount that, when administered alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents, in one or more doses, is effective to improve the cancer (i.e., slow the growth of the cancer, stop the growth of the cancer, reverse the growth of the cancer, kill cancer cells (including tumor cells, or the like) in the individual being treated. For example, an effective amount of a subject a VH domain or a VH domain containing molecule can reduce a cancer growth rate or reduce a cancer size in an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or more, compared to in the absence of treatment with the VH domain.

[0120] In some instances, a subject may be treated systemically, including with the subject VH domain, with or without one or more additional reagents. By “systemic treatment”, as used herein, is meant a treatment that is not directed solely to target a specific tumor (such as e.g., a primary tumor or a defined secondary tumor) or a specific cancer containing tissue (such as e.g., the liver in the case of liver cancer, the blood in the case of a blood cancer, etc.). Systemic treatments will generally be directed to thesubject’s body as a whole and may include but are not limited to e.g., systemic radiation therapy, systemic chemotherapy, systemic immunotherapy, combinations thereof and the like.

[0121] In some instances, a subject may be treated locally, including with the subject multispecific a VH domain or a VH domain containing molecule, with or without one or more additional reagents. By “local treatment”, as used herein, is meant a treatment that is specifically directed to the location of a tumor (such as e.g., a primary tumor or a defined secondary tumor) or specifically directed to a cancer containing tissue (such as e.g., the liver in the case of liver cancer, the blood in the case of a blood cancer, etc.). In some instances, local treatment may also be administered in such a way as to affect the environment surrounding a tumor, such as tissue surrounding the tumor, such as tissue immediately adjacent to the tumor. Local treatment will generally not affect or not be targeted to tissues distant from the site of cancer including the site of a tumor, such as a primary tumor. Useful local treatments that may be administered in addition to or in combination with a subject multi-specific antibody, e.g., include but are not limited to surgery, local radiation therapy, local cryotherapy, local laser therapy, local topical therapy, combinations thereof, and the like.

[0122] In some embodiments, a subject treatment method involves administering a subject a VH domain or a VH domain containing molecule and one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, chemotherapeutic agents, radiation therapy reagents, immunotherapy reagents, other antibody or multi-specific antibody agents, and the like. Additional therapies that may be administered to a subject before, during or after a subject administering a multi-specific antibody of the present disclosure will vary depending on numerous factors including e.g., the type of cancer, the subject’s medical history, general state of health and / or any co-morbidities, and the like. Useful cancer therapies include but are not limited to e.g., radiation therapy, chemotherapy, immunotherapy, and the like.

[0123] Radiation therapy includes, but is not limited to, x-rays or gamma rays that are delivered from either an externally applied source such as a beam, or by implantation of small radioactive sources.

[0124] Individuals suitable for treatment using a method of the present disclosure include an individual having a cancer; an individual diagnosed as having a cancer; an individual being treated for a cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc.); an individual who has been treated for a cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.), and who has failed to respond to the treatment; an individual who has been treated for a cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.), and who initially responded to the treatment but who subsequently relapsed, i.e., the cancer recurred.

[0125] The methods of the present disclosure may be employed to target and treat a variety of cancers, including e.g., primary cancer, secondary cancers, re-growing cancers, recurrent cancers,refractory cancers and the like. For example, in some instances, the methods of the present disclosure may be employed as an initial treatment of a primary cancer identified in a subject. In some instances, the methods of the present disclosure may be employed as a non-primary (e.g., secondary or later) treatment, e.g., in a subject with a cancer that is refractory to a prior treatment, in a subject with a cancer that is regrowing following a prior treatment, in a subject with a mixed response to a prior treatment (e.g., a positive response to at least one tumor in the subject and a negative or neutral response to at least a second tumor in the subject), and the like.

[0126] The methods of the present disclosure may find use in treating various cancers including but not limited to, e.g., Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers (e.g., Kaposi Sarcoma, Lymphoma, etc.), Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid / Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer (Extrahepatic), Bladder Cancer, Bone Cancer (e.g., Ewing Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma, etc.). Brain Stem Glioma, Brain Tumors (e.g., Astrocytomas, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniopharyngioma, Ependymoma, etc.), Breast Cancer (e.g., female breast cancer, male breast cancer, childhood breast cancer, etc.), Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor (e.g., Childhood, Gastrointestinal, etc.), Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Central Nervous System (e.g., Atypical Teratoid / Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumor, Lymphoma, etc.), Cervical Cancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Duct (e.g., Bile Duct, Extrahepatic, etc.), Ductal Carcinoma In Situ (DOS), Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer,Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer (e.g.. Intraocular Melanoma, Retinoblastoma, etc.), Fibrous Histiocytoma of Bone (e.g., Malignant, Osteosarcoma, etc.), Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumor (e.g., Extracranial, Extragonadal, Ovarian, Testicular, etc.), Gestational Trophoblastic Disease, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis (e.g., Langerhans Cell, etc.), Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors (e.g., Pancreatic Neuroendocrine Tumors, etc.), Kaposi Sarcoma, Kidney Cancer (e.g., Renal Cell, Wilms Tumor, Childhood Kidney Tumors, etc.), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia (e.g., Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML), Hairy Cell, etc.), Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer (e.g., Non-Small Cell, Small Cell, etc.), Lymphoma(e.g., AIDS-Related, Burkitt, Cutaneous T-Cell, Hodgkin, Non-Hodgkin, Primary Central Nervous System (CNS), etc.), Macroglobulinemia (e.g., Waldenstrom, etc.), Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma / Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic / Myeloproliferative Neoplasms, Myelogenous Leukemia (e.g., Chronic (CML), etc.). Myeloid Leukemia (e.g., Acute (AML), etc.). Myeloproliferative Neoplasms (e.g., Chronic, etc.), Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer (e.g., Lip, etc.), Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer (e.g., Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor, etc. ), Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (e.g., Ewing, Kaposi, Osteosarcoma, Rhabdomyosarcoma, Soft Tissue, Uterine, etc.), Sezary Syndrome, Skin Cancer (e.g., Childhood, Melanoma, Merkel Cell Carcinoma, Nonmelanoma, etc.), Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer (e.g., with Occult Primary, Metastatic, etc.), Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Ureter and Renal Pelvis Cancer, Urethral Cancer, Uterine Cancer (e.g., Endometrial, etc.), Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, Wilms Tumor, and the like.Dosages

[0127] A suitable dosage can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently. A subject VH domain may be administered in amounts between 1 ng / kg body weight and 20 mg / kg body weight per dose, e.g., between 0.1 mg / kg body weight to 10 mg / kg body weight, e.g., between 0.5 mg / kg body weight to 5 mg / kg body weight; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Ifthe regimen is a continuous infusion, it can also be in the range of 1 pg to 10 mg per kilogram of body weight per minute.

[0128] In addition to body-wcight-bascd dosing schedules, a VH domain or a VH domain containing molecule disclosed herein can also be administered by fixed dosing, where the foxed dose and dosing schedule are determined and adjusted depending on the target disease.

[0129] Those of skill will readily appreciate that dose levels can vary as a function of the specific a VH domain or a VH domain containing molecule, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the ait by a variety of means.Routes of administration

[0130] In the subject methods, a subject VH domain can be administered to the host using any convenient means capable of resulting in the desired therapeutic effect or diagnostic effect. Thus, a subject a VH domain or a VH domain containing molecule is administered to an individual using any available method and route suitable for ding delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.

[0131] The main routes of administration for the a VH domain or a VH domain containing molecule herein incudes intravenous (IV), subcutaneous (SC), intramuscular (IM), and pulmonary administration. Thus, the subject a VH domain or a VH domain containing molecule may, for example, be delivered by intravenous infusion, subcutaneous injection, or by intranasal delivery, but other administration routes, such as oral delivery are also possible.

[0132] In particular, the subject VH domain may be administered by intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In one embodiment, the a VH domain or a VH domain containing molecule arc administered by intr avenous administration. For such purposes, the formulation may be injected using a syringe or via an IV line, for example. In one embodiment, the formulation is administered by subcutaneous administration.

[0133] The subject VH domain may be administered by intravenous infusion typically in about 30 to 90 minutes, every week, biweekly or every three weeks. Alternatively, or in addition, the VH domain or a VH domain containing molecule may be administered subcutaneously, as a fixed dose or body weight-adjusted dose over a shorter period of time, such as 2 to 20 minutes, or 2 to 10 minutes, or 2 to 5 minutes, weekly, once every two weeks or once every three weeks. The advantage of subcutaneous injections is that it allows the medical practitioner to perform it in a rather short intervention with the patient. Moreover, the patient can be trained to perform the subcutaneous injection by himself. Such self-administration is particularly useful during maintenance dosing because no hospital care is needed (reduced medical resource utilization). Usually, injections via the subcutaneous route are limited to approximately 2 ml. For patients requiring multiple doses, several unit dose formulations can be injected at multiple sites of the body surface. The subject VH domain or a VH domain containing molecule can be co-administered with other therapeutic agents, such as, for example, chemotherapeutic agents. By "coadministering" is meant administering two (or more) drugs during the same administration, rather than sequential administration of the two or more drugs. In the case of intravenous administration, this will generally involve combining the two (or more) drugs into the same IV bag prior to co-administration but co-administration from different, separate formulations is also included.

[0134] The VH domain and one or more additionally drugs may also be administered concurrently. A drug that is administered "concurrently" with one or more other drugs is administered during the same treatment cycle, on the same day of treatment as the one or more other drugs, and, optionally, at the same time as the one or more other drugs. For instance, for cancer therapies given every 3-weeks, the concurrently administered drugs are each administered on day-1 of a 3-week cycle.Kits for Diagnosis

[0135] Kits for the detection of ENPP1 are also provided. Kits will typically comprise one or more VH domains and antibodies comprising the VH domains. The VH domains and antibodies comprising the VH domains can optionally be labeled. In addition, the kits will typically include instructional materials disclosing means of use VH domains and antibodies comprising the VH domains, e.g., in the detection of ENPP1. The kits may also include additional components to facilitate the particular application for which the kit is designed. The kit can additionally contain means of detecting the label (e.g., enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a sheep anti-human antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents arc well known to those of skill in the art.Examples of Non-Limiting Aspects of the Disclosure

[0136] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:1. A variable heavy (VH) domain that specifically binds to Ectodomain phosphatase / phosphodiesterase- 1 (ENPP 1 ) , the VH domain comprising:(i) a heavy chain complementarity determining region 1 (HCDR 1) comprising the sequence: DIYYSYYI (SEQ ID NO:1); a heavy chain complementarity determining region 2 (HCDR 2) comprising the sequence: RISPSYGSTS (SEQ ID NO:2); and a heavy chain complementarity determining region 3 (HCDR 3) comprising the sequence: FAY / APW / FYVADDAL (SEQ ID NOG);(ii) a HCDR 1 comprising the sequence: AIYYYDI (SEQ ID NO:4); a HCDR 2 comprising the sequence: RISPYSGSTY (SEQ ID NOG); and a HCDR 3 comprising the sequence: AHWDGYGYVDDWVAI (SEQ ID NOG);(iii) a HCDR 1 comprising the sequence: DIYYSEI (SEQ ID NO:7); a HCDR 2 comprising the sequence: RISPYYGSTS (SEQ ID NO:8); and a HCDR 3 comprising the sequence: FDPYHYFSDY YGI (SEQ ID NO:9); or(iv) a HCDR 1 comprising the sequence: AIYSYEI (SEQ ID NO: 10); a HCDR 2 comprising the sequence: RISPYSSSTY (SEQ ID NO: 11); and a HCDR 3 comprising the sequence: FWGPWFSSAL (SEQ ID NO: 12).2. The VH domain of aspect 1, wherein the ENPP1 is conjugated to a Fc chain.3. The VH domain of aspect 1 or 2, wherein the ENPP1 is present on the cell surface of a mammalian cell.4. The VH domain of aspect 3, wherein the mammalian cell is a cancer cell, optionally, wherein the cancer cell is a human cancer cell.5. The VH domain of any one of aspects 1-4, comprising:(i) a HCDR 1 comprising the sequence: DIYYSYYI (SEQ ID NO:1); a HCDR 2 comprising the sequence: RISPSYGSTS (SEQ ID NO:2); and a HCDR 3 comprising the sequence: FAYPWYVADDAL (SEQ ID NO: 13) or FAAPWYVADDAL (SEQ ID NO: 14) or FAYPFYVADDAL (SEQ ID NO: 15).6. The VH domain of aspect 5, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% identity to the amino acid sequence set forth inEVQLVESGGGLVQPGGSLRLSCAASGFDIYYSYYIGWVRRAPGKGEELVARISPSYGSTS YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFAYPWYVADDALDYWGQGTLVTV SS (VH27, SEQ ID NO: 16) orEVQLVESGGGLVQPGGSLRLSCAASGFDIYYSYYIGWVRRAPGKGEELVARISPSYGSTS YADSVKGRFTISADISKNTAYLQMNSLRVEDTAVYYCARFAYPWYVADDALDYWGQGTLVTVS S (VH27.2, SEQ ID NO: 17).7. The VH domain of aspect 5 or 6, wherein the amino acid sequence of the VH domain comprises the amino acid Isoleucine at position 75 with reference to the amino acid sequence set forth in SEQ ID NO: 16.8. The VH domain of any one of aspects 5-7, wherein the amino acid sequence of the VH domain comprises the amino acid Valine at position 89 with reference to the amino acid sequence set forth in SEQ ID NO: 16.9. The VH domain of any one of aspects 1-8, wherein the HCDR3 according to (i) comprises the sequence FAYPWYVADDAL (SEQ ID NO: 13) and wherein the VH domain inhibits hydrolysis of ATP and cGAMP by ENPP1.10. The VH domain of any one of aspects 1-8, wherein the HCDR3 according to (i) comprises the sequence FAAPWYVADDAL or (SEQ ID NO: 14) or FAYPFYVADDAL (SEQ ID NO: 15) and wherein the VH domain inhibits hydrolysis of cGAMP by ENPP1.1 1 . The VH domain of any one of aspects 1 -4, comprising:(ii) a HCDR 1 comprising the sequence: a HCDR 1 comprising the sequence: AIYYYDI (SEQ ID NO:4); a HCDR 2 comprising the sequence: RISPYSGSTY (SEQ ID NO:5); and a HCDR 3 comprising the sequence: AHWDGYGYVDDWVAI (SEQ ID NO:6).12. The VH domain of aspect 11, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% identity to the amino acid sequence set forth inEVQLVESGGGLVQPGGSLRLSCAASGFAIYYYDIGWVRRAPGKGEELVARISPYSGSTYY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARAHWDGYGYVDDWVAIDYWGQGTLV TVSS (VH24, SEQ ID NO: 18).13. The VH domain of any one of aspects 1-4, comprising:(iii) a HCDR 1 comprising the sequence: DIYYSEI (SEQ ID NO:7); a HCDR 2 comprising the sequence: RISPYYGSTS (SEQ ID NO:8); and a HCDR 3 comprising the sequence: FDPYHYFSDYYGI (SEQ ID NO:9).14. The VH domain of aspect 13, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% identity to the amino acid sequence set forth inEVQLVESGGGLVQPGGSLRLSCAASGFDIYYSE1GWVRRAPGKGEELVAR1SPYYGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFDPYHYFSDYYGIDYWGQGTLVTVSS (VH31, SEQ ID NO: 19).15. The VH domain of any one of aspects 1-4, comprising:(iv) a HCDR 1 comprising the sequence: AIYSYEI (SEQ ID NO: 10); a HCDR 2 comprising the sequence: R1SPYSSSTY (SEQ ID NO:11); and a HCDR 3 comprising the sequence: FWGPWFSSAL (SEQ ID NO: 12).16. The VH domain of aspect 15, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% identity to the amino acid sequence set forth inEVQLVESGGGLVQPGGSLRLSCAASGFAIYSYEIGWVRRAPGKGEELVARISPYSSSTYYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFWGPWFSSALDYWGQGTLVTVSS(VH3 8, SEQ ID NO:20).17. The VH domain of any one of aspect 1-16, fused to a Fc domain.18. The VH domain of aspect 17, wherein the Fc domain is a human immunoglobulin Fc domain.19. The VH domain of aspect 18, wherein the human immunoglobulin Fc domain is a human IgG Fc domain, optionally, wherein the IgG is IgG2 or IgG4.20. The VH domain of any one of aspects 1-19, associated with a variable light domain.21. The VH domain of any one of aspects 1-19, wherein the VH domain is a first VH domain that is associated with a second VH domain.22. The VH domain of aspect 21, wherein the HCDR1-3 of the second VH domain have the same sequence as that of the first VH domain.23. The VH domain of aspect 21, wherein the HCDR1-3 of the second VH domain are different in sequence from that of the first VH domain.24. The VH domain of any one of aspects 21-23, wherein the second VH domain further comprises a Fc domain, optionally wherein Fc domain is a human immunoglobulin Fc domain or a human IgG Fc domain, optionally, wherein the IgG is IgG2 or IgG4.25. The VH domain of any one of aspects 23 or 24, wherein the second VH domain binds to a epitope of ENPP1 different from the epitope bound by the first VH domain.26. The VH domain of any one of aspects 23 or 24, wherein the second VH domain binds to an immune checkpoint marker and prevents activation of the immune checkpoint marker, optionally wherein the immune checkpoint marker is PD-1, LAG-3, TIM-3, CTLA-4, or PD-L1.27. The VH domain of any one of aspects 23 or 24, wherein the second VH domain binds to a T cell.28. The VH domain of aspect 27, wherein the second VH domain binds to a T cell receptor.29. The VH domain of aspect 27 or 28, wherein the T cell is a cytotoxic T cell.30. The VH domain of any one of aspects 27-29, wherein the second VH domain binds to CD3.31. The VH domain of any one of aspects 1-24, wherein VH domain is fused to a T cell receptor to generate a chimeric antigen receptor (CAR).32. The VH domain of aspect 31, wherein the CAR is present on a T cell.33. The VH domain of any one of aspects 1-24, wherein VH domain is fused to a proteolysis targeting chimera (PROTAC) pay load.34. The VH domain of aspect 33, wherein the PROTAC payload comprises a ubiquitin ligase.35. The VH domain of any one of aspects 1-34, formatted as an antibody that is a single chain Fv (scFv), IgG, Fab, (Fab)i, or (scFv)2-36. The VH domain of any one of aspects 1-35, wherein the VH domain or an antibody comprising the VH domain is labeled.37. The VH domain of any one of aspects 1-36, wherein the VH domain or an antibody comprising the VH domain is attached to a solid support.38. An isolated nucleic acid comprising a nucleotide sequence encoding the VH of any one of aspects 1-37 or an antibody comprising the VH domain.39. A vector comprising the isolated nucleic acid of aspect 38.40. A cell comprising the nucleic acid of aspect 38 or the vector of aspect 39.41 . The cell of aspect 40, wherein the cell is a prokarytoic cell or a eurkaryotic cell, optionally the cell is a mammalian cell, e.g., a human cell, a human T cell.42. A method for binding ENPP1 in a sample, comprising: contacting the VH domain of any one of aspects 1-37 with the sample, determining binding of the VH domain to ENPP1 in the sample.43. The method of aspect 42, wherein the VH domain is labeled.44. The method of aspect 43, wherein the label is a fluorescent label, a chemiluminescent label, a radiolabel, an enzyme, or a chromogenic label.45. The method of any one of aspects 42-44, wherein the sample is tissue or blood.46. A method of admistering an anti-EPPPl antibody to a subject in need thereof, the method comprising administering the VH domain of any one of aspects 1 -37 in a therapeutically effective amount to the subject.47. A method for treating cancer in a subject, the method comprising: administering the VH domain of any one of aspects 1-37 in a therapeutically effective amount to the subject.48. A method for treating insulin resistance and / or Type 2 diabetes in a subject, the method comprising: administering the VH domain of any one of aspects 1 -37 in a therapeutically effective amount to the subject.49. Use of a VH domain according to any one of aspects 1-37 in the preparation of a medicament for the treatment of cancer in a subject.50. Use of a VH domain according to any one of aspects 1-37 in the preparation of a medicament for the treatment of insulin resistance and / or Type 2 diabetes in a subject.51. The method of aspect 47 or use of aspect 49, wherein the method comprises administering the VH domain in combination with at least one additional active agent, wherein the at least one additional active agent comprises a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapy agent, or a combination thereof.52. A pharmaceutical composition comprising the VH domain of any one of aspects 1-37 and a pharmaceutically acceptable excipient.EXAMPLES

[0137] The following examples are offered to illustrate, but not to limit any embodiments provided by the present disclosure.Abstract

[0138] Ectodomain phosphatase / phosphodiesterase-1 (ENPP1) is an extracellular enzyme that is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing cyclic guanosine monophosphate adenosine monophosphate (cGAMP), an immune-stimulant. There are small molecule ENPP1 inhibitors that are in early phase clinical trials, however biologic inhibitors of ENPP1 have not yet been generated. A significant therapeutic advantage of biologies is that they can be recombinantly engineered into multi-functional formats for greater tissue selectivity and efficacy as immunotherapies. Here we utilized phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1. We discovered a VH that allosteric ally inhibited the hydrolysis of cGAMP and ATP. We solved a 3.2 A-resolution cryo-electron microscopy (cryo-EM) structure for the VH inhibitor complexed with ENPP1 that confirmed its novel allosteric binding pose. Finally, we engineered the VH into multi-specific formats, including a bispecific fusion with an anti-PD-Ll checkpoint inhibitor that showed potent cellular activity. Our studies demonstrate that anti-ENPPl VH allosteric binders can be modularly engineered into multi-functional and potent inhibitors that arc non-competitive with cGAMP levels, and could have important benefits for immunotherapeutic development.Introduction

[0139] Ectodomain phosphatase / phosphodiesterase-1 (ENPP1) is an extracellular enzyme that promotes immune evasion in the context of cancer progression and metastasis. ENPP1 is an immune checkpoint that modulates innate and adaptive immune pathways by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP) and ATP(l-4). cGAMP is produced by cyclic GMP-AMP synthase (cGAS) in response to the detection of nucleic acid in the cytosol, which occurs after viral infection but also in tumor cells. Cells export and import cGAMP, triggering intercellular activation of type 1 interferons that coordinates a plciotropic immune response and recruitment of immune cells(l, 3-7). Simultaneously, cGAMP acts as negative regulator of metastasis by suppressing epithelial-mesenchymal transition pathways in tumor cells and promoting antitumor responses in immune cells(4-6, 8, 9). The tumor microenvironment (TME) can have high concentrationsof extracellular cGAMP, and ENPP1 is frequently up-regulated on tumor cells to evade trans cGAMP signaling, potentiating immune evasion and metastasis(l, 3-8).

[0140] Accordingly, there is significant interest in inhibiting ENPP1 to overcome therapeutic challenges for cancers that exclude lymphocytes from the TME (immune ‘cold’ tumors) (1-3, 6, 10-14). Immunotherapies such as checkpoint inhibitors that target CTLA-4 or PD-1 / PD-L1 and ionizing radiation require tumor infiltrating lymphocytes (TILs) and are less effective against immune ‘cold’ tumors(15- 20). Small molecule inhibitors of ENPP1 have set therapeutic precedent for blocking the hydrolysis of endogenous cGAMP in the TME to sensitize tumor cells to immunotherapy and radiation(l-3, 6, 10-14). Stimulator of interferon genes (STING) and toll-like receptor (TLR) activators similarly aim to inflame ‘cold’ tumors through type 1 interferon signaling but require direct intra-tumoral administration to prevent cytokine-mediated toxicities in healthy tissues(21-26). Extending the half-life of endogenous cGAMP via ENPP1 inhibition is an alternative strategy. However, there is still evidence of hyperactive immune responses when ENPP1 is targeted systemically, underscoring the need to optimize the selectivity of ENPP1 inhibitors for tumor cells(3).

[0141] ENPP1 is expressed and secreted by normal tissues and controls multiple physiological pathways creating concerns for off-tumor toxicities and obstacles for on-tumor drug deli very (27-29). ENPP1 metabolizes nucleotide substrates and is a central regulator of purinergic signaling and inorganic phosphate levels(29, 30). Genetic deletions of ENPP1 in mice and variants of ENPP1 discovered in genome-wide association studies have connected ENPP1 activity to musculoskeletal mineralization and cardiovascular- calcification discascs(27, 30-35). ENPP1 levels have also been associated with insulin resistance, plasma cell survival, cell motility, and pre-mature aging emphasizing its multi-functional biology(27, 30, 36-39). Additionally, ENPP1 on healthy tissues and in plasma sequesters systemically administered inhibitors and interferes with the delivery of drug to tumor cells, affecting therapeutic potency and minimum dose concentrations. Therefore, therapeutic ENPP1 inhibitors require optimal selectivity for tumor cells and ideally would not be competitive with high levels of ATP and cGAMP in the TME, properties that can be difficult to design using a small molecule approach.

[0142] Biologies such as antibodies are a class of therapeutics that are ideal for recombinant engineering into multivalent formats and have strong pharmacokinetic profiles. In contrast to small molecules, an antibody-based inhibitor of ENPP1 could be optimized for tumor cell selectivity by reformatting into multivalent constructs that recognize a second tumor-specific antigen. Importantly, such antibodies arc immune handles for antibody-dependent cellular cytotoxicity (ADCC) responses by effector cells, and for T cell-directed killing when formatted as a T cell-engager. Therefore, an antibodybased inhibitor of ENPP1 could synergize cGAMP activation with targeted immunotherapy to amplify efficacy.

[0143] Here, we describe the first reported antibody campaign to generate biologies targeting the enzymatic activity of ENPP1. We isolated high affinity binders to ENPP1 including a variable heavy (VH) domain that allostcrically inhibits cGAMP and ATP hydrolysis. We formatted the inhibitor into biparatopic and bispecific molecules with potent cellular activities. Finally, we solved a 3.2 A-resolution cryo-electron microscopy (cryo-EM) structure of the VH inhibitor in complex with ENPP1, elucidating the allosteric mechanism of inhibition and providing insights for structure-guided designs of nextgeneration therapeutics.ResultsExample 1: Single-domain VH-phage display library generated high affinity binders to native ENPP1 on PDX-derived osteosarcoma cells.

[0144] ENPP1 is a type 2 membrane protein with a nuclease-like domain, phosphodiesterase domain (PDE), and two somatomedin B-like domains (SMB1 and SMB 2) orientated toward the extracellular space (Figure lA)(40-43). ENPP1 forms a homo-dimer on the cell membrane, but is also secreted as an enzymatically active monomer(43). To express antigen for phage display, the ectodomain of human ENPP1 was modified by truncating the SMB1 and SMB2 domains (ASMB) and mutating the catalytic threonine to alanine (T256A). For bead-based phage display selections, the C-terminus of the ENPP1 antigen was fused to an Fc domain (ENPPl-Fc) with a C-terminal AviTag via a linker containing a tobacco etch virus (TEV) protease site (Figure IB). The biotinylated antigen was immobilized on streptavidin beads for iterative rounds of phage display.

[0145] We used our synthetic single-domain VH library based on a stabilized trastuzamab scaffold(44). VH domains have three complementarity determining regions (CDRs) and generally have weaker affinities than Fabs having six CDRs. However, the smaller size of the VH domain (15kDa) could have significant advantages over a Fab (60kDa) to bind cryptic or sterically challenging sites. Moreover, VH binders can be affinity matured or linked into multivalent formats as a single chain to significantly improve their affinities(44).

[0146] We performed parallel VH-phage selections using two elution strategies to generate binders globally to ENPP1 and specifically to inhibitory epitopes. In the first standard selection, the bound VH-phage were released using TEV protease treatment to capture any VH binding to ENPP1 ectodomain (Figure 1C). In the second substrate-specific selection, the bound VH-phage were eluted using excess ATP to isolate clones that are competitive with substrate binding or are trapping the enzyme in an inactive conformation (Figure ID). Four rounds of selection were performed by increasing positive and negative selection stringencies as indicated (Figure 1C,D).

[0147] The TEV and ATP elution strategies isolated non-overlapping sets of VH clones that were triaged to a panel of four VH candidates based on affinity and cellular staining (TEV elution: VH24 and VH38, ATP elution: VH27 and VH31; CDR sequences: Figure 6A). First, we expressed bivalent VH-Fc constructs by fusing the C-terminus of the VH directly to the Fc hinge. The binding constants of the VH-Fc were measured to be in the single-digit nanomolar range using biolayer interferometry (Figure 1E,F). On-cell binding was assayed using a patient-derived xenograft (PDX)-derived osteosarcoma (OS) cell line with high ENPP1 expression (OS384)(45, 46). For a paired isogenic comparison, OS384 cells were engineered with CRISPR knockout (KO) of ENPP1 or safe-guide (SG) control. VH-Fc constructs containing VH24, 27, 31, and 38 all recognized epitopes on ENPP1 in its native dimeric form on OS384 cells. VH31-Fc and VH38-Fc were strong on-cell binders with 7.5-fold and 2.5-fold greater staining of SG over ENPP1 KO cell lines. VH24-Fc and VH27-Fc had lower SG over ENPP1 KO staining ratios of 1.3 and 1.5 despite all the binders having similar affinities and dissociation kinetics (Figure 1G, Figure 6C). It is possible that the epitopes recognized by VH24-Fc and VH27-Fc are less accessible when native ENPP1 is dimerized on the cell membrane compared to the modified ENPPl-Fc antigen. Several factors that might contribute to steric effects specific to the native context are the presence of SMB1 and SMB2 domains, the orientation of ENPP1 as an N-terminal dimer on the membrane (vs. ENPPl-Fc as a C- terminal dimer), or obstructions from protein interactors on the membrane such as the insulin receptor(36, 37).

[0148] Next, we expressed catalytically active (WT T256) ENPPl-Fc and performed luciferasebased functional assays monitoring ATP or cGAMP hydrolysis to identify VH candidates in the panel that could inhibit ENPP1. VH27-Fc was the only molecule found to functionally inhibit recombinant ENPP1 with Michaelis-Men ten Ki values for ATP and cGAMP of 220nM (95% confidence interval (CI95) 160 to 300 nM) and 130 nM (CI95 97 to 160 nM), respectively (Figure 1H,I). Interestingly, Vappin the presence of VH27-Fc did not reach the Vmaxof the enzyme as substrate concentration was increased, indicating that VH27-Fc is non-competitive with substrate and inhibits ENPP1 by an allosteric mechanism. This was interesting because VH27 was isolated by elution with excess ATP, yet it was neither directly nor conformationally competitive with substrate therefore its discovery may have been serendipitous.

[0149] Finally, to confirm that VH27-Fc could also inhibit native soluble ENPP1 we tested the inhibitor in donor human plasma. VH27-Fc inhibited the hydrolysis of cGAMP with IC50 values of approximately 3.8 and 3.9 uM over 90 minutes and 24 hours, respectively (Figure 1J). Others have reported small molecule ENPP1 inhibitors with IC50 values that arc higher when tested using plasma or plated cells than the value measured with recombinant enzyme(2, 12). The shift in potency reflects nonspecific protein binding in more biologically complex samples. Differences in antigen avidity and epitope accessibility are additional variables that will affect the efficacy of VH27-Fc.Example 2: Yeast display of VH27 yielded a more thermostable mutant with improved KD, K;, and IC50.

[0150] Single-chain VH are well-suited for affinity maturation to evolve even stronger binders. Having validated VH27 as an ENPP1 inhibitor in recombinant enzyme and plasma assays, we aimed to improve its binding kinetics and stability using autonomous hypermutation yeast surface display (AHEAD)(47). As previously described, the AHEAD yeast strain utilizes an orthogonal error-prone polymerase that exclusively replicates and mutagenizes an orthogonal plasmid encoding chosen genes without affecting genomic DNA (OrthoRep system)(47, 48). The AHEAD platform enabled soft- randomization in cellulo, which simplified the cloning compared to traditional error-prone PCR library preparation and would allow for re-diversification should selection bottlenecks occur. We incorporated the VH27 gene into this plasmid with a HA tag to quantify expression. In iterative rounds of selection, yeast cells continuously-diversifying VH27 sequences were stained with fluorescently-labeled antigen and anti-HA antibody and analyzed by fluorescence-activated cell sorting (FACS) (Figure 2A, Figure 7A).

[0151] After five rounds of the AHEAD process, several VH27 mutants emerged and were detected with next-generation sequencing (NGS) read abundances over 1%. T75I and A89V were top ranked mutations located in the VH framework (Figure 2B). Residue T75I is in a scaffold loop adjacent the tyrosine-rich CDR Hl; this preference for isoleucine could indicate hydrophobic interactions with aromatic CDR Hl residues. The T75I substitution decreased the dissociation rate supporting the hypothesis that it might be extending effects to the CDR configuration (Figure 2C). Residue A89V is in spatial proximity to the VH domain C-terminus and might affect compaction and stability. The A89V mutation enhanced the association rate, which can be related to gains in stability that increase the effective concentration of folded protein (Figure 2C).

[0152] We then built a consensus mutant (VH27 / T75I / A89V-Fc) that had improved association and dissociation rates and a sub-nanomolar KD value (Figure 2C, Figure 7B-7D). These gains corresponded to lower Ki values for ATP and cGAMP hydrolysis, 120 nM (CI95 83 to 160 nM) and 43 nM (CI95 30 to 57 nM), respectively (Figure 2D,E). VH27 / T75I / A89V-Fc was also a three-fold stronger inhibitor of cGAMP degradation in plasma with IC50 values between 1.2 uM and 1.4 uM for three donors (Figure 2F). Additionally, we tested the inhibitor for cross-reactivity with mouse ENPP1 (mENPPl), which has -80% homology to human ENPP1 (hENPPl)(42). VH27 / T75I / A89V-Fc inhibited the hydrolysis of ATP and cGAMP by recombinant mENPPl monomer and blocked cGAMP degradation by native mENPPl in ex vivo C57BL / 6J mouse plasma (Figure 7E-7G).

[0153] Aligned with our hypothesis that these scaffold changes might impact domain stability, we determined that the double mutant had enhanced biophysical properties. The thermal melting temperature for single-domain VH27 was dramatically increased from 48°C to 63°C with the T75I / A89V mutations under non-reducing conditions (Figure 2G). When the VH disulfide was reduced with BME, the double mutant was still significantly more stable than WT VH27, with melting temperatures of 50°C and 45°C, respectively (Figure 2G). Finally, incorporation of the two mutations decreased the level of aggregation observed by size exclusion chromatography (SEC) when expressed in the VH-Fc format (Figure 2H). While aggregated WT VH27-Fc produced some non-specific cellular stickiness, VH27 / T751 / A89V-Fc showed dramatically less background staining on OS384 ENPP1 KO cells (Figure 7B, Figure 8G). We used VH27 / T75I / A89V for the rest of the experiments and refer to it as VH27.2 for brevity.Example 3: Bi-paratopic and bispecific VH constructs improved cellular inhibition by increasing membrane localization.

[0154] We tested if the VH inhibitor could be engineered into multivalent formats to enhance potency and tumor specificity. As examples, we built bi-paratopic and bispecific tetravalent Fc constructs that append a second recognition arm onto the C-terminus of the Fc via a 15 amino acid glycine-serine linker (Figure 3A,F). These Fc formats are symmetric and do not require specialized ‘knob-into-hole" expression or assembly pipelines; the multi- valent constructs could be produced in high yields in mammalian cell culture and are biophysically stable (Figure 8A,8B).

[0155] To generate a bi-paratopic inhibitor, we performed epitope binning for the VH-Fc panel and found that each could simultaneously engage ENPP1 in the presence of VH27-Fc, indicating the epitopes were non-overlapping and compatible to combine with VH27-Fc (Figure 3B,C). We created the VH27.2 bi-paratopic inhibitor using VH31 because it displayed the best cellular staining of OS384; we hypothesized that the VH31 moiety would increase the local concentration of inhibitor on ENPP1 on the membrane (Figure 1G). Biolayer interferometry verified that the tetravalent bi-paratopic molecule had a sub-nanomolar binding constant to ENPPl-Fc in vitro, and its IC50 in plasma was measured to be between 0.96 uM and 1.2 uM for three donors (Figure 3D,E). Next, we stained OS384 SG and ENPP1 KO cells and demonstrated that valency with VH31 increased the specific binding by 69%, with SG over ENPP1 KO ratios of 1.6 and 2.7 for VH27.2-Fc and bi-paratopic inhibitor, respectively (Figure 3J, Figure 8C). Finally, EC50 values for cellular binding on MDA-MB-231 cells were determined for VH27.2-Fc, VH31- Fc, and bi-paratopic inhibitor. The EC50 values for VH27.2-Fc and VH31-Fc were estimated to be >10 uM and 0.65 uM, respectively, and the bi-paratopic molecule had a decreased EC50 fit relative to VH27.2- Fc (Figure 3K).

[0156] The inhibitory potency of the bi-paratopic inhibitor and VH27.2-Fc were compared in MDA-MB-231 cellular assays using two substrates of ENPP1, cGAMP (Figure 3L,M) and p- Nitrophcnyl thymidine 5'-monophosphatc (pNP-TMP) (Figure 3N,O). While pNP-TMP is a synthetic substrate hydrolyzed by other extracellular phosphodiesterase enzymes, it provides a direct colorimetric read-out enabling higher throughput and lower noise than a cGAMP ELISA. These cellular assays demonstrated equivalent trends for cGAMP and pNP-TMP substrates. VH27.2-Fc inhibited cellular ENPP1 with IC50 values between 0.73 and 0.78 uM, which was lower than the IC50 value for soluble ENPP1 in plasma (Figure 3L-O, Figure 2F). This is consistent with there being higher avidity for the homo-dimeric membrane species compared to the secreted monomer, which should increase the ability to specifically target the membrane bound form of ENPP1. The bi-paratopic inhibitor was approximately twice as potent as VH27.2-Fc with a cellular IC50 value between 0.30 to 0.40 uM (Figure 3L,3N).

[0157] Compared to the parent VH27 / T75I / A89V-Fc inhibitor, the bi-paratopic inhibitor greatly improved the IC50 against membrane-bound ENPP1 but only moderately decreased the IC50 in plasma. These results are consistent with previous flow cytometry data that demonstrated VH27.2-Fc had a lower capacity to bind native ENPP1 in membrane environments. Valency with the highly accessible VH31 epitope therefore distinctly rescued cellular binding (Figure 1G, Figure 3J,K). Interestingly, there was a hook effect for the highest concentration of bi-paratopic molecule in the cell-based assays that was not observed in plasma (Figure 3E,L,N). There may be steric limitations when the VH31 epitope becomes saturated on both subunits of the ENPP1 homo-dimer.

[0158] Next, we generated a bispecific ENPP1 inhibitor using the clinically approved PD-L1 checkpoint inhibiting nanobody, Envafoliniab (Figure 3F)(49, 50). Several reports have shown that cotreating small molecule ENPP1 inhibitors with an immune checkpoint blockade significantly enhances tumor regression(l l, 13, 14). Bispecific biologies combine these two synergizing therapies into a single molecule. Further, overexpressed PD-L1 on various cancers should increase the local concentration of VH27.2 on tumor cell membranes to augment potency and tumor selectivity.

[0159] We constructed the bispecific VH27.2 / Envafolimab inhibitor and confirmed its binding to both ENPPl-Fc and PD-LLFc antigens (Figure 3G,H). The bispecific molecule also retained its inhibitory function in plasma assays (Figure 31). PD-L1 is highly expressed on MDA-MB-231 cells therefore we measured on-cell binding of the bispecific molecule relative to VH27.2-Fc and Fc- Envafolimab (Envafolimab fused to the C-terminus of the Fc via a 15 amino acid glycine-serine linker). While VH27.2-Fc was a weak cellular- binder, Fc-Envafolimab was a robust cellular binder with an EC50 value of ~1 nM. Compared to VH27.2-Fc, the bispecific molecule had a dramatically decreased EC50 value of ~4 nM (Figure 3K). The bispecific molecule and Fc-Envafolimab showed comparable cellularstaining indicating that we could effectively leverage PD-L1 recognition to boost inhibitor concentrations on the cell membrane.

[0160] The orders of magnitude increase in cellular localization of the bispccific inhibitor relative to VH27.2-Fc corresponded in seven- to ten-fold improvements in the cellular IC50 values for pNP-TMP and cGAMP substrates, measured to be 0.11 and 0.07 uM, respectively (Figure 3L-O). As observed for the bi-paratopic inhibitor, there was distinct improvement in the inhibition of membrane ENPP1 but not the secreted form in plasma samples. This further validated that engineering multi-specific recognition of a second tumor epitope or antigen can improve cellular localization to optimize potency. As a result, co-targeting PD-L1 yielded our most effective molecule by driving significantly higher inhibitor concentrations on the tumor cell membrane.Example 4: Reformatted VH domains function as immunotherapy scaffolds and next-generation protein degraders.

[0161] ADCC is a fundamental mechanism of clinically approved therapeutic antibodies(51-53). In addition to inhibiting ENPP1, constructs containing Fc domains engage Fc receptors such as CD 16 on NK cells and macrophages to activate ADCC(54, 55). As expected, the bivalent and tetravalent Fc inhibitors were high affinity binders of CD 16, whereas the single-domain VH27.2 did not bind to Fc receptor (Figure 4A).

[0162] VH domains can be engineered into bispecific T cell -engagers (BiTEs) that incorporate a CD3 recognition arm. We generated a BiTE by linking (15 amino acid glycine-serine linker) the singledomain VH27.2 with OKT3, a well-established anti-CD3 scFv (Figure 4B)(56, 57). In activated T cells, nuclear factor of activated T cells (NFAT) is a family of transcription factors that regulate the expression of cytokines and growth factors for immune responses. Jurkat T cells that express GFP under the control of a NFAT response element (NFAT-GFP reporter) were robustly activated after a 20 hr incubation with 10 nM BiTE and ENPPl-Fc immobilized on streptavidin magnetic beads. There was no NFAT response when Jurkat cells were cultured without the BiTE or without ENPPl-coupled beads, demonstrating the high specificity of T cell activation by the VH27.2 BiTE (Figure 4C, Figure 8D).

[0163] The therapeutic space for biologies and immunotherapies continues to expand and the VH from our panel can be directly ported into novel formats. For example, protein degraders are a new class of bispecific molecules with degradation-inducing arms(58-60). AbTACs incorporate an IgG arm that recruits the membrane-bound E3 ligase RNF43 to ubiquitinate lysine residues on the cytosolic domain of a target protein and designate it for lysosomal degradation(58). KineTACs are fusions with a cytokine or chemokine that bind internalizing receptors and mediate lysosomal degradation of the target protein(59). While only one VH in our panel recognized a functional epitope on ENPP1, it is possible thatany VH can be constructed into an AbTAC or KineTAC to hijack degradation pathways and inactivate ENPP1.

[0164] There arc three lysine residues in the cytosolic domain of ENPP1 suggesting it could be a viable target for RNF43 ubiquitination. We constructed ‘knob-into-hole’ bispecific VH-IgG AbTACs for VH24, VH27.2, VH31, and VH38 (Figure 4D). Whereas bispecific molecules with two Fab arms require complicated expression and assembly protocols to prevent light chain mismatching, the VH lacks a light chain permitting facile co-expression of ‘knob’ and ‘hole’ pieces in a single mammalian transfection. These were purified using a His tag introduced on the ‘knob’ arm to exclude contaminating ‘hole -hole’ homo-dimers.

[0165] Degradation by formation of a tertiary complex of E3 ligase, bispecific degrader, and target protein is epitope dependent(59, 61 ). We treated MDA-MB-231 cells, which express RNF43 and have been used for RNF43-mediated degradation of target proteins previously, with the AbTACs and discovered that the VH27.2 AbTAC was the most potent ENPP1 degrader(58, 61). 44% of ENPP1 was degraded after 24 hr incubation with 500 nM VH27.2 AbTAC. Treatment with 500 nM VH27.2-Fc did not decrease ENPP1 levels, validating that the AbTAC is inducing protein degr adation opposed to internalization (Figure 4E,4F, Figure 8E). It is common for AbTACs show this level of maximal degradation (Dmax) as it is a composite of synthesis and degradation rates, and additionally the Dmax can be underestimated when measuring bulk lysates that include the intracellular pool(58, 59, 61).

[0166] Finally, we pre-treated MDA-MB-231 cells with 1 uM VH27.2 AbTAC, VH27.2-Fc and isotype controls, or PBS for 24 hr. The solution containing the AbTAC or control molecules was removed, cells were washed twice, and cells were incubated with pNP-TMP substrate for 5 hr. After washout, cells primed with VH27.2-AbTAC exhibited -36% less pNP-TMP hydrolysis relative to the PBS condition, a comparable level of inhibition to that observed when cells were incubated with 3 uM bispecific molecule (Figure 4G, Figure 3N). In contrast, the cells pre-treated with VH27.2-Fc and isotype controls were not statistically different from the PBS condition (Figure 4G). These results further validate the mechanism of ENPP1 degradation and demonstrate it can effectively inhibit ENPP1 hydrolytic activity.Example 5: Cryo-EM structure of VH27.2 in complex with ENPP1 elucidated the allosteric binding pose.

[0167] To reveal the epitope and mechanism of allosteric inhibition we solved a 3.2 A-global resolution cryo-EM structure of VH27.2 in complex with T256A ENPPl-Fc (Figure 5A, Figure 9A- H)(PDB 8HGR, EMD-40047). Not surprisingly, the density for the Fc domains was not observed due to the flexible hinge and linker. The cryo-EM structure showed the expected 2:1 VH:ENPP1 homo-dimerstoichiometry, with a VH bound to each ENPP1 ectodomain configured in a head-to-head dimer (Figure 5A, Figure 9C,9D). The VH-ENPP1 structure was aligned to the crystal structure of full length ENPP1 ectodomain including SMB domains (PDB 6wfj) and the global RMSD was 0.972A (Figure 9E)(42). Overlaying these structures, it was apparent that the VH CDR H3 bound close to the SMB domains but without clashing. Notably, there would be insufficient space to accommodate a light chain indicating our original motivation to use small VH domains to target cryptic epitopes was imperative. The VH-ENPP1 structure was also aligned to the mENPPl crystal structure (PDB 4gtw) (Figure 9F)(40). While overall homology between mENPPl and hENPPl is approximately 80%, the catalytic domain shares close to 90% homology(42). As expected, key interface interactions were preserved explaining species crossreactivity (Figure 9F).

[0168] All three VH CDRs engaged ENPP1 with impressive shape-complementarity, binding a region close to the active site, but not occluding the site or interacting with the residues in the nucleotide binding pocket (Figure 5B-D)(40, 41). Density resembling a nucleotide monophosphate, which we modeled as AMP, was observed in the active site (likely left from purification since no ligand was supplied exogenously), demonstrating that VH27.2 and a nucleotide such as AMP can bind simultaneously. Substrates like ATP and cGAMP bind in the same nucleotide pocket as AMP, the product of catalysis. This suggests that these substrates would also be able to bind to the ENPP1-VH complex, consistent with the observed non-competitive inhibition. The VH was skewed toward the guanosine- adjacent site, and overlaying the VH-ENPP1 structure with the crystal structure of mENPPl complexed with 3 ’-5 ’-linked pApG (a cGAMP hydrolysis linear intermediate) (PDB 6aek) exhibited that the VH would clash with the guanosine base (Figure 9G)(41). However, a previous report found that the guanosine base has a degree of mobility within the pocket, such that mutations to these guanosine- adjacent resides did not completely disrupt cGAMP binding and hydrolysis(3). Based on this positional flexibility, the VH overlap with these residues would not necessarily prevent cGAMP binding, as is supported by our Michaelis-Menten characterizations.

[0169] We performed epitope analysis by alanine scanning residues that formed specific interactions between VH and ENPP1 (Figure 5E-G). These structure-function characterizations identified two hot spots on VH27.2 that contribute to the binding and allosteric inhibition of ENPP1: (1) CDR Hl Y30 / Y31 and (2) CDR H3 Y102 / W104 / Y105.

[0170] The VH CDR Hl loop is internally structured through pi-stacking of Y31 and Y30, which arc both within range of cation-pi interactions with ENPP1 K528. Furthermore, VH Y31 pi-stacks with ENPP1 H380 (Figure 5B). VH27.2-Fc binding to ENPP1 was abolished when Y31 or Y30 were mutated to alanine (Figure 5F). WT VH27.2-Fc did not bind to ENPP1 H38OA however retained affinity for ENPP1 K528A, indicating H38O is the essential contact and its aromatic interaction with VH Y31 isenergetically indispensable (Figure 5H). Together, ENPP1 H38O, VH Y31, VH Y30, and ENPP1 K528 connect an extensive aromatic-cation network that greatly contributes to VH affinity.

[0171] The pi-stacking between VH Y31 and ENPP1 H380, a residue in the catalytic core of ENPP1 that coordinates Zn2+, may contribute to the allosteric inhibition and three-fold selectivity for cGAMP inhibition over ATP (Figure 5B). Because the hydrolytic activity of ENPP1 is highly dependent on Zn2+coordination of substrates, we hypothesize that VH Y31 pi-stacking with H38O could exert an inhibitory effect. Although lack of binding between VH27.2-Fc Y31A and ENPP1 prevented enzymatic inhibition assays, we note that mENPPl H362 corresponding to hENPPl H380 was previously discovered to be essential for cGAMP but not ATP degradation, and mutant mENPPl H362A was unable to coordinate Zn2+for productive cGAMP hydrolysis(3). There was Zn2+density in the VH-ENPP1 complex with AMP in the nucleotide pocket, therefore the pi-stacking network involving H38O may lower the pKa of the histidine residue without completely diminishing its affinity for Zn2+like was observed for the more extreme histidine-to-alanine mutation(62). Another possibility is that VH engagement at this site may perturb reaction geometry or conformational trajectory during catalysis.

[0172] Structure-guided mutagenesis uncovered a second hot spot, CDR H3, that is critical to binding affinity as well as inhibition of ATP hydrolysis specifically. VH CDR H3 residues Y102, W104, and Y 105 pi-stack to form a hydrophobic pocket that makes several contacts with ENPP1 residues: weak polar interaction between VH Y102 and ENPP1 S377 sidechain; hydrogen bond between VH W104 and ENPP1 G342 backbone; pi-stacking between VH W104 and ENPP1 F346; and cation-pi interaction between VH Y105 and ENPP1 R395, requiring the R395 sidechain to break its salt bridge with ENPP1 E347 and rotate compared to its register in non-VH-bound ENPP1 (PDB 6wjf) to prevent clashing with Y105 (Figure 5D,E). The major energetic contributions to binding of these three aromatic residues were validated by alanine-scanning. VH27.2-Fc W 104A did not bind to ENPP1, and VH27.2-Fc Y 102A and Y105A had weakened affinity towards ENPP1 by two orders of magnitude (Figure 5F, Figure 9H).

[0173] Remarkably, 500 nM VH27.2-Fc Y102A failed to inhibit ATP hydrolysis by ENPP1, but still inhibited cGAMP and pNP-TMP hydrolysis (Figure 5G). Comparing VH27.2-Fc Y102A to WT, the Michaelis-Menten Ki for cGAMP increased approximately five -fold to 220 nM (CI95 170 to 290 nM). However, the Ki for ATP was greater than 4.5 uM (CI95 2.8 to 11 uM), indicating the Y102A mutation conferred extraordinary substrate selectivity (Figure 91, J). This ATP-specific effect for the VH27.2-Fc Y102A mutant could implicate the weak polar- interaction with ENPP1 S377, or alternatively that the tyrosine residue supports the structural integrity of the hydrophobic pocket with VH W104 and Y 105. To decouple these effects, we generated a phenylalanine mutant for Y102 and found it rescued the affinity and inhibition of ATP hydrolysis (Figure 51, J). These results indicated that the polar contact is dispensable but the hydrophobic interactions are critical for shaping the CDR conformation. Similarly,VH27.2-Fc Y105F and W104F mutants restored affinity compared to the respective alanine mutants, confirming the importance of this hydrophobic filling at the ENPP1 interface (Figure 51).

[0174] Intriguingly, the VH27.2-Fc W 104F mutation recapitulated the substrate bias of the Y102A mutation, where VH27.2-Fc W104F was much less potent towaids inhibition of ATP hydrolysis compared to cGAMP or pNP-TMP hydrolysis (Figure 5J). The Ki value for cGAMP was 270 nM (CI95 240 to 300 nM) but the Ki value for ATP was 2.3 uM (CI95 1-2 to 10 uM) (Figure 9L,M). While WT VH27.2-Fc was 2.7-fold selective for inhibiting cGAMP hydrolysis over ATP hydrolysis, VH-Fc Y 102A and W104F were -20.6- and -8.5-fold selective, respectively (Figure 5K). Even though WT, Y102A, and W104F VH-Fc had dramatically different binding affinities (KD= 0.7, 111, and 14.2 nM, respectively) there was no correlation (R2=0.17) between VH-Fc affinity and the Ki value for cGAMP. In contrast, there was strong correlation (R2=0.85) when comparing VH-Fc affinity and the Ki value for ATP, indicating destabilization of CDR H3 disproportionately de-tuned the potency for ATP (Figure 5L, Figure 9H-M). Mechanistically, the W104F mutation disrupts the hydrogen bond to ENPP1 G342 backbone carbonyl. Phenylalanine is also unable to fill the hydrophobic hole from the tryptophan and might weaken the aromatic interaction with ENPP1 F346. WT VH27.2-Fc had reduced affinity for ENPP1 F346A mutant, validating that the pi-stacking between ENPP1 F346 and VH W104 is an important energetic component (Figure 5H).

[0175] Comprehensively, these mutagenesis results suggested that VH W 104 is imperative for the ATP inhibitory mechanism, and the hydrophobic filling of Y 102 and Y105 support the correct conformation of W104. Further, VH-Fc Y102A and W104F mutations minimally affected the Kifor cGAMP, implying that CDR Hl engagement with ENPP1 H380 has a dominant role in the inhibitory mechanism for cGAMP. Therefore, structure-activity analysis revealed that the VH CDRs engage distinct sites on ENPP1 that separately modulate the hydrolysis of cGAMP or ATP. These discoveries may inspire designs of new small molecule and biologic inhibitors that interrogate the same allosteric epitopes as our VH domain.Discussion

[0176] Using display technologies and protein engineering we generated biologies and immunotherapies targeting ENPP1, including a first-in-class allosteric VH inhibitor with cellular IC50 as low as 70-110 nM in a bispecific format. The cryo-EM structure verified that the VH epitope is noncompetitive with substrate and functional analyses revealed novel mechanisms of inhibition at CDRs Hl and H3. The structure and cellular binding data confirmed that the inhibitory epitope is sterically challenging, substantiating our rationale to use a single-domain library to produce binders and inhibitors. The VH domains were recombinantly engineered into various stable formats combining nanobodies,scFv, and IgG arms, exemplifying their utility and modularity for building diverse, multi-specific molecules. As examples we constructed biologies that combine ENPP1 inhibition with 1) PD-L1 blockade, 2) ADCC function, 3) T cell activation, and 4) targeted ENPP1 protein degradation. These novel anti-ENPPl VH can readily be repurposed into other next-generation biologies and immunotherapies as they are developed.

[0177] As potent small molecule inhibitors for ENPP1 begin to show exciting translational results, biologies provide a complementary mode of inhibition with different strengths(2). First, drug localization to tumor cells is important for minimizing potential off-tumor toxicities. Additionally, strategies to optimize delivery of the inhibitor to tumor cells mitigates the impact of soluble ENPP1 in plasma that could sequester drug and decrease therapeutic potency. Small molecules typically distribute systemically and it can be challenging to engineer them for greater tumor specificity. In contrast, we recombinantly expressed the VH inhibitor as a bispecific molecule dual-targeting high PD-L1 and ENPP1 expression on MDA-MB-231 cells. This bispecific approach robustly increased the concentration of inhibitor on the membrane of double -positive tumor cells and significantly improved the cellular IC50 value without changing the IC50 value in plasma. Co-targeting other up-regulated biomarkers on the tumor membrane, such as HER2 or EGFR, can similarly bias tumor cell localization to address various cancer types.

[0178] Second, studies have indicated that combining ENPP1 inhibitors with checkpoint inhibitors increases then- therapeutic index against immune ‘cold’ tumorsfl l, 13, 14). It is difficult to build drug-likc large bifunctional small molecules. In contrast, straightforward protein engineering approaches allowed construction of a bispecific with VH27.2 and Envafolimab to synergize dual checkpoint therapies into a single molecule. ENPP1 is also an anti-metastatic target and could be utilized in bifunctional formats with other therapeutics(4-6, 8, 9). For example, OS is a cancer that becomes fatal when the disease progresses to metastasis(63). We showed that our VH inhibitor binds to PDX-derived OS cells with up-regulated ENPP1. As HER2 is also overexpressed in OS, a bispecific molecule combining ENPP1 inhibitor with second clinical antibody such as trastuzamab could be another opportunity to dual-target mesenchymal transition pathways and malignant signaling to treat this aggressive cancer.

[0179] Finally, ENPP1 biologic inhibitors containing Fc domains or formatted into BiTEs are also antigen-directed immunotherapies. NK cells and macrophages recruited to the TME by cGAMP trans signaling will target tumor cells bound with the Fc effector active forms for ADCC. Similarly, wc demonstrated that the ENPP1 inhibitor in BiTE format robustly activated T cells. Additionally, VH domains are exceptionally useful because of their stable expression as chimeric antigen receptor-T cells (CAR-Ts). We hypothesize that layering immune cell trafficking with antigen-directed killing couldpotentiate the therapeutic efficacy of biologic inhibitors of ENPP1 compared to small molecules that have only a single mechanism of action. As the VH inhibitor is cross-reactive with mouse ENPP1, these immunotherapeutic synergies will be evaluated using immunocompetent mice in future studies.

[0180] There are considerations for ENPP1 -directed immunotherapies because ENPP1 is secreted and found on several healthy tissues. Soluble ENPP1 may have a decoy effect preventing immune cells from engaging tumor-bound ENPP1. However, the VH inhibitor was more potent in cellbased assays than in plasma assays suggesting avidity is a factor that biases binding to membrane ENPP1 over the monomeric soluble species. As ENPP1 is also expressed on some normal tissues, the potential off-tumor toxicities of anti-ENPPl immunotherapies will require investigation. Based on our data for the anti-ENPPl / anti-PD-Ll bispecific molecule, bispecific CAR or BiTE modalities co-targeting a second biomarker could enhance localization to tumor cells over healthy cells expressing ENPP1.

[0181] Inhibition via targeted degradation is another therapeutic approach enabled by the panel of anti-ENPPl VH. The VH27.2 AbTAC effectively mediated the ubiquitination of ENPP1 by RNF43. The VH27.2 AbTAC still retains function as an inhibitor, so we believe degradation will be additive. Previously, AbTACs recognizing multiple known sites on EGFR suggested that epitopes closer to the membrane induced greater degradation, probably due to the relatively short ectodomain of RNF43(61). Consistently, the cryo-EM structure verified that VH27.2 binds a membrane -proximal epitope on the phosphodiesterase domain. Optimizing AbTAC flexibility, valency, and tertiary complex orientation through basic reformatting can enhance degradation levels, as can co-opting more abundant E3 ligases or cytokinc / chcmokinc receptors (KincTACs)(59, 61). Additionally, KincTACs could be promising for degrading soluble ENPP1 in the TME as the technology does not require a cytosolic domain on the target protein(59). We have focused on inhibiting ENPP1 biochemical activity, however ENPP1 is known to negatively regulate insulin signaling through direct binding to the insulin receptor(36, 37, 40). It is possible that degrading ENPP1 to eliminate this interaction could have therapeutic relevance to insulin resistance and Type 2 diabetes.

[0182] We solved the cryo-EM structure of the VH-ENPP1 complex and used mutagenesis to validate interactions and investigate the inhibitory mechanism. There was a significant pi-stacking network engaging CDR Hl Y31 and Y30 and ENPP1 H38O that we predict is affecting catalysis. Based on the substantial role of CDR H3 in blocking ATP but not cGAMP hydrolysis, we suspect that the CDR Hl interaction with H38O is significant to the mechanism of inhibition for cGAMP degradation, as would be consistent with previous rcports(3). Excitingly, the structure-function analysis demonstrated that minor perturbations to the VH CDR H3 residues could modulate ENPP1 substrate preferences thr ough a novel mechanism. Y102A and W104F mutants were poor inhibitors of ATP hydrolysis but only slightly affected the K; of cGAMP relative to WT VH27.2-Fc. With further affinity maturation and optimization,these cGAMP-selective VH mutants will be valuable reagents for studying cGAMP biology in cancer and other diseases. The discovery of separate allosteric pockets that decouple ATP and cGAMP hydrolytic mechanisms can aid future designs of small molecule and biologic inhibitors with greater potencies and specialized activities.MethodsCell culture

[0183] PDX-dcrivcd OS384 cells were engineered with plasmid containing safe-guide RNA (SG) or ENPP1 guide RNA (KO). Cells were cultured in DMEM containing 10% FBS, lx glutamine, and 1% pen / strep at 37°C and 5% CO2. Jurkat cells expressing NFAT-GFP reporter were cultured in RPMI containing 10% FBS, 1% pen / strep, and 2 mg / mL Geneticin at 37°C and 5% CO2. MDA-MB-231 cells were cultured in DMEM containing 10% FBS and 1% pen / strep at 37C and 5% CO?. The OS384 PDX- derived and MDA-MB-231 cell lines were validated by STR testing and NFAT-GFP Jurkat cell line was purchased from Thermo.Mammalian expression of proteins (ENPPl-Fc, VH-Fc, BiTE, AbTAC)

[0184] HEK-293 EXPI (Expi293) cells were cultured in Expi293 media (Gibco) at 37°C and 8% humidity with orbital shaking at 250 rpm. ENPPl-Fc or VH-Fc were cloned into pFUSE vector (Invivogcn) with upstream IL-2 secretion signal. Cells were transfected at 3M / mL density using Expifectamine Transfection kit (Gibco) according to manufacturer protocols. To biotinylate the AviT g of ENPPl-Fc Expi293 cells expressing BirA were used and 0.05 mM biotin was included in the media at the time of transfection. 72-120h after addition of enhancers the supernatant was harvested (30min, 4000g) and filtered using 0.45 micron filters. Proteins were purified with Protein A affinity chromatography or Nickel resin and buffer exchanged into PBS using appropriate MW spin filters (Amicon). Protein purity was assessed using SDS-pagc and proteins were stored at -80°C.Bacterial expression of proteins (single-domain VH)

[0185] VH were expressed in E. Coli C43(DE3) Pro-i- pTUM-i- using optimized autoinduction media. Bacterial pellets were lysed with B-PER (Thermo) containing protease inhibitors (Millipore Sigma) and lysates were purified using Protein A affinity chromatography and buffer exchanged into PBS using 3 or 10 MW spin filters (Amicon). Protein purity was assessed using SDS-page and proteins were stored at -80°C.Phage Selection with VH-phage library

[0186] Phage display using a VH-phagemid library was performed according to previously established protocols(44).

[0187] For the standard selection, biotinylated antigen or biotinylated Fc were immobilized to streptavidin-coated magnetic beads (Promega). For each round, the phage library was first cleared by incubating with Fc to remove non-specific binders, and subsequently the positive selection was performed using the antigen. Phage binding antigen were washed three times and eluted by treatment with 2 ug / mL tobacco etch virus (TEV) protease. XL-1 blue E. Coli were infected with eluted phage and the allowed to propagate overnight. In total four rounds of selection were performed with increasing antigen and clear stringency, as shown in Figure 1C. All steps were carried out in TBS buffer containing 0.02% TWEEN- 20, 0.2% BSA, 200uM zinc chloride, 2mM calcium chloride, and 75mM sodium chloride. After four rounds, single XL-1 blue colonies were picked and evaluated by ELISA and sanger sequencing.

[0188] For the substrate competitive selection, the procedure above was performed but instead of TEV treatment the bound phage were eluted by incubating with lOmM ATP for 30 minutes at room temperature, as shown in Figure ID.VH maturation via autonomous hypermutation yeast surface display

[0189] VH27 was integrated into the AHEAD yeast strain (yAW301) as previously reported(47). VH27 was first subcloned into the pAW240 integration plasmid, linearized via restriction digest, and transformed into chemically competent yAW301 using Frozen-EZ Yeast Transformation II kit (Zymo). After 3 days of growth on selective SC-HLUWMC dropout media (US Biologies) single colonies were grown to saturation in SC-HLUWMC supplemented with 2% glucose. Incorporation of VH27 was validated by sanger sequencing. VH27 was autonomously diversified by passaging transformed yAW301 through 2-3 lOOOx fold dilutions in SC-HLUW with 2% glucose. For FACS experiments, VH27 expression was induced in yeast by switching to SC-HLUW with 2% galactose for 48 hrs. Yeast were labeled with biotinylated ENPPl-Fc for 1-2 hrs, with round 1 starting at 65 nM and every subsequent round at half the previous concentration. To prevent ligand depletion and allow binding reactions to reach equilibrium, yeast cell counts, reaction volumes, and incubation times were further adjusted. After washing, yeast were stained with lOOOx dilution of anti-HA-AF488 (Thermo) and streptavidin-647 (Thermo), washed again, and subjected to FACS using an Aria II instrument. During each round ~2e7 cells were stained and used to sort out 200-2,000 cells. To avoid selection of mutants with mutations in VH27’s HA-tag (fused to VH27 in order to measure display level by anti-HA-AF488) a strict floor on HA signal was set such that only the top -15% of clones on the HA signal axis were sorted for ENPPl-Fc labeling. Omitting HA-tag mutants was important since these falsely appear as improved ENPP1 bindersbecause they disable the HA-tag display level signal to which the ENPPl-Fc signal is normalized. Cells were sorted into 3 mL of SC-HLUW with 2% glucose, grown to saturation (~3 days), and then subjected to the next round.Biolayer Interferometry ( BLI)

[0190] BLI data was collected using an OctetRED384 (ForteBio) instrument. To analyze the association and dissociation kinetics of purified binders, streptavidin biosensor tips (Sartorius) were loaded with antigen, blocked with 5 uM biotin, dipped into protein analyte in solution for 600 seconds (association), and finally dipped into buffer for 900 seconds (dissociation). For epitope binning experiments, biosensor loaded with ENPPl-Fc was blocked with 5 uM biotin, dipped into solution containing 25 nM binder 1 until saturation (600 seconds), and then immediately dipped into solution containing 25 nM binder 1 and 25 nM binder 2 (600 seconds). Experiments for ENPP1 antigen were performed using TBS buffer containing 0.02% TWEEN-20, 0.2% BSA, 200uM zinc chloride, 2mM calcium chloride, and 75mM sodium chloride. All other antigens were assayed using PBS containing 0.02% TWEEN-20 and 0.2% BSA. Data were analyzed using ForteBio Octet analysis software and kinetic parameters were determined based on a 1:1 monovalent binding model.Flow Cytometry

[0191] Cells were lifted with versene, washed PBS, and resuspended in PBS with 3% BSA. Cells were stained with binder at indicated concentrations in PBS with 3% BSA for 1 hr at 4°C. Cells were washed twice with PBS with 3% BSA. Cells were then incubated with ProtA-647 secondary (Thermo) at 1:1000 dilution in PBS with 3% BSA for 30 min at 4°C. Cells were washed three times in PBS with 3% BSA cells and resuspended in PBS for flow cytometry analysis using a Beckman Coulter Cytoflex Flow Cytometer. Data were processed with FlowJo vl 0.8.1 software, and EC50 curves were calculated using GraphPad PRISM 7.0 ‘Sigmoidal, 4PL’ non-linear fit with Hill coefficient set to greater than 1.Recombinant hENPPl and mENPPl Inhibition Assays

[0192] WT ENPPl-Fc was cloned and used for recombinant enzyme activity assays. The Michaelis-Menten reaction conditions used were 5 nM WT ENPPl-Fc, 300 nM inhibitor (or PBS), and a titration of 500, 50, 5, or 0.5uM ATP (Thermo) or cGAMP (Invivogcn). The reaction buffer was TBS containing 0.02% TWEEN-20, 0.2% BSA, 200 uM zinc chloride, 2 mM calcium chloride, and 75 rnM sodium chloride. Every 2 min for a total of 8 min the reactions were quenched by heating to 95°C for lOmin. Cell-Titer Gio (Promega) or AMP-Glo (Promega) were performed according to manufacturerprotocol to determine amount of ATP remaining or AMP produced from cGAMP hydrolysis. The initial velocity was determined by averaging the velocities calculated over 4-8 min (ATP) and 2-8min (cGAMP) at each substrate concentration. The Michaelis-Menten K; was analyzed using GraphPad PRISM 7.0 ‘Enzyme kinetics-Noncompetitive inhibition’ model.

[0193] To assess single -point inhibition values for alanine and phenylalanine mutants, 5nM WT ENPPl-Fc (or catalytically dead control), 500 nM inhibitor (or PBS), and substrate (ATP = 2.5 mM, cGAMP = 1 mM, pNP-TMP= ImM) were incubated in reaction buffer at 25°C for 30 min, quenched by heating to 95°C for lOmin, and assayed by Cell-Titer Gio (ATP), AMP-Glo (cGAMP), or absorbance at 405 nm (pNP-TMP).

[0194] Mouse ENPP1 (mENPPl) was purified as previously described(2, 40). 10 nM mENPPl and 500 nM VH27 / T75I / A98I-Fc / Fc isotype / PBS was incubated with 2.5 mM ATP or 1 mM cGAMP in reaction buffer at 25°C for 30 min. Reactions were quenched by heating at 95°C for lOmin and ENPP1 activity was measured by Cell-Titer Gio (ATP) or AMP Gio (cGAMP).Human and Mouse Recovered Plasma ICso Assay

[0195] Recovered human plasma (sodium heparin anti-coagulant) from three donors was purchased from ZenBio (Donor E031620A, Donor 2:PL070720AC, Donor 3:PL070720X). Plasma from three male C57BL / 6J mice was prepared with sodium heparin anti-coagulant as previously described(2). Final reactions were 10 uL and contained 65% plasma, ImM cGAMP, and inhibitor or control molecule at indicated concentration. A reaction without cGAMP was used as a control to subtract baseline AMP signal. Reactions were incubated for 90 min (or 24 hr when specified). Samples were diluted 10-fold in PBS and either measured immediately or quenched and stored for later analysis by centrifuging in a 10 MW spin filter. AMP produced through cGAMP hydrolysis was measured using AMP-Glo assay. Values were corrected by subtracting the background signal of plasma without supplemented cGAMP and data were normalized to the PBS condition. IC50 value for each donor was analyzed using GraphPad PRISM 7.0 ‘Dose-response-inhibition (four parameters)’.Differential Scanning Fluorimetry (DSF)

[0196] Purified VH protein was diluted to 1 uM in PBS buffer containing 4x Sypro Orange (Invitrogen). BME was supplemented to 6.25% final dilution for samples assayed in reducing conditions. Samples were heated from 30°C to 95°C with 0.3C / 30 second ramp rate and fluorescent emissions at 490 nm and 575 nm were continuously recorded. Roche LC480 LightCycler and associated software were used for data collection and Tmcalculations. Two to four technical replicates were analyzed for N=4 independent replicates.Size Exclusion Chromatography (SEC)

[0197] SEC was performed using an Agilent HPLC 1260 Infinity II LC System and either AdvanceBio column (300A, 2.7 um, Agilent) or TSKgel SuperSW mAh HTP column (4 uin, Tosoh Biosciences). Fluorescence was detected using excitation 285 nm and emission 340 nm.Extracellular cGAMP IC50 ELISA

[0198] MDA-MB-231 cells were plated at 2K cell / well 24 hr before cell treatments. Assay was performed in serum-free DMEM / F-12 media that contains zinc sulfate. Cells were washed with media and resuspended in media containing the inhibitor (or Fc isotype or PBS) and allowed to incubate for 5 min before cGAMP was added at a final concentration of 50 uM. cGAMP was also added to a well containing only media without cells as a maximum concentration control. Cells were placed at 37°C and 5% CO2 for 12 hr. To harvest, 2 uL of media was diluted 50-fold in PBS and centrifuged in a 10 MW spin filter. To assay cGAMP remaining in the media by cGAMP ELISA (Cayman Chemical), the sample was diluted another 75x using Immunoassay Buffer-C (Cayman Chemical) and the ELISA was performed according to the manufacturer protocol. The level of cGAMP remaining in each sample was normalized to the range between the PBS control (full enzymatic activity) and cGAMP in media without cells (no enzymatic activity). Data were analyzed using GraphPad PRISM 7.0 ‘Dose-response-inhibition (four parameters)’. For the bi-paratopic molecule the 3 uM concentration was excluded from the IC50 calculation.Extracellular pNP-TMP IC50 Assay

[0199] MDA-MB-2 1 cells were plated at 2K cell / well 36 hr before cell treatments. Assay was performed in serum-free and phenol red-free DMEM / F-12 media. Cells were washed with media and resuspended in media containing the inhibitor (or Fc isotype or PBS) and incubated for 10 min. pNP-TMP substrate (Sigma Aldrich) was added at a final concentration of 200 uM. Cells were placed at 37°C and 5% CO2 for 5 hr and absorbance at 405 nm was measured. Data were normalized using the PBS treatment. Data were analyzed using GraphPad PRISM 7.0 ‘Dose-response-inhibition (four parameters)’. For the bi-paratopic molecule the 3 uM concentration was excluded from the IC50 calculation.NFAT-GFP Jurkat Assay

[0200] Jurkat cells expressing NFAT-GFP reporter were used to measure T cell activation with BiTE treatments. Streptavadin coated magnetic beads (Promega) were incubated with 0, 10, or 100 nM ENPPl-Fc antigen for 30 min at room temperature followed by three washes. In each assay well, 50KJurkat NFAT-GFP cells were plated in serum-free RPMI and treated with 20 uL of beads and 10 nM BiTE. Conditions with no beads and without BiTE (PBS) were included for controls. After incubating for 20 hr at 37°C and 5% CO2 magnetic beads were removed and cellular GFP expression was analyzed by flow cytometry.AbTAC

[0201] 8K MDA-MB-231 cells were plated in 48-well culture plates and incubated at 37°C for 24 hr. Cells were treated with the indicated concentration of AbTAC or control molecule in serum-free DMEM / F12 media and incubated at 37°C and 5% CO2 for 24 hr. Cells were washed with PBS and lysates were harvested with RIPA (Sigma Aldrich) containing protease inhibitor. Samples for gel electrophoresis were prepared by boiling lysate in NuPAGE loading dye containing BME. Proteins were transferred from the gel to a PVDF membrane using iBlot reagenets and instrument (Thermo). Membranes were blocked and subsequently stained with anti-ENPPl antibody (Abeam 223268) and anti-Actin antibody (Santa Cruz Biotechnologies) at 1:1000 dilutions at 4°C overnight. Membranes were then stained with LICOR secondary antibodies (Goat anti-Rat 680 and Goat anti-Mouse 800) at 1:5000 dilutions for 1 hr at room temperature and imaged using the LICOR Odyssey instrument.

[0202] To measure the effect of ENPP1 degradation on extracellular pNP-TMP hydrolysis, 4K MDA-MB-231 cells were plated in culture media in 96-well plates and incubated at 37°C and 5% CO2 for 24 hr. Cells were treated with 1 uM VH27.2 AbTAC or control molecule prepared in serum-free and phenol red-free DMEM / F12 media and incubated at 37°C and 5% CO2 for 24 hr. The solution containing the AbTAC or control molecules was removed, cells were washed twice with fresh serum-free and phenol red-free DMEM / F12 media, and serum-free and phenol red-free DMEM / F12 media containing 200 uM pNP-TMP substrate was added. Cells were placed at 37°C and 5% CO2 for 5 hr and absorbance at 405 nm was measured. Data were normalized using the PBS treatment.Structural sample preparation, data collection, and processing

[0203] T256A ENPPl-Fc and lOx VH27 / T75I / A89V were incubated at 4°C for 1 hr. The complex was injected onto an Akta Pure system (GE Healthcare) and the peak was isolated by SEC using a Superdex 200 Increase 10 / 300 GL column (Figure 9A). Presence of both VH and antigen in the eluted peak was confirmed by SDS-PAGE gel (Figure 9B). Stoichiometry of the complex was characterized by Refeyn mass photometry (Figure 9C,D).

[0204] For cryo-EM grids preparation, 2 mg / ml T256A ENPPl-Fc was mixed with 10X VH27 / T75I / A89V and the mixture was directly applied to glow-discharged 300 mesh gold grids (Quantifoil Rl.2 / 1.3) and vitrified using a FEI Vitrobot Mark IV (Thermo Fisher Scientific). Data werecollected on a Titan Krios (SLAC / Stanford) operated at 300 keV using a Gatan K3 direct electron detector in counting mode, with 0.8677 A pixel size. A total of 4426 movies were obtained. Each stack movie was recorded for a total of 2.5 s with 0.05 s per frame. The dose rate was 1.14 electrons / A2 / subframe, resulting in an accumulated dose of 57 electrons per A2. The data were collected using SerialEM(64).

[0205] Data processing was done in cryoSPARC2v3.3.2. Dose-fractionated movies were subjected to beam-induced Patch Motion correction followed by Patch-Based CTF parameters estimation. After that, 3414 movies with CTF fit better than 4 A were selected. Particle autopicking with Blob Picker, 2D classification, and 3D refinements were performed in cryoSPARC. The autopicked particles were first subjected to 2D classification; 2D classes that look like protein complex were selected for ab initio reconstruction and heterogenous refinement. The best 3D class (highest estimated resolution and good orientation distribution) was selected and the particles are subjected to non-uniform refinement3. Resolution of the final map for the ENPP1-VH complex was 3.2A (masked), as determined by the gold- standard Fourier shell correlation cutoff of 0.143 (Figure 9E).

[0206] The structure of human ENPP1 (PDB 6WFJ) and a homology model of VH27 (based on PDB 7JWB) were rigid body fit into the local resolution estimated map using UCSF Chimera(65), then manually adjusted using Coot and real space refined in PHENIX(66, 67). Geometries of the structure models were validated using MolProbity(68). The Fourier shell correlation curves were calculated between the refined models and full maps using PHENIX (Figure 9F). Structural figures were generated using PyMOL and UCSF Chimcra(69). 3DFSC analysis was done using the online server (https: / / 3dfsc.salk.edu / ) (Figure 9G)(70). Density was present in the active site of ENPP1 that resembled a nucleotide monophosphate, even though no ligand was included in the experiment. This density was fit to AMP based on previous knowledge that ENPP1 binds adenosine more tightly than the other nucleosides (Figure 9H)(40).

[0207] The following parameters were used for data collection and processing:complex (EMDB-40047) (PDB 8GHR)Data collection and processingMagnification 130,000Voltage (kV) 300Electron exposure (e- / A2) 57Defocus range (pm) 0.7-2Pixel size (A) 0.8677Symmetry imposed C2Initial particle images (no.) 1709573Final particle images (no.) 77023Map resolution masked (A)* 3.2FSC threshold 0.143Map resolution range (A) 2.73-30RefinementInitial model used (PDB code) 6WFJModel resolution masked (A) 3.23FSC threshold 0.5Model resolution range (A) 3.2-31.89Map sharpening B factor (A2) -107.54Model compositionNon-hydrogen atoms 13864Protein residues 1716Ligands 20B factors (A2)Protein 45.44Ligand 40.18R.m.s. deviationsBond lengths (A) 0.002Bond angles (°) 0.576ValidationMolProbity score 1.53Clashscore 5.07Poor retainers (%) 0Ramachandran plotFavored (%) 96.14Allowed (%) 3.86Disallowed (%) 0*Unmasked map resolution = 3.8 AReferences1. J. A. 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[0208] While the subject VH domains, method, and composition have been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A variable heavy (VH) domain that specifically binds to Ectodomain phosphatase / phosphodies terase- 1 (ENPP 1 ) , the VH domain comprising:(i) a heavy chain complementarity determining region 1 (HCDR 1) comprising the sequence: DIYYSYYI (SEQ ID NO:1); a heavy chain complementarity determining region 2 (HCDR 2) comprising the sequence: RISPSYGSTS (SEQ ID NO:2); and a heavy chain complementarity determining region 3 (HCDR 3) comprising the sequence: FAY / APW / FYVADDAL (SEQ ID NOG);(ii) a HCDR 1 comprising the sequence: AIYYYDI (SEQ ID NO:4); a HCDR 2 comprising the sequence: RISPYSGSTY (SEQ ID NOG); and a HCDR 3 comprising the sequence: AHWDGYGYVDDWVAI (SEQ ID NO: 6);(iii) a HCDR 1 comprising the sequence: DIYYSEI (SEQ ID NO:7); a HCDR 2 comprising the sequence: RISPYYGSTS (SEQ ID NO:8); and a HCDR 3 comprising the sequence: FDPYHYFSDYYGI (SEQ ID NO:9); or(iv) a HCDR 1 comprising the sequence: AIYSYEI (SEQ ID NO: 10); a HCDR 2 comprising the sequence: RISPYSSSTY (SEQ ID NO:11); and a HCDR 3 comprising the sequence: FWGPWFSSAL (SEQ ID NO: 12).

2. The VH domain of claim 1, wherein the ENPP1 is conjugated to a Fc chain.

3. The VH domain of claim 1 or 2, wherein the ENPP1 is present on the cell surface of a mammalian cell.

4. The VH domain of claim 3, wherein the mammalian cell is a cancer cell, optionally, wherein the cancer cell is a human cancer cell.

5. The VH domain of any one of claims 1-4, comprising:(i) a HCDR 1 comprising the sequence: DIYYSYYI (SEQ ID NO:1); a HCDR 2 comprising the sequence: RISPSYGSTS (SEQ ID NO:2); and a HCDR 3 comprising the sequence: FAYPWYVADDAL (SEQ ID NO: 13) or FAAPWYVADDAL (SEQ ID NO: 14) or FAYPFYVADDAL (SEQ ID NO: 15).

6. The VH domain of claim 5, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% identity to the amino acid sequence set forth inEVQLVESGGGLVQPGGSLRLSCAASGFDIYYSYYIGWVRRAPGKGEELVARISPSYGSTS YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFAYPWYVADDALDYWGQGTLVTV SS (VH27, SEQ ID NO:16) orEVQLVESGGGLVQPGGSLRLSCAASGFDIYYSYYIGWVRRAPGKGEELVARISPSYGSTS YADSVKGRFTISADISKNTAYLQMNSLRVEDTAVYYCARFAYPWYVADDALDYWGQGTLVTVS S (VH27.2, SEQ ID NO: 17).

7. The VH domain of claim 5 or 6, wherein the amino acid sequence of the VH domain comprises the amino acid Isoleucine at position 75 with reference to the amino acid sequence set forth in SEQ ID NO: 16.

8. The VH domain of any one of claims 5-7, wherein the amino acid sequence of the VH domain comprises the amino acid Valine at position 89 with reference to the amino acid sequence set forth in SEQ ID NO: 16.

9. The VH domain of any one of claims 1-8, wherein the HCDR3 according to (i) comprises the sequence FAYPWYVADDAL (SEQ ID NO: 13) and wherein the VH domain inhibits hydrolysis of ATP and cGAMP by ENPP1.

10. The VH domain of any one of claims 1-8, wherein the HCDR3 according to (i) comprises the sequence FAAPWYVADDAL or (SEQ ID NO: 14) or FAYPFYVADDAL (SEQ ID NO: 15) and wherein the VH domain inhibits hydrolysis of cGAMP by ENPP1.

11. The VH domain of any one of claims 1-4, comprising:(ii) a HCDR 1 comprising the sequence: a HCDR 1 comprising the sequence: AIYYYDI (SEQ ID NO:4); a HCDR 2 comprising the sequence: RISPYSGSTY (SEQ ID NO:5); and a HCDR 3 comprising the sequence: AHWDGYGYVDDWVAI (SEQ ID NO:6).

12. The VH domain of claim 11, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% identity to the amino acid sequence set forth inEVQLVESGGGLVQPGGSLRLSCAASGFAIYYYDIGWVRRAPGKGEELVARISPYSGSTYY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARAHWDGYGYVDDWVAIDYWGQGTLV TVSS (VH24, SEQ ID NO: 18).

13. The VH domain of any one of claims 1-4, comprising:(iii) a HCDR 1 comprising the sequence: DIYYSEI (SEQ ID NO:7); a HCDR 2 comprising the sequence: RISPYYGSTS (SEQ ID NO:8); and a HCDR 3 comprising the sequence: FDPYHYFSDYYGI (SEQ ID NO:9).

14. The VH domain of claim 13, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% identity to the amino acid sequence set forth inEVQLVESGGGLVQPGGSLRLSCAASGFDIYYSEIGWVRRAPGKGEELVARISPYYGSTSY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFDPYHYFSDYYGIDYWGQGTLVTVSS (VH31, SEQ ID NO: 19).

15. The VH domain of any one of claims 1-4, comprising:(iv) a HCDR 1 comprising the sequence: AIYSYEI (SEQ ID NO: 10); a HCDR 2 comprising the sequence: RISPYSSSTY (SEQ ID NO: 11); and a HCDR 3 comprising the sequence: FWGPWFSSAL (SEQ ID NO: 12).

16. The VH domain of claim 15, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% identity to the amino acid sequence set forth inEVQLVESGGGLVQPGGSLRLSCAASGFAIYSYEIGWVRRAPGKGEELVARISPYSSSTYYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFWGPWFSSALDYWGQGTLVTVSS(VH3 8, SEQ ID NO:20).

17. The VH domain of any one of claim 1-16, fused to a Fc domain.

18. The VH domain of claim 17, wherein the Fc domain is a human immunoglobulin Fc domain.

19. The VH domain of claim 18, wherein the human immunoglobulin Fc domain is a human IgG Fc domain, optionally, wherein the IgG is IgG2 or IgG4.

20. The VH domain of any one of claims 1-19, associated with a variable light domain.

21. The VH domain of any one of claims 1-19, wherein the VH domain is a first VH domain that is associated with a second VH domain.

22. The VH domain of claim 21, wherein the HCDR1-3 of the second VH domain have the same sequence as that of the first VH domain.

23. The VH domain of claim 21, wherein the HCDR1-3 of the second VH domain are different in sequence from that of the first VH domain.

24. The VH domain of any one of claims 21-23, wherein the second VH domain further comprises a Fc domain, optionally wherein Fc domain is a human immunoglobulin Fc domain or a human IgG Fc domain, optionally, wherein the IgG is IgG2 or IgG4.

25. The VH domain of any one of claims 23 or 24, wherein the second VH domain binds to a epitope of ENPP1 different from the epitope bound by the first VH domain.

26. The VH domain of any one of claims 23 or 24, wherein the second VH domain binds to an immune checkpoint marker and prevents activation of the immune checkpoint marker, optionally wherein the immune checkpoint marker is PD-1, LAG-3, TIM-3, CTLA-4, or PD-L1.

27. The VH domain of any one of claims 23 or 24, wherein the second VH domain binds to a T cell.

28. The VH domain of claim 27, wherein the second VH domain binds to a T cell receptor.

29. The VH domain of claim 27 or 28, wherein the T cell is a cytotoxic T cell.

30. The VH domain of any one of claims 27-29, wherein the second VH domain binds toCD3.

31. The VH domain of any one of claims 1-24, wherein VH domain is fused to a T cell receptor to generate a chimeric antigen receptor (CAR).

32. The VH domain of claim 31 , wherein the CAR is present on a T cell.

33. The VH domain of any one of claims 1-24, wherein VH domain is fused to a proteolysis targeting chimera (PROTAC) pay load.

34. The VH domain of claim 33, wherein the PROTAC payload comprises a ubiquitin ligase.

35. The VH domain of any one of claims 1-34, formatted as an antibody that is a single chainFv (scFv), IgG, Fab, (Fab)2, or (scFv -36. The VH domain of any one of claims 1-35, wherein the VH domain or an antibody comprising the VH domain is labeled.

37. The VH domain of any one of claims 1-36, wherein the VH domain or an antibody comprising the VH domain is attached to a solid support.

38. An isolated nucleic acid comprising a nucleotide sequence encoding the VH of any one of claims 1-37 or an antibody comprising the VH domain.

39. A vector comprising the isolated nucleic acid of claim 38.

40. A cell comprising the nucleic acid of claim 38 or the vector of claim 39.

41. The cell of claim 40, wherein the cell is a prokarytoic cell or a eurkaryotic cell, optionally the cell is a mammalian cell, e.g., a human cell, a human T cell.

42. A method for binding ENPP1 in a sample, comprising: contacting the VH domain of any one of claims 1-37 with the sample,determining binding of the VH domain to ENPP1 in the sample.

43. The method of claim 42, wherein the VH domain is labeled.

44. The method of claim 43, wherein the label is a fluorescent label, a chemiluminescent label, a radiolabel, an enzyme, or a chromogenic label.

45. The method of any one of claims 42-44, wherein the sample is tissue or blood.

46. A method of admistering an anti-EPPPl antibody to a subject in need thereof, the method comprising administering the VH domain of any one of claims 1-37 in a therapeutically effective amount to the subject.

47. A method for treating cancer in a subject, the method comprising: administering the VH domain of any one of claims 1-37 in a therapeutically effective amount to the subject.

48. A method for treating insulin resistance and / or Type 2 diabetes in a subject, the method comprising: administering the VH domain of any one of claims 1-37 in a therapeutically effective amount to the subject.

49. Use of a VH domain according to any one of claims 1-37 in the preparation of a medicament for the treatment of cancer in a subject.

50. Use of a VH domain according to any one of claims 1-37 in the preparation of a medicament for the treatment of insulin resistance and / or Type 2 diabetes in a subject.

51. The method of claim 47 or use of claim 49, wherein the method comprises administering the VH domain in combination with at least one additional active agent, wherein the at least one additional active agent comprises a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapy agent, or a combination thereof.

52. A pharmaceutical composition comprising the VH domain of any one of claims 1-37 and a pharmaceutically acceptable excipient.

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