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WO2025166339A1 - Reprogramming the cd112-cd112r axis for optimized cancer immunotherapy - Google Patents

Reprogramming the cd112-cd112r axis for optimized cancer immunotherapy

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Publication number
WO2025166339A1
WO2025166339A1 PCT/US2025/014297 US2025014297W WO2025166339A1 WO 2025166339 A1 WO2025166339 A1 WO 2025166339A1 US 2025014297 W US2025014297 W US 2025014297W WO 2025166339 A1 WO2025166339 A1 WO 2025166339A1
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WO
WIPO (PCT)
Prior art keywords
cd112r
substitution
target binding
binding molecule
isolated receptor
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PCT/US2025/014297
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French (fr)
Inventor
Patrick Hwu
Vincent Luca
Estefania JULIA
Srishti Singh
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H Lee Moffitt Cancer Center and Research Institute Inc
Original Assignee
H Lee Moffitt Cancer Center and Research Institute Inc
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Publication of WO2025166339A1 publication Critical patent/WO2025166339A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • pembrolizumab and nivolumab antibodies that block programmed cell death protein-1 (PD- 1), were effectively used to treat a variety of tumors, such as renal cell carcinoma (RCC), advanced melanoma, non-small-cell lung cancer (NSCLC), breast cancer, and advanced ATTORNEY DOCKET NO.10110-461WO1 hepatocellular carcinoma.
  • RCC renal cell carcinoma
  • NSCLC non-small-cell lung cancer
  • breast cancer breast cancer
  • ATTORNEY DOCKET NO.10110-461WO1 hepatocellular carcinoma advanced ATTORNEY DOCKET NO.10110-461WO1 hepatocellular carcinoma.
  • the anti-PD-1 drugs demonstrated better survival rates than conventional therapies when used for cancer immunotherapy.
  • a substantial number of cancer patients either failed to respond to these single-agent immune checkpoint inhibition therapeutics or demonstrated an initial response followed by acquired resistance.
  • Effector T cells exhibit the exhaust phenotype and malfunction within the tumor microenvironment (TME).
  • Dysfunctional T cells have increased numbers of co-inhibitory receptors, such as CTLA-4 and PD-1, on their surfaces. Based on these facts, identifying new immune checkpoint pathways might improve the immunotherapy response rates and broaden the efficacy of the treatment provided. Given the limitations described above, there is a need to explore new therapies that target other co-inhibitory receptor pathways. SUMMARY Disclosed here are isolated receptors of CD112 and CD112 binding molecules and methods of their use in the treatment, inhibition, reduction, amelioration, and/or prevention of cancer.
  • isolated receptors or target binding molecules that binds CD112, wherein the isolated receptor or target binding molecule (such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells) comprises a CD112R extracellular domain variant. Also disclosed herein are isolated receptors or target binding molecules of any preceding aspect, wherein the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N-terminal end of SEQ ID NO: 1.
  • isolated receptors or target binding molecules of any preceding aspect wherein the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 50 (such as, for example, a methionine to lysine substitution (M50K)), 92 (such as, for example, a histidine to glutamine substitution (H92Q)), 138 (such as, for example, a serine to threonine substitution (S138T)), 69 (such as, for example a serine to asparagine (S69N), serine to tyrosine (S69Y), serine to proline (S69P), serine to histidine (S69H), serine to threonine (S69T), serine to aspartic acid (S69D) or serine to alanine (S69A) substitution), 82 (such as, for example, a glycine to histidine (G82H), glycine to arginine (G82R), gly
  • the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 2); or a M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 5).
  • isolated receptors or target binding molecules of any preceding aspect wherein the isolated receptor binds an epitope on tumor cells comprising CD112.
  • the CD112R is a human CD112R.
  • compositions comprising the isolated receptor or target binding molecule of any preceding aspect, and pharmaceutically acceptable carrier.
  • the pharmaceutical composition can comprise at least one additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB.
  • a cancer and/or metastasis such as for example, breast cancer including, but not limited to triple negative breast cancer
  • a subject in need thereof, comprising administering to a subject an isolated receptor or target binding molecule that binds CD112, wherein the isolated receptor or target binding molecule (such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells) comprises a CD112R extracellular domain variant or administering to the subject a pharmaceutical composition comprising said isolated receptor or target binding molecule.
  • the isolated receptor or target binding molecule such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells
  • the pharmaceutical composition can further comprise at least one ATTORNEY DOCKET NO.10110-461WO1 additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB.
  • ATTORNEY DOCKET NO.10110-461WO1 additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB.
  • the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N-terminal end of SEQ ID NO: 1.
  • the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 50 (such as, for example, a methionine to lysine substitution (M50K)), 92 (such as, for example, a histidine to glutamine substitution (H92Q)), 138 (such as, for example, a serine to threonine substitution (S138T)), 69 (such as, for example a serine to asparagine (S69N), serine to tyrosine (S69Y), serine to proline (S69P), serine to histidine (S69H), serine to threonine (S69T), serine to aspartic acid (S69D) or serine to alanine (S69A) substitution), 82 (such as, for example, a glycine to histidine (G), 92 (such as, for example, a histidine to glutamine substitution (H92Q)), 138 (such as, for example, a serine to thre
  • the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 2); or a M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 5). Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect, wherein the isolated receptor binds an epitope on tumor cells comprising of CD112.
  • FIG. 1 shows CD112R and CD112 expression in health and tumor microenvironment. In normal human peripheral blood cell subsets, CD112R is predominantly expressed in NK cells and CD8+ T cells, but not in CD4+ T cells, DCs, neutrophils, monocytes and B cells.
  • CD112R+CD8+ T cells are mainly detected in effector memory T (CD45RA ⁇ CCR7 ⁇ ) cells in phenotype, and rarely in na ⁇ ve T (CD45RA+CCR7+) cells.
  • CD112R is expressed abundantly in NK cells, CD4+ T cells and CD8+ T cells.
  • CD112 was detectable primarily in DCs, tumor-associated macrophages (TAMs) or monocytes, and tumors cells.
  • FIG. 2 represents TCGA cancer types with corresponding GTEx normal tissues. The GTEx normal and TCGA normal are combined. The gene expression unit is log2(TPM+1).
  • FIG. 3 shows a molecule of CD112R and 1 molecule of CD112 D1 crystallographic unit. CD112 D1 and CD112R have 9 antiparallel ⁇ -strands.
  • FIG.4 shows complex structure design of Interface remodeling and Velcro engineering libraries to isolate a CD112R mutant with high affinity for CD112 ligand.
  • FIG. 5 represents a new variant as CD112RIV which is a result of combining all the mutations from Interface and Velcro library.
  • FIG. 6 represents staining in MDA-MB-468 triple negative breast cancer cell line. A binding test is performed where cell lines are stained with CD112RWT, CD112R IV and TIGIT extracellular domain recombinant proteins.
  • CD112R is the dominant receptor of CD112 ligand and TIGIT (second receptor) also binds to CD112 but with very low affinity.
  • CD112R IV binds specifically with CD112 ligands only.
  • FIG.7 depicts affinity maturation of the CD112R IV mutant to enhance affinity. Random mutagenesis resulted in a panel of CD112R mutants with different affinities.
  • FIG.8 represents CD112R IV and CD112R IVE as the two best binders out of the 9 clones.
  • FIG.9 depicts a combined mutation table from CD112R IV affinity maturation.
  • FIG.10 shows a flowchart of engineering the CD112R mutants. ATTORNEY DOCKET NO.10110-461WO1 FIG.
  • FIG. 11 shows CD112 is a ligand in the PVR-like protein network.
  • A Schematic depicting known interactions between PVR-like proteins.
  • B CD112 is overexpressed in several cancers (TCGA RNAseq database) compared to corresponding normal tissues (GTEx and TCGA RNAseq databases combined).
  • FIG. 12 shows purification of CD112R, TIGIT, and CD112 ECDs. Representative chromatograms show that all three proteins elute from the SEC column as monodisperse peaks, which is indicative of favorable biochemical behavior. Protein purity was evaluated by SDS PAGE (insets).
  • FIG. 13 depicts CD112R binding disrupting CD112 homodimerization.
  • CD112 and CD112R were purified individually or in complex on a SEC column. CD112 eluted as a dimer, CD112R eluted as a monomer, and CD112:CD112R eluted as a 1:1 complex. Stoichiometries were estimated based on MW standards.
  • FIG. 14 represents a comparison of experimentally determined CD112-CD112R structure to published CD112-TIGIT structure.
  • A Crystal structure of CD112R (purple) bound to CD112 D1 (beige).
  • B Crystal structure of TIGIT (pink) bound to CD112 D1 (beige, PDB 5V52). In both (A) and (B), inset zoom windows depict the lock and key motif at the binding interface.
  • FIG. 15 shows the structure-guided engineering of CD112R mutant libraries.
  • the interface library was generated by mutating residues at the CD112-CD112R binding interface (red) as indicated in the table.
  • the velcro library was generated by introducing a randomized 5 amino acid peptide insertion (red) at the N-terminus of CD112R.
  • FIG. 16 depicts CD112R IV consensus variant binds to CD112 with high affinity. Mutations (red spheres) from the interface and velcro libraries were combined to generate CD112R IV .
  • FIG.17 depicts specific binding of CD112R IV to CD112 + TNBC cells.
  • CD112R IV binds to the CD112-expressing TNBC cell line MDA-MB-468, but not to cells lacking CD112. Knockout of CD155 did not affect CD112R IV binding. In both the parent cell line and the CD155 knockout line, CD112R IV bound more robustly than CD112R WT .
  • FIG. 18 represents CD112 blockade increases CAR-T cell cytotoxicity.
  • CAR-T cells were co-cultured with CD112+ MDA-MB-468 cells in the presence of IgG1 isotype control or Fc-CD112R IV at a 1:1 E: T ratio. After 24 hours, killing was measured by luciferase assay.
  • FIG.19 shows CD112R variants as soluble inhibitors construct design.
  • FIG. 20 shows CD112R library design and selection strategy.
  • A Flow chart and structural representation of the CD112R affinity-maturation scheme. The interface library was generated by mutating hotspot residues at the CD112-CD112R interface (red spheres), the velcro library was generated by introducing a randomized 5 amino acid peptide insertion at the N-terminus of CD112R (orange spheres), and the unbiased library was generated using error- prone PCR (cyan spheres).
  • FIG. 21 shows comparison of CD112-CD112R and CD112-TIGIT structures.
  • A TIGIT (pink) and CD112R (light purple) ectodomains from CD112-bound structures were superimposed and shown in cartoon representation to compare their binding modes.
  • D1 of CD112 is shown in surface representation (beige). The right panels indicate the CD112R- and TIGIT-binding interfaces. The table below compares various structural parameters for each interface.
  • FIG. 22 shows molecular interactions at THE CD112R/CD112 interface. Cartoon representation of the CD112R-CD112 structure (left) and a table indicating contact residues between the two proteins (right). The zoom panels highlight various H-bonds, salt bridges, and van der Waals interactions listed in the table. H-bonds and salt bridges are shown as dotted lines, and H-bond cutoffs were defined as contact distances of less than 3.5 ⁇ .
  • FIG. 23 shows selection strategy for CD112R libraries.
  • FIG. 1 Flow charts depict the selection strategies used to isolate high-affinity CD112 variants from the interface, velcro, and unbiased libraries. Red arrows and text indicate negative selections.
  • FIG. 1 The gating strategy used to isolate yeast singlets is shown (bottom). The gate in the SSC/FSC plot (left) was drawn to select for the bulk yeast population, and these yeast were again gated in the FSCH/FSCA plot (middle) to isolate singlets. Yeast singlets were then analyzed based on surface expression ATTORNEY DOCKET NO.10110-461WO1 (FL-1, staining with myc-488 antibody) and CD112 (FL-4, staining with biotinylated CD112 bound to SA-647).
  • FIG.24 shows expression yield of CD112R and CD112R variants.
  • the table (right) indicates the yield of CD112R, CD112RIV, or CD112RIVE from 1L of infected insect cell cultures.
  • FIG. 25 shows AlphaFold3 model of CD112R IVE bound to D1 of CD112.
  • the Alphafold3 model of CD112RIVE (light purple, cartoon representation) bound to D1 of CD112 (beige surface representation with positive/negative charges colored). Zoom windows highlight mutations from the interface (red), velcro (orange), and unbiased (cyan) libraries.
  • FIG. 26 shows CD112R variants have increased binding to recombinant CD112 and CD112-expressing cells.
  • A SPR was used to determine the binding affinities of CD112R, CD112RIV, and CD112RIVE. Site-specifically biotinylated CD112R proteins were immobilized on a Streptavidin (SA)-coated sensor chip and 3-fold dilutions of the full-length CD112 ECD were injected over the surface, starting at a concentration of 20,000 nM. Representative SPR sensograms from a single experiment are shown, and the KD and s.d. were determined by averaging the values from two-independent experiments.
  • FIG. 27 shows MDA-MB-468 TNBC cell line variants utilized for functional assays.
  • FIG.28 shows CD112R variants are effective CD112 traps and CD112-targeting T cell engagers.
  • a competition assay was used to measure the blockade of CD112-CD112R interactions by CD112R IV and CD112R IVE .
  • Yeast expressing CD112 were stained with a fixed 100nM tetramer concentration of fluorescently labeled CD112R in the presence of increasing concentrations of CD112R IV or CD112R IVE Fc fusion proteins, and CD112-CD112R binding was monitored by flow cytometry.
  • B Illustration of TCEs incorporating CD112R variants in ATTORNEY DOCKET NO.10110-461WO1 co-culture with MDA-MB-468 cells and T cells.
  • FIG.29 shows TCE purification and analysis of TCE antigen specificity.
  • A Overlaid size exclusion chromatograms show the purification of CD112R, CD112R IV and CD112R IVE TCEs from a size-exclusion column.
  • composition refers to any agent that has a beneficial biological effect.
  • beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition.
  • composition also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • composition includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • compositions CD112 is part of the PVR-like protein co-signaling network.
  • CD112 also known as Nectin-2 or PVRL2
  • PVR-like Polio Virus Receptor-like network of immune co-signaling molecules
  • the transmembrane CD112 protein is predominantly localized to the adherens junction in epithelial cells, but it is also expressed in endothelial cells, neurons, and fibroblasts.
  • Several human tumors overexpress CD112, including breast cancer, ovarian cancer, and pancreatic cancer, making it an intriguing target for immunomodulatory drugs (FIG.11B).
  • Interface remodeling and Velcro engineering libraries were used to isolate a CD112R mutant with high affinity for CD112 ligand.
  • Use of the “Interface library” introduced mutations at interface hotspots to improve surface complementarity to CD112 and have the binding energetics of CD112R.
  • isolated receptors or target binding molecules that binds CD112, wherein the isolated receptor or target binding molecule (such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells) comprises a CD112R extracellular domain variant.
  • the isolated receptor or target binding molecule such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells
  • the isolated receptor or target binding molecule comprises a CD112R extracellular domain variant.
  • “Velcro engineered” libraries are assessed for high affinity binding between CD112 variants and CD112 ligand expressed by cancer cells.
  • the Velcro library is designed by introducing a randomized nine-residue peptide extending from the N- terminus of CD112R.
  • isolated receptors or target binding molecules wherein the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N-terminal end of SEQ ID NO: 1.
  • technical and scientific terms used herein have the same meaning as commonly under- stood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY 35 AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).
  • CD112R polypeptide CD112R protein
  • CD112R CD112R
  • CD112R CD112R protein
  • CD112R CD112R
  • the CD112R described herein may be isolated from a variety of sources, such as from human or from a nonhuman organism, or prepared by recombinant or synthetic methods.
  • CD112R is a polypeptide capable of binding to CD112 and having at least 90% sequence identity to at least a 10 amino acid consecutive sequence to SEQ ID NO: 1.
  • CD112R is a polypeptide capable of binding to CD112 and having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least a 10 amino acid consecutive sequence of SEQ ID NO: 1. All disclosures in this specification which refer to the "CD112R" refer to each of the polypeptides individually as 6 well as jointly.
  • CD112R polypeptide refers to each polypeptide of the ATTORNEY DOCKET NO.10110-461WO1 disclosure individually.
  • CD112R polypeptide refers to each polypeptide of the ATTORNEY DOCKET NO.10110-461WO1 disclosure individually.
  • CD112R polypeptide refers to each polypeptide of the ATTORNEY DOCKET NO.10110-461WO1 disclosure individually.
  • CD112R polypeptide CD112R protein
  • CD112R also include variants of the CD112R disclosed herein or known in the art.
  • isolated receptors or target binding molecules wherein the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 50 (such as, for example, a methionine to lysine substitution (M50K)), 92 (such as, for example, a histidine to glutamine substitution (H92Q)), 138 (such as, for example, a serine to threonine substitution (S138T)), 69 (such as, for example a serine to asparagine (S69N), serine to tyrosine (S69Y), serine to proline (S69P), serine to histidine (S69H), serine to threonine (S69T), serine to aspartic acid (S69D) or serine to alanine (S69A) substitution), 82 (such as, for example, a glycine to histidine (G82H), glycine to arginine (G82R), glycine to asparag
  • residues 50 such as,
  • the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 2); or a M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 5).
  • the present disclosure provides a CD112R protein, or a mutant CD112R protein.
  • mutant CD112R is a polypeptide capable of binding to CD112 and having at least 90% sequence identity to at least a 10 amino acid consecutive sequence to SEQ ID NO: 2 or SEQ ID. NO: 5.
  • mutant CD112R is a polypeptide capable of binding to CD112 and having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence.
  • CD112R WT refers to naturally occurring receptor molecules with single pass membrane structure. The structure includes one transmembrane spanning region, a long intracellular domain, and a single extracellular immunoglobulin variable-like (IgV) domain encoded inside the CD112R.
  • IgV immunoglobulin variable-like domain encoded inside the CD112R.
  • ATTORNEY DOCKET NO.10110-461WO1 The terms “CD112R” refers to human CD112R present on T cells and NK cells, unless otherwise specifically indicated (e.g.
  • mouse CD112R mouse CD112R, cynomolgus CD112R, etc.
  • the term includes full -length, unprocessed CD112R as well as any form of CD112R that results from processing in the cell.
  • the term encompasses naturally occurring variants of human CD112R, e.g., splice variants allelic variants.
  • External ID's for CD112R gene includes Entrez Gene: 79037, Ensembl: ENSG00000213413, OMIM; 617012, and UniProtKB: Q6DKI7.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid 50 sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • Affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a target).
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., receptor and target).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kn). Affinity can be measured by common methods known in art, including those described herein.
  • An “affinity matured” receptor refers to a receptor with one or more alterations in extracellular domain, compared to a parent receptor which does not possess such alterations, such alterations optionally resulting in an improvement in the affinity of the receptor for target.
  • An “Extracellular immunoglobulin variable-like (IgV) domain” refers to the extracellular domain of the 36kD single-pass transmembrane protein CD112R. Also disclosed herein are isolated receptors or target binding molecules, wherein the isolated receptor binds an epitope on tumor cells comprising CD112.
  • compositions comprising the isolated receptor or target binding molecule of any preceding aspect, and pharmaceutically acceptable carrier.
  • the pharmaceutical composition can comprise at least one additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB.
  • compositions and methods for modulating T-cell mediated immune response in a subject in need thereof includes administering to the subject an effective amount of a CD112R protein, or a mutant CD112R protein of the present disclosure, which are collectively referred to herein as “CD112 targeting molecules ” or “ anti-CD112 binding molecules ” or “ high affinity CD112R mutants” .
  • CD112 targeting molecules or “anti-CD112 binding molecules ” or “ high affinity CD112R mutants”
  • high affinity CD112R mutants high affinity CD112R mutants
  • lymphomas such as B cell lymphoma and T cell lymphoma; mycosis fungoides; Hodgkin’s Disease; myeloid leukemia (including, but not limited to acute myeloid leukemia (AML) and/or chronic myeloid leukemia (CML)); bladder cancer; brain cancer (including, but not limited to brain lower grade glioma); nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer (including, but not limited to kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, and kidney chomophobe); lung cancers such as small cell lung cancer, non- small cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LUAD); neuroblastoma/glioblastoma; adrenocortical carcinoma, ovarian cancer; pancreatic cancer; prostate cancer; skin
  • ATTORNEY DOCKET NO.10110-461WO1 In one aspect, disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis (such as for example, breast cancer including, but not limited to triple negative breast cancer) in a subject in need thereof, comprising administering to a subject an effective amount of the isolated receptor, target binding molecules, or pharmaceutical compositions of any preceding aspect.
  • a cancer and/or metastasis such as for example, breast cancer including, but not limited to triple negative breast cancer
  • a subject in need thereof, comprising administering to a subject an isolated receptor or target binding molecule that binds CD112, wherein the isolated receptor or target binding molecule (such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells) comprises a CD112R extracellular domain variant or administering to the subject a pharmaceutical composition comprising said isolated receptor or target binding molecule.
  • the isolated receptor or target binding molecule such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells
  • the pharmaceutical composition can further comprise at least one additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB.
  • the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N-terminal end of SEQ ID NO: 1.
  • the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 50 (such as, for example, a methionine to lysine substitution (M50K)), 92 (such as, for example, a histidine to glutamine substitution (H92Q)), 138 (such as, for example, a serine to threonine substitution (S138T)), 69 (such as, for example a serine to asparagine (S69N), serine to tyrosine (S69Y), serine to proline (S69P), serine to histidine (S69H), serine to threonine (S69T), serine to aspartic acid (S69D) or serine to alanine (S69A) substitution), 82 (such as, for example, a glycine to histidine (G82H), g
  • the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 2); or a M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 5). Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect, wherein the isolated receptor binds an epitope on tumor cells comprising of CD112.
  • an "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • ATTORNEY DOCKET NO.10110-461WO1 By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth).
  • tumor growth means reducing the rate of growth of a tumor relative to a standard or a control.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented.
  • the term “subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific ATTORNEY DOCKET NO.10110-461WO1 therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • Biocompatible generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.
  • a “control” is an alternative subject or sample used in an experiment for comparison purposes.
  • a control can be "positive” or "negative.”
  • Effective amount of an agent refers to a sufficient amount of an agent to provide a desired effect.
  • the amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.
  • an “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a "pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
  • “Pharmaceutically acceptable carrier” means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions ATTORNEY DOCKET NO.10110-461WO1 (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • carrier encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
  • “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
  • “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer).
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • therapeutic agent e.g., sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium stea, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate
  • a composition comprising an agent refers to an amount that is effective to achieve a desired therapeutic result.
  • a desired therapeutic result is the control of type I diabetes.
  • a desired therapeutic result is the control of obesity.
  • Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.
  • the term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief.
  • the precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
  • a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
  • block in the context of an interaction between two or more molecules, is used herein to refer to inhibition or prevention of said interaction between the two or more molecules, wherein the inhibition or prevention of said interaction between the two or more molecules is complete or nearly complete under at least one condition.
  • a “nearly complete” inhibition is a percent inhibition of about 70-99.9%, and a “complete” inhibition is 100%.
  • a molecule is said to "block” an interaction between two or more other molecules if it completely or nearly completely inhibits such interaction at certain concentrations in a dose dependent manner.
  • the term “cancer” is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth.
  • a cancer may be benign (also referred to as a benign tumor), pre-malignant, or malignant.
  • Cancer cells may be solid cancer cells or leukemic cancer cells.
  • tumor is used herein to refer to a cell or cells that comprise a cancer.
  • tumor growth is used herein to refer to proliferation or growth by a cell or cells that comprise a cancer that leads to a corresponding increase in the size or extent of the cancer.
  • chimeric receptor refers to a receptor in which a portion of the extracellular immunoglobulin variable-like (IgV) domain is mutated via random mutations derived from Interface library and Velcro library.
  • class of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • IgA immunoglobulins
  • IgD immunoglobulins
  • IgE immunoglobulins
  • IgG immunoglobulins
  • IgM immunoglobulins
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, ' E, y, and ⁇ , respectively.
  • Administration "in combination with” one or more further therapeutic agents include simultaneous (concurrent) and consecutive (sequential) administration in any order.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, 112s, y9o, Re1s6, R Sm1s3, Bi212, p32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anti-cancer agents disclosed below.
  • radioactive isotopes e.g., At211, 11
  • ATTORNEY DOCKET NO.10110-461WO1 "Effector functions" refer to those biological activities attributable to the Fe region of an antibody, which vary with the antibody isotype. Examples of receptor effector functions include: Clq binding and complement dependent cyto- toxicity (CDC); Fe receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor/ T cell surface receptor); and T cell activation.
  • An "effective amount” of an agent e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • full length antibody is used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fe region as defined herein.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell and may contain mutations.
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of human antibody specifically excludes a humanized antibody comprising non- human antigen-binding residues.
  • the term "variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs).
  • FRs conserved framework regions
  • HVRs hypervariable regions
  • An "immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression ATTORNEY DOCKET NO.10110-461WO1 of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors.”
  • “immune checkpoint receptors” are checkpoint blockade inhibitors used in cancer therapies that target the extracellular domains (ECDs) of immune inhibitory receptors. These successful drugs reverse T cell exhaustion by blocking ligand- mediated activation of receptor signaling.
  • Current invention focuses on novel immunotherapies targeting the next-generation checkpoint protein CD112.
  • high affinity mutant CD112R are provided.
  • the high affinity mutant CD112R shares certain structural and/or functional features.
  • the high affinity mutant CD112R includes a parent receptor with random mutation induced via Interface or Velcro methods in the extracellular domain and affinity matured thereof.
  • the affinity matured variants comprise variants CD112IVA3 and CD112IVA5.
  • the affinity matured variants comprise receptors with random mutations as compared to wild type receptor.
  • a pharmaceutical composition comprising the variant receptor of CD112R and a pharmaceutically acceptable carrier, wherein the composition optionally comprises an opsonizing agent, a regulatory T cell depleting agent, chemotherapy, and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB.
  • the variant receptor of CD112R or pharmaceutical composition is used for treating cancer in a subject, wherein the cancer is optionally carcinoma, lymphoma, blastoma, sarcoma, or leukemia, or wherein the cancer is optionally squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulva, cancer, thyroid cancer, hepatic carcinoma, salivary
  • a pharmaceutical composition comprising the variant receptor of CD112R for use in treating cancer, wherein the use further comprises administering a second ATTORNEY DOCKET NO.10110-461WO1 therapy, wherein the second therapy is optionally radiotherapy, surgery or administration of a second agent, wherein the second agent is optionally an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3 or TIM-3, or an antagonist of TIGIT or CD96, or an antagonist of PVRLl, PVRL2, PVRL3, PVRL4, and CD155, or is an antagonist of CD47, or is an antagonist of CD39, or is an antagonist of IL-27, or is STING agonist, wherein the second agent is optionally an antagonist receptor.
  • the second agent is optionally an antagonist receptor.
  • the second agent can be an agonist for a stimulatory receptor on a T cell including, but not limited to OX40L (such as, for example, OX40), CD40L (such as, for example, CD40), CD28 (such as, for example, CD80/CD86), Inducible costimulatory (ICOS)(such as, for example, ICOS ligand), lymphocyte function associated antigen 1 (LFA-1) (such as, for example, intracellular adhesion molecule 1 (ICAM-1)), and T cell immunoglobulin and mucin domain 1 (TIM-1) (such as, for example, TIM-4), 4-1BB (such as, for example, 4-1BBL).
  • OX40L such as, for example, OX40
  • CD40L such as, for example, CD40
  • CD28 such as, for example, CD80/CD86
  • ICM-1 lymphocyte function associated antigen 1
  • TIM-1 T cell immunoglobulin and mucin domain 1
  • 4-1BB such as, for example,
  • a method of producing the variant receptor of CD112 comprises culturing the yeast cells of under conditions wherein the receptor is expressed, optionally further comprising purifying the receptor.
  • the additional therapeutic agent or second agent is a chemotherapeutic agent, an opsonizing agent, a regulatory T cell ("Treg") depleting agent, an antagonist of a target other than CD112R, or an agonist of a target other than CD112R.
  • the second agent is a chemotherapeutic agent described herein or any known chemotherapeutic agent.
  • the second agent is an opsonizing agent, wherein the opsonizing agent is an antibody other than an anti-CD112R antibody that targets cancer or tumor cells.
  • the second agent is a Treg depleting agent described herein or any known Treg depleting agent.
  • the second agent is an antagonist of a target other than CD112R.
  • the second agent is an agonist of a target other than CD112R.
  • a variant of CD112R provided herein is a multispecific T cell engager, e.g. a bispecific T cell engager.
  • one of the binding specificities is for CD112 and the other is for any other target.
  • one of the binding specificities is for CD112 and the other is for selected independently from one (in the case of bispecific) or more (in the case of multispecific) of OX40, CD28, CD40L, LFA-1, ICOS, 4-1BB, PD-1, PD-Ll, CTLA-4, Lag- 3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, STING, CD47, CD39, and IL-27.
  • bispecific antibodies may bind to two different epitopes of ATTORNEY DOCKET NO.10110-461WO1 CD112R.
  • BiTEs Bispecific T cell engagers
  • BiTEs may also be used to localize cytotoxic agents to cells which express CD112.
  • BiTEs can be prepared as full-length receptor or fragments thereof.
  • amino acid sequence variants of CD112R provided herein are contemplated. For example, it is desirable to improve the binding affinity and/or other biological properties of the receptor.
  • Amino acid sequence variants of the receptor may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the receptor, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the receptor.
  • any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., target binding.
  • antibody variants having one or more amino acid substitutions are provided.
  • one or more bispecific T-cell engagers of the disclosure are used in combination with one or more anti-CD112 binding antibodies, one or more cancer vaccines, Freund's Adjuvant, one or more CAR-T cell therapies, one or more stem cell therapies, and/or one or more scFv therapies.
  • the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (P
  • the treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD- 1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT-011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for
  • CD112 is part of the PVR-like protein co-signaling network.
  • CD112 is part of the PVR-like protein co-signaling network.
  • CD112 (also known as Nectin-2 or PVRL2) is a ligand in the Polio Virus Receptor-like (PVR-like) network of immune co-signaling molecules (FIG. 11A).
  • the transmembrane CD112 protein is predominantly localized to the adherens junction in epithelial cells, but it is also expressed in endothelial cells, neurons, and fibroblasts.
  • Several human tumors overexpress CD112, including breast cancer, ovarian cancer, and pancreatic cancer, making it an intriguing target for immunomodulatory drugs (FIG. 11B).
  • CD112 was first characterized as a ligand for the immunostimulatory receptor CD226 and the immunosuppressive receptor TIGIT.
  • the protein CD112R (formerly known as PVRIG) was recently determined to be the dominant receptor for CD112 ATTORNEY DOCKET NO.10110-461WO1 (FIG.11A).
  • CD112R-CD112 interactions are > 100-fold higher affinity than CD112 interactions with CD226 or TIGIT.
  • SPR surface plasmon resonance
  • CD112 engagement of CD112R induces the phosphorylation of ITIM motifs in the CD112R intracellular domain (ICD), initiating a signaling cascade that inhibits T cell function by suppressing expression of the transcriptional activator NFAT.
  • ICD CD112R intracellular domain
  • the PVR-like protein network includes the inhibitory receptor CD96, and the TIGIT/CD96-activating ligand CD155 (FIG.11A).
  • Example 2 Determine structures of CD112 bound to inhibitory (CD112R) and activating receptors (CD226), and quantitatively measure the binding affinities between all PVR- like proteins. Establish the molecular basis for CD112 interactions with activating and inhibitory receptors. Several proteins in the PVR-like network are promiscuous and form cis or trans interactions to regulate immune signaling. For example, structural studies have revealed that TIGIT dimerizes upon engagement of CD155 homodimers to form 2:2 heterotetramers, whereas TIGIT binding disrupts CD112 dimerization and forms a 1:1 complex. TIGIT has also been shown to inhibit CD226 signaling through the formation of cis TIGIT-CD226 complexes on the cell surface.
  • CD112, CD112R, and TIGIT were purified to facilitate the structural and biophysical studies. These domains contain disulfide bonds and N-linked glycans, so they are not amenable to expression in prokaryotic systems such as E. coli.
  • the constructs were expressed in insect (T. Ni) cells using recombinant Baculovirus. Each protein was purified ATTORNEY DOCKET NO.10110-461WO1 from cell culture supernatants using nickel affinity and size-exclusion chromatography (SEC).
  • SEC nickel affinity and size-exclusion chromatography
  • CD112 homodimers (76.8 kD) have a larger predicted MW than the CD112-CD112R complex (54 kD), a rightward shift corresponding to a decrease in MW was observed if CD112R binding disrupts homodimerization. A right shift in the peak was observed when CD112 was co-purified with CD112R, indicating that both TIGIT and CD112R disrupt CD112 dimerization upon binding (FIG.13). Crystal structure of CD112 D1 bound to CD112R. It was determined that the initiating step in CD112R signaling was to visualize the crystal structure of a CD112-CD112R complex.
  • the ECD of CD112R was co-purified, and the receptor-binding D1 domain of CD112 and subjected the proteins to sparse matrix screening.
  • Optimized crystals of the complex diffracted to 2.2 ⁇ resolution and the structure was solved by molecular replacement.
  • the Rwork/Rfree were 0.20/0.24 (FIG. 14A), which is within the publishable range for a 2.2 ⁇ resolution structure.
  • Analysis of the structure revealed that the CD112R ECD is a prototypical Ig-like fold that interacts with CD112 D1 (also an Ig domain) in an antiparallel orientation (FIG. 14A).
  • CD112R interacts with the core beta-sheet of CD112 through a lock-and-key motif that has been characterized in CD155-TIGIT and CD112- TIGIT complexes (FIG. 14A and 14B).
  • the binding mode of CD112-CD112R resembles that of a previously determined CD112-TIGIT structure.
  • the CD112- CD112R complex is more extensive both concerning buried surface area (884 ⁇ 2 vs 790 ⁇ 2) and polar contacts (8 H-bonds, 2 salt bridges vs 9 H-bonds, 0 salt bridge) (FIG. 14B and 14C).
  • the antiparallel binding mode of CD112R-CD112 resembles those adopted by other PVR-like receptor-ligand pairs, including TIGIT-CD112 (FIG. 21A) and a recently published structure of CD112R-CD112.
  • the interacting domains of PVR-like proteins contain complementary lock (T/S, F/Y, P) and key (AX6G) motifs that stabilize the binding interface (FIG.21B).
  • T/S, F/Y, P complementary lock
  • AX6G key
  • the aromatic residue, F139 inserts into a concave hydrophobic pocket formed by the lock motif (AAFHPKMG (SEQ ID NO: 30), residues 83 to 90) of CD112 (FIG.14A).
  • CD112R interacts with CD112 via a broad interface centered around the ⁇ -sheet formed by the C, D, G, and H strands (FIG.22).
  • Soluble receptor ECDs compete with endogenous receptors for ligand binding, making them appealing low-molecular weight alternatives to monoclonal antibodies.
  • CD112R-CD112 the velcro peptide is predicted to enhance binding by creating new contacts with a patch of CD112 surface residues in proximity to the CD112R N-terminus (FIG.14A).
  • FIG.15A To design the CD112R interface library we introduced conservative mutations at nine hotspot residues (S69, L72, G82, A83, T87, V90, A137, F139 and S143) that contact CD112 (FIG.15A).
  • the library was generated using assembly PCR with degenerate primers and has a theoretical diversity of 6x10 7 unique clones.
  • Purified library DNA was then transformed into ATTORNEY DOCKET NO.10110-461WO1 yeast such that the number of transformants was > 6x10 7 to ensure full coverage of the sequence space.
  • CD112R IV a consensus variant containing interface and velcro mutations
  • the gene encoding CD112R IV was then subjected to error-prone PCR to generate an unbiased mutant library.
  • this approach could indirectly enhance affinity by stabilizing the global protein fold or the ⁇ -sheet comprising the binding interface.
  • M50K, H92Q and S138T additional affinity-enhancing mutations from the unbiased mutant library.
  • the variant containing the interface, velcro, and error-prone PCR mutations was named CD112R IVE (FIGS.22A, 25 and 8).
  • CD112R ECDs To purify the recombinant CD112R ECDs, we expressed CD112R and each variant in insect cells using Baculovirus. Although the yield of each protein was sufficient for downstream experiments, we observed that the CD112 IV and CD112R IVE variants had particularly high levels of expression. To quantitatively measure the expression levels, we purified CD112R, CD112R IV , and CD112R IVE from 1L cultures of Baculovirus-infected insect cells. Following the purification, the yields of CD112R, CD112R IV , and CD112R IVE were 3 mg/L, 8 mg/L, and 13 mg/L, respectively (FIG.24).
  • CD112R IV and CD112R IVE mutations are biochemically well-tolerated and may have serendipitously improved protein folding or secretion.
  • Yeast expressing CD112 with recombinant CD112R, CD112R I , CD112R V , CD112R IV or CD112R IVE was stained to directly compare the binding affinities of each variant (FIG.20B). It was found that the binding level was highest for CD112R IVE , followed by CD112R IV , CD112R I , CD112R V , and CD112R.
  • V90L, S138T, and S143T are predicted to create new van der Waals contacts at the interface through their gain of an additional side chain carbon atom (FIG. 25).
  • S69N, G82H, and H92Q gained bulky or charged side chains that may engage in polar contacts with opposing CD112 residues (FIG. 25).
  • the M50K and A83G mutations do not directly affect the interface but may improve protein solubility or favor the receptor-bound conformation, respectively.
  • the velcro peptide was predicted to make only glancing contacts with CD112, which is consistent with its relatively minor effect on binding affinity (FIGS.22B and 25). Determine the structure of a CD112-CD226 complex.
  • CD112 transduces inhibitory or activating signals depending on the receptor subtype it engages.
  • the crystal structure of CD112 bound to the activating receptor CD226 was determined to directly compare the structural features of each class of receptor.
  • CD226 contains two tandem Ig domains that have each been implicated in ligand binding. Therefore, the D1-D2 region of CD226 was co-purified with the full ECD (D1-D2-D3) of CD112 for use in the structural studies. Co-crystallization and structure determination were carried out as described above for CD112-CD112R.
  • the structure was analyzed to assess how multidomain interactions, receptor assembly, and ligand-induced conformational changes contribute to signal transduction. Perform a comparative structural analysis of CD112 signaling complexes.
  • the structures of CD112-CD112R and CD112-CD226 were determined and a detailed computational analysis was performed to identify conserved and receptor-specific features of the binding interfaces. This data ultimately led to improved selectivity in drugs targeting conserved epitopes of this receptor family, as there is a high degree of conservation among CD112-receptor binding interfaces. Given that there is also a CD112-TIGIT structure available, this therapeutically important checkpoint receptor complex was included in the analysis.
  • CD112 and CD155 interact with multiple receptors to induce activating and inhibitory signals.
  • the binding affinities of these interactions have been individually measured using various techniques, but the full interactome has not been systematically characterized using a single standard methodology.
  • CD112R was only recently discovered, it was important to understand whether binding partners outside of CD112 regulate its function. There is also growing evidence that both cis- and trans-interactions can influence immune checkpoint signaling. Therefore, surface plasmon resonance (SPR) was used to determine the kinetics and affinity of interactions among CD112, CD155, CD96, CD112R, TIGIT, and CD226.
  • SPR surface plasmon resonance
  • the affinity was enhanced by ⁇ 7-fold for CD112R IV and ⁇ 14-fold for CD112 IVE .
  • inspection of the SPR sensograms suggest that the affinity ATTORNEY DOCKET NO.10110-461WO1 improvement in each variant is largely due to a decrease in off-rate (FIG. 26).
  • CD112R-CD112 KD 526 nM
  • CD112R-Fc fusion protein 88nM
  • CD112R variants have increased affinity for CD112, it is unclear whether their mutations affect binding to the CD112 paralog CD155. To address this question, we used a cell-based binding assay to evaluate the CD112R IV and CD112R IVE binding specificity.
  • TNBC triple negative breast cancer
  • CD112R IVE and CD112R IV bound more robustly to cells expressing CD112 (CD112 + CD155 + and CD112 + CD155-) compared to CD112R, which is consistent with their higher affinities.
  • CD112- CD155 + and CD112-CD155- CD112- CD155-
  • CD112R tetramers were used to enhance CD112R avidity because monomeric CD112R binding to CD112 was undetectably weak.
  • IC50 41.9 and 24.5 nM for CD112R IV -Fc, and CD112R IVE -Fc respectively.
  • the dimeric Fc-fusion proteins blocked CD112 interactions with higher-avidity CD112R tetramers, suggesting that they may have even greater inhibitory potency than could be measured in this assay (FIG.28A).
  • Incorporation of CD112R variants into CD112-targeting TCEs In contrast to checkpoint inhibitors, which block T cells from receiving immunosuppressive signals, TCEs function by tethering T cells to tumor antigens while ATTORNEY DOCKET NO.10110-461WO1 activating their T cell receptors (TCRs).
  • TCRs T cell receptors
  • TCEs We generated three TCEs by fusing CD112R, CD112R IV , or CD112R IVE to an anti-CD3 single-chain antibody variable fragment (scFv) using a flexible (GGGGS) 4 linker (SEQ ID NO: 31) (FIGS.30B and 31A).
  • scFv single-chain antibody variable fragment
  • GGGGS flexible 4 linker
  • tumor cell killing (FIG.28C) and interferon gamma (IFN ⁇ ) levels (FIG.28D) were evaluated using a Luciferase-based assay and ELISA, respectively.
  • TCEs incorporating the CD112R IV and CD112R IVE variants each triggered a dose-dependent activation of T cells and both had greatly increased potency compared to the CD112R TCE.
  • the highest-affinity CD112R IVE variant exhibited the highest potency, both in terms of tumor cell killing and IFN ⁇ secretion.
  • Example 3 To engineer high-affinity CD112R decoys and assess their ability to enhance T cell function via blockade of CD112 signaling. Clinical development of TIGIT and CD112R inhibitors.
  • TIGIT and CD112R are the most potent immunosuppressive receptors in the PVR-like protein network, and both TIGIT and CD112R inhibitors are under development as cancer immunotherapies.
  • TIGIT and CD112R inhibitors are under development as cancer immunotherapies.
  • co-administration of anti-TIGIT and anti-CD112R antibodies boosts NK and CD8+ T cell function.
  • TIGIT or CD112R inhibitors are most commonly co-administered or given in combination with PD-1 inhibitors or chemoradiotherapy.
  • TIGIT antagonist antibodies are now in phase 3 trials for the treatment of non-small cell lung cancer (NSCLC), esophageal cancer, and squamous cell carcinoma, and a CD112R inhibitor recently entered phase 2 trials for the treatment of a variety of solid tumors.
  • NSCLC non-small cell lung cancer
  • esophageal cancer esophageal cancer
  • squamous cell carcinoma a CD112R inhibitor
  • CD112R inhibitor recently entered phase 2 trials for the treatment of a variety of solid tumors.
  • CD112R inhibitor recently entered phase 2 trials for the treatment of a variety of solid tumors.
  • ATTORNEY DOCKET NO.10110-461WO1 Evolve high-affinity CD112R decoys and assess their ability to enhance T-cell responses.
  • monoclonal antibodies MW ⁇ 150 kD
  • Camelid-derived nanobodies and soluble receptor ECDs also known as ligand traps
  • the smaller size of these proteins is associated with increased tissue penetrance and thermostability, and their single-chain structure allows for more straightforward genetic manipulation.
  • Nanobodies and ligand traps have already been approved by the FDA for the treatment of thrombotic thrombocytopenic purpura and myelodysplastic syndrome, and several nanobodies and ligand traps are under active clinical development.
  • structure-guided engineering was utilized to generate high-affinity CD112R variants that function as potent CD112 traps. The ability of these lower molecular weight inhibitors was tested to enhance the antitumor activity of CAR-T cells in TNBC.
  • Blockade of CD112 was especially beneficial given that CD112 signals through both major inhibitory receptors (CD112R and TIGIT) in the PVR-like family, and it was anticipated that CD112 traps boost the efficacy of T cell therapy against a wide range of CD112-expressing tumors.
  • the yeast display affinity-maturation process begins with the generation of a mutant library of the target protein (CD112R). The library is then stained with a fluorescently labeled ligand (CD112) and subjected to several rounds of MACS or FACS to enrich for high-affinity binders.
  • the high-resolution CD112-CD112R structure allowed us to take a structure-guided approach to CD112R library generation.
  • the first library the “interface library”
  • conservative mutations were introduced at interface hotspots to improve surface complementarity and bind energetics (FIG. 15A).
  • the second library the “velcro library”
  • a randomized 5-residue peptide insertion extending from the N-terminus of CD112R (FIG. 15B) was introduced.
  • Structural analysis revealed that the N-terminus of CD112R was adjacent to the CD112 binding interface, so the introduction of this velcro-like peptide is predicted to strengthen interactions by creating additional interface contacts.
  • the interface and peptide libraries each contained >107 unique variants were determined.
  • ATTORNEY DOCKET NO.10110-461WO1 Selection of high-affinity CD112R variants from yeast display libraries.
  • the libraries above were subjected to several rounds of MACS or FACS selection against the recombinant CD112 ECD. Decreasing concentrations of CD112 were used in subsequent rounds to increase stringency and retain only the highest affinity binders. Once selections were completed, individual clones were sequenced to identify affinity-enhancing mutations.
  • CD112R IV was then cloned into a Baculovirus expression vector as a fusion to human IgG1 Fc (Fc- CD112R IV ) and purified from insect cells as described herein. The yield from the purification was approximately 8mg/L, which is sufficient for downstream in vivo experiments. Characterization of the binding specificity of CD112RIV on a TNBC cell line. To generate a model to study the antigen specificity of the novel CD112R variants, MDA-MB-468 was selected as a representative TNBC cell line that expresses high levels of CD112 and CD155.
  • CD112R WT wild-type CD112R
  • CD112R IV showed higher binding to CD112+ CD155+ cells than CD112R WT , and the binding was abrogated in the ATTORNEY DOCKET NO.10110-461WO1 absence of CD112 (CD112- CD155+) (FIG.17).
  • CD112+ CD155- high specificity of CD112R IV for CD112.
  • Affinity-mature CD112R variants using an unbiased mutagenesis strategy Receptor-ligand affinity was altered directly through modifications to interface residues, or indirectly through allosteric effects or global stabilization.
  • an unbiased mutant library of CD112R IV using error-prone PCR was generated. The library was selected against decreasing concentrations of fluorescently labeled CD112 to isolate additional affinity- enhancing mutations. Clones isolated from the library were purified and dissociation constants were determined by SPR as described herein.
  • CD112R V , CD112R I , or CD112R IV were titrated. The decrease in binding signal was monitored as concentrations of the variants increased, and the maximum signal at each inhibitor concentration was plotted and fitted to obtain an IC50 for each variant.
  • the CD112h ⁇ TNBC cell line MDA-MB-468 (FIG.17) was used to evaluate the CD112R decoys. The cells were stained with a fixed 50 nM concentration of fluorescently CD112R tetramers alone or in the presence of increasing concentrations of inhibitor, and then the binding was detected by flow cytometry. The mean fluorescence intensity (MFI) of binding at each inhibitor concentration was plotted and fitted to obtain IC50 values.
  • CD112R IV and CD112R IVE function as soluble CD112 traps and TCEs.
  • Different binding affinities are associated with distinct functional properties.
  • CAR single-chain variable fragments (scFvs) with moderate affinities have optimal therapeutic efficacy. In part, this is because this reduced affinity helps to minimize off-target toxicity and T cell exhaustion from sustained ATTORNEY DOCKET NO.10110-461WO1 receptor activation.
  • IFN ⁇ signaling is important for CAR T cell killing of solid, but not liquid, tumors, likely due to the increased adhesion of CAR T cells to tumors following IFN- ⁇ stimulation.
  • CD112R-based TCEs it is found that wild-type CD112R induced very low levels of tumor killing. This finding agrees with previous studies showing that higher affinity target-binding arms are associated with improved TCE efficacy.
  • the increased affinity of CD112R IV and CD112R IVE TCEs drove high levels of IFN- ⁇ . Therefore, affinity-optimization should be considered for the development of TCEs and other isolated receptors incorporating engineered receptor ECDs.
  • both CD112R IV and CD112R IVE had greatly increased expression yields compared to the wild-type CD112R ECD.
  • yeast display selections isolate clones with the highest levels of bound ligand, irrespective of whether this binding is due to an increase in affinity, or the number of receptors displayed on the yeast. Therefore, we speculate that increased purification yields are a serendipitous byproduct of our selections enriching for both CD112 binding and surface expression. Additionally, the inclusion of a velcro peptide at the N-terminus of the CD112R protein may facilitate more efficient cleavage of the signal peptide, leading to increased levels of secretion. Regardless, these data suggest that yeast display affinity-tuning may have unexpected manufacturing benefits beyond their increased binding potency.
  • CD112 expression profile indicates that it is often overexpressed in cancer, including ovarian, breast, pancreatic and colorectal cancers, compared to normal tissues. However, lower levels of CD112 are also expressed on healthy cells, so careful evaluation of toxicity due to CD112 inhibition, or T cell killing of CD112 + cells will be important for optimal therapeutic development. Affinity-tuned biologics can provide one possible approach to mitigating such toxicities in future studies. MATERIALS AND METHODS ATTORNEY DOCKET NO.10110-461WO1 Protein expression and purification.
  • Human CD112 D1 (residues 32-160) [uniprot: Q92692] was cloned into a pET26b(+) vector containing a N-terminal pelB periplasmic signal peptide and a C-terminal 6xHis tag.
  • the recombinant protein was expressed in Escherichia coli strain BL21 (DE3). Cells were grown to optical density 600 nm of 0.6 to 0.8 and expression was induced through the addition of 0.4 mM isopropyl ⁇ -D thiogalactoside (IPTG), followed by an overnight incubation at 16 oC.
  • the cells were harvested by centrifugation and the pellet was stored at -20 oC for one hour. The frozen pellet was then thawed, and the pellet was resuspended in SET buffer (0.5 M sucrose, 0.5 mM EDTA and 0.2 M Tris pH 8.0), stirred for 30 minutes, then an equal volume of buffer containing 150 nM NaCl, and 20 mM MgCl2 and 2 ⁇ L benzonase, was added and the mixture was stirred for an additional 45 minutes.
  • SET buffer 0.5 M sucrose, 0.5 mM EDTA and 0.2 M Tris pH 8.0
  • the lysate was then centrifuged at 20,000g for 20 mins and the protein was purified from the supernatant using nickel affinity (Nickel-NTA resin, Qiagen) chromatography followed by size-exclusion chromatography (SEC) on an Superdex 75 Increase 10/300 GL column (GE).
  • Nickel-NTA resin Nickel-NTA resin, Qiagen
  • SEC size-exclusion chromatography
  • the protein Prior to purification on the size-exclusion column, the protein was treated with bovine carboxypeptidase A (1:100) and bovine carboxypeptidase B (1:200) to remove the C- terminal 6xHis tag.
  • the full-length CD112 ECD D1 to D3
  • the CD112R ECD, CD112R variants, and CD112R-based TCEs were all expressed in insect cells using Baculovirus.
  • the ECD of CD112 (D1 to D3, residues 32 to 350) and the ECD of human CD112R (residues 41-172) [Uniprot ID: Q6DKI7] contained a C-terminal 8xHis tag.
  • CD112R, CD112R I , CD112R V , CD112R IV , and CD112R IVE were modified to include a C-terminal biotin acceptor peptide (BAP) tag upstream of their C-terminal 6xHis tag for site-specific labeling with biotin.
  • CD112R IV and CD112R IVE Fc fusion proteins contained C-terminal human IgG1 Fc domains followed by 6xHis tags.
  • the CD112R-based TCEs were generated by fusing CD112R, CD112R IV , or CD112R IVE to the N-terminus of a single-chain antibody variable fragment (scFv) derived from the anti-CD3 antibody OKT3 via a (GGGGS)4 linker.
  • the TCEs contained a C-terminal Myc tag and 8xHis tag. All of the above CD112R proteins, CD112R variants, or TCEs were expressed by infecting Trichoplusia ni cells (Expression Systems) at a density of 2 ⁇ 10 6 cells/mL with Baculovirus followed by a 72-hour incubation at 27 °C.
  • the proteins were then purified from culture supernatants using nickel and size-exclusion chromatography.
  • ATTORNEY DOCKET NO.10110-461WO1 Protein purity was analyzed by SDS–PAGE using TGX 12% Precast gels (Bio-Rad). All proteins were flash-frozen in liquid nitrogen and stored at ⁇ 80 °C for later use.
  • CD112R protein preps that would be used for crystallography, 5 ⁇ M kifunensine was also added to sensitize the protein for deglycosylation.
  • the purified CD112R ECD were incubated overnight at 4 °C with 1:200 (w/w) endoglycosidase F1, 1:100 (w/w) bovine carboxypeptidase A (Sigma-Aldrich) and 1:200 (w/w) bovine carboxypeptidase B (Sigma- Aldrich) to remove N-linked glycans and disordered residues at the C terminus of the protein, respectively.
  • the enzymatically processed CD112R was mixed with CD112 D1 in 1:1 ratio and applied to a Superdex 200 Increase 10/300 GL column (GE) and fractions corresponding to the co-eluted complex were pooled and concentrated to 14.7 mg/mL. Crystallization and structure determination.
  • Crystals of the concentrated, deglycosylated CD112 D1-CD112R complex were grown by sitting drop vapor diffusion. Drops containing 0.1 ⁇ L of protein were combined with 0.1 ⁇ L of mother liquor from JCSG1-4 sparse matrix screens (Qiagen). Diffracting crystals were grown in 0.1M CHES pH 9.5, 20% w/v PEG 8000. Crystals were cryoprotected by adding ethylene glycol to the condition to a final concentration of 25% before plunge freezing in liquid nitrogen. Diffraction data of the crystals were collected at Advanced Photon Source beamline 22- BM.
  • the data sets were collected at the detector at a crystal-to-detector distance ranging from 300 mm with a 1° oscillation angle and an exposure time of 1 second per image. A total of 360 images were collected.
  • the data was indexed, integrated, scaled and merged with XDS.
  • the complex structure was solved by Molecular replacement (MR) in Phenix.
  • the MR model for CD112R was generated with AlphaFold2 using the Colabfold server and we used the previously determined structure of CD112 D1 (PDB ID: 4DFH) as the model for CD112. Following molecular replacement, the structure was subjected to rigid body refinement using Phenix.refine followed by iterative rounds of building and refinement in COOT and Phenix.refine, respectively.
  • the final 2.2 ⁇ -resolution structure of CD112 D1-CD112R has an R work of 20.8% and R free of 24.5%.
  • the complex structure has unmodeled gaps from Ser 121 to Ser 125 and Ser 154 to Leu 172 in CD112R.
  • the extracellular domain of CD112R was cloned into a modified pCT vector as an N-terminal fusion to a c-Myc epitope tag (EQKLISEEDL, (SEQ ID NO: 32) and the yeast cell wall protein Aga2.
  • the plasmid was transformed into EBY100 cells by electroporation and recovered in SD-CAA growth medium at 30 °C.
  • Yeast cultures were allowed to grow overnight and then induced in SG-CAA medium at 20°C for 24-48 hours.
  • ATTORNEY DOCKET NO.10110-461WO1 After a 48-hour induction, expression of CD112R was detected by flow cytometer with Alexafluor-488-conjugated Myc antibody (Cell Signaling) or Alexafluor-647-conjugated Myc antibody (Cell Signaling).
  • Mutant library generation To generate all three libraries (the interface library, velcro library, and unbiased library), we transformed 50 ⁇ g of insert DNA and 10 ⁇ g of linearized pCT vector DNA into electrocompetent yeast as previously described.
  • the inserts each contained 40-50 bp overlaps with the ends of the vector DNA to allow for in vivo recombination of the vector and insert.
  • 9 interface residues were selected for mutagenesis, and then 12 overlapping primers with degenerate codons at the desired positions were used to perform assembly PCR (SEQ ID NOS: 15-29).
  • Flanking primers (CD112Rlib_F1 and CD112Rlib_R6, SEQ ID NOS: 15-29) were used to introduce the 40-50 bp overhangs between the library insert and the linearized vector.
  • the theoretical diversity of the interface library is 6x10 7 , and we obtained 1x10 8 transformants following electroporation.
  • velcro library To generate the velcro library, we used a primer containing five NNK codons upstream of the mature CD112R N-terminus to amplify the CD112R ECD gene (SEQ ID NOS: 15-29). The Velcro peptide-containing insert was subjected to a second round of PCR amplification using the flanking primers described above. The theoretical diversity of the velcro library is 3.2x10 6 and we obtained 5x10 7 transformants. The unbiased CD112R IV mutant library was generated through PCR amplification of the CD112R IV gene from the pCT vector.
  • PCR was performed using an error-prone polymerase (Genemorph II kit, Agilent) and the CD112R IV and modified pCT vector overhang flanking primers (SEQ ID NOS: 15-29).
  • SEQ ID NOS: 15-29 modified pCT vector overhang flanking primers
  • Yeast display library selections. For each round, a number of yeasts representing >5- to 10-fold excess of the library diversity were pelleted and used for selections. Yeast were pre- washed with HBM buffer (20mM HEPES+ 150 mM NaCl+0.1% bovine serum albumin plus 5mM maltose to prevent yeast flocculation).
  • yeast were washed with HBM buffer and mixed with 250 ⁇ L SA-coated magnetic microbeads (Miltenyi Biotech), incubated for 30 minutes, and then flowed over a magnetic-activated cell sorting (MACS) LS column (Miltenyi Biotech) to remove non-specific binders.
  • MCS magnetic-activated cell sorting
  • yeast were incubated with 100 nM SA-647 for 30 min, washed, incubated ATTORNEY DOCKET NO.10110-461WO1 for 30 min with 50 ⁇ M of anti-647 microbeads (Miltenyi), washed, and then flowed through an LS column to remove non-specific binders. Interface library.
  • 250 ⁇ L SA-coated microbeads were coated with biotinylated CD112 to a final concentration of 450 nM. The beads were then mixed with the negatively selected yeast in a 10 ml volume of HBM incubated for 2 hours at 4°C.
  • the yeasts were washed with HBM buffer and passed through the MACS LS column.
  • the column-bound yeasts were then recovered in SD-CAA media for 24 hours and then induced in SG-CAA media for 24-48 hours prior to the round 2 selection.
  • the second selection was performed using a 100nM concentration of CD112 tetramers. Tetramers were formed by mixing biotinylated CD112 with SA-647 in a 4.5: 1 ratio followed by a 10-minute incubation on ice. After a 2-hour incubation at 4 Co, yeast were washed and then incubated with 50 ⁇ L anti-647 microbeads (Miltenyi Biotech) for 30 minutes.
  • yeast were washed and CD112 binders were captured by flowing the mixture over a magnetic LS column. This selection process was then repeated for 3 additional rounds using CD112 protein concentrations of 250 nM, 50 nM and 5nM, respectively. Velcro library.
  • the positive selection for round 1 was performed as described above for the interface library.
  • yeast were incubated with 1 ⁇ M biotinylated CD112 for two hours, washed and incubated with SA-647 (1:50 dilution) for 30 minutes. After 30 minutes the yeast was washed and incubated with 50 ⁇ L anti-647 microbeads (Miltenyi Biotech) for 30 minutes.
  • yeast were washed and flowed over the LS column to isolate binders. This selection process was repeated for 3 rounds at 20nM, 15nM and 5nM concentrations of CD112, respectively. Sequencing of yeast clones following selections. Upon completion of selections, yeast isolated from the final round of selection from each library were plated on SD-CAA plates. Individual colonies from the libraries were picked, cultured in SD-CAA, and induced in SG- ATTORNEY DOCKET NO.10110-461WO1 CAA. Plasmids from the selected clones were extracted using the Zymoprep Yeast Plasmid Miniprep II Kit (Zymo Research) and sequenced to identify affinity-enhancing mutations. SPR binding studies.
  • the SPR experiment was performed on a BiacoreT200 instrument (GE Healthcare). Approximately 150 resonance units (RUs) of biotinylated CD112R WT , CD112R IV and CD112R IVE were immobilized on streptavidin-coated sensor chips (series S SA-coated sensor chip; GE healthcare). An increasing concentration of recombinant His-tagged CD112 was flowed over the chip in HBS buffer (HEPES) supplemented with 0.005% P20 surfactant at 20°C. Binding and dissociation phases were performed at 45 ⁇ L/min for 120s and 120s, respectively. The chip was regenerated after every injection with 30 second washes of 0.5 MgCl2+10% Ethylene glycol.
  • HBS buffer HBS buffer
  • the curves were reference-subtracted from the flow cell containing negative control protein (extracellular domain of human carbonic anhydrase IX).
  • the maximum RU for each experiment was normalized to 100 RU and plotted as a function of concentration using PRISM 9 (GraphPad).
  • Steady-state binding curves were fitted using T200 BIAcore evaluation software (Cytiva) to a 1:1 Langmuir model to determine K D .
  • Competition assay measuring CD112R variant inhibition of CD112-CD112R interactions.
  • the extracellular domain of CD112 (D1) was cloned into a modified pCT vector as an N-terminal fusion to myc epitope tag and the yeast cell wall protein Aga2.
  • the plasmid was transformed into EBY100 cells by electroporation, recovered in SD-CAA and induced in SG-CAA.
  • CD112R tetramers 200 nM were formed by mixing biotinylated CD112R with SA- 647 at a 4.5:1 ratio on ice for 10 minutes. During the incubation, CD112R IV -Fc and CD112R IVE -Fc proteins were serially diluted from 1 ⁇ M to 0.5nM.
  • CD112R tetramers were mixed 1:1 with the serially diluted CD112R IV -Fc CD112R IVE -Fc proteins, resulting in final concentration of CD112R 100nM tetramer plus 500nM to the CD112R variants.
  • CD112R tetramer binding to the yeast was then detected by flow cytometry. Flow cytometry acquisitions were performed on a BD Accuri analyzer unless specified otherwise, and analyzed with FlowJo software (BD Biosciences). Cell Line and Crispr-Cas9 gene targeting.
  • MDA-MB-468 (ATCC HTB-132) cell line was acquired from ATCC, authenticated by short tandem repeat (STR), and regularly tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection kit (Lonza).
  • Cells were grown at 37°C, 5% CO2 with RPMI-1640 Medium supplemented with 2mM L- glutamine, 1X MEM Non-Essential Amino Acids, 1X Sodium Pyruvate, 55 ⁇ M 2- Mercaptoethanol, 1X Penicillin- Streptomycin (from Gibco), and 10% fetal bovine serum ATTORNEY DOCKET NO.10110-461WO1 (FBS, GeminiBio).
  • GFP green fluorescent protein
  • FFLuc green fluorescent protein-firefly luciferase
  • MDA-MD-468 cells were transfected with the 10 ⁇ l Neon transfection system (Invitrogen) using 2 pulses at 1400 V and 20ms width.
  • the efficiency of the gene deletion was at least 95% as measured by flow cytometry after surface staining with PE anti-human CD112 (clone TX31, Biolegend) or PE/Dazzle 594 anti-human CD155 (clone SKII.4, Biolegend) (FIG. S7A).
  • Flow cytometry was performed on a LSRII flow cytometer using FACSDiva software (BD Biosciences) at the Moffitt Cancer Center Flow Cytometry Core. MDA-MB-468 cell line staining.
  • MDA-MB-468 cells from each cell variant (CD112+ CD155+; CD112- CD155+; CD112+ CD155-; CD112- CD155-) were stained with 1 ⁇ M biotinylated recombinant CD112R, CD112R IV and CD112R IVE for 60min at 4°C, washed 2 times, followed by staining with SA-647. Binding was detected by flow cytometry. Peripheral blood mononuclear cell and T cell isolation. De-identified healthy donor Buffy Coats were obtained from Lifesouth Community Blood Centers.
  • PBMC Peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • DMSO dimethyl sulfoxide
  • T cells were isolated from PBMC using EasySep Human T Cell Isolation Kit (Stemcell) following the manufacturer’s instructions and cultured in RPMI-1640 Medium supplemented with 2mM L-glutamine, 1X Penicillin- Streptomycin, and 10% FBS (cRPMI) at 37°C, 5% CO2.
  • RPMI-1640 Medium supplemented with 2mM L-glutamine, 1X Penicillin- Streptomycin, and 10% FBS (cRPMI) at 37°C, 5% CO2.
  • cRPMI FBS
  • TCE activity 2x10 5 T cells were co-cultured with 8x10 4 MDA-MB-468 cells (2.5:1 E:T ratio) in 200ul cRPMI, and the different TCE were added to the media in the concentrations indicated in the FIG.. After 48hr, supernatant was collected and kept at -80°C for cytokine measurement. Cells were incubated with Bright-Glo Luciferase Assay System (Promega) for 5min to allow complete cell lysis and the lysed mixture was then transferred into a plate compatible with luminescence measurements. Relative luminescence intensity (RLI) was measured in a GloMax Discover ATTORNEY DOCKET NO.10110-461WO1 microplate reader (Promega).
  • the percentage of cytotoxicity was calculated as: 100- (RLI sample/ RLI control* 100), where the control was target cells without effector cells.
  • PVRIG is Expressed on Stem-Like T Cells in Dendritic Cell–Rich Niches in Tumors and Its Blockade May Induce Immune Infiltration in Non-Inflamed Tumors. Cancer Immunol. Res.12, 876–890 (2024). • Bailey, S. R., Vatsa, S., Larson, R. C., Bouffard, A. A., Scarf ⁇ , I., Kann, M. C., Berger, T. R., Leick, M. B., Wehrli, M., Schmidts, A., et al. Blockade or Deletion of IFN ⁇ Reduces Macrophage Activation without Compromising CAR T-cell Function in Hematologic Malignancies.
  • Nectin ectodomain structures reveal a canonical adhesive interface. Nat. Struct. Mol. Biol.19, 906–915 (2012). • He, C., Mansilla-Soto, J., Khanra, N., Hamieh, M., Bustos, V., Paquette, A. J., Garcia Angus, A., Shore, D. M., Rice, W. J., Khelashvili, G., et al. CD19 CAR antigen engagement mechanisms and affinity tuning. Sci. Immunol.8, eadf1426 (2023). • Ho, C. C. M., Guo, N., Sockolosky, J. T., Ring, A.
  • Poliovirus receptor (PVR)-like protein cosignaling network new opportunities for cancer immunotherapy. J. Exp. Clin. Cancer Res.40, 267 (2021).
  • ATTORNEY DOCKET NO.10110-461WO1 • Xu, F., Sunderland, A., Zhou, Y., Schulick, R. D., Edil, B. H. & Zhu, Y. Blockade of CD112R and TIGIT signaling sensitizes human natural killer cell functions. Cancer Immunol. Immunother.66, 1367–1375 (2017). • Xu, F., Sunderland, A., Zhou, Y., Schulick, R. D., Edil, B. H. & Zhu, Y.
  • ATTORNEY DOCKET NO.10110-461WO1 SEQUENCES SEQ ID. NO: 1 > CD112R WT TPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIR QWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDP GLSAPPTPAPILRADL SEQ ID.

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Abstract

Disclosed are compositions and methods for the treatment of cancer through immune checkpoints inhibitors. CD112R is being presented as a new immune checkpoint inhibitor with high potential in cancer immunotherapy and cancer prognosis. Also disclosed herein are methods of treating cancer in a subject by administering an isolated receptor or target binding molecule such as CD112R extracellular domain variant that binds CD112 or administering a pharmaceutical composition comprising CD112R extracellular domain variant to a subject with a cancer.

Description

ATTORNEY DOCKET NO.10110-461WO1 REPROGRAMMING THE CD112-CD112R AXIS FOR OPTIMIZED CANCER IMMUNOTHERAPY CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of, U.S. Provisional Patent Application No. 63/548,722, filed February 01, 2024, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government Support under Grant No. R35GM133482 awarded by the National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING The sequence listing submitted on February 03, 2025, as an .XML file created on January 30, 2025, is entitled “10110-461WO1_ST26.xml”, and is 48,039 bytes in size is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). FIELD The present disclosure relates to harnessing the potential of CD112-CD112R axis in cancer immunotherapy. BACKGROUND Immune checkpoint blockade (ICB) has revolutionized the landscape of cancer therapy, although a substantial number of patients either fail to respond or acquire resistance to these treatments. Current ICB therapies focus on PD-1 or CTLA-4 and have a response rate of 20- 30%, which has spurred the development of “next generation” inhibitors targeting receptors such as LAG3 and TIGIT. The FDA’s approval of Ipilimumab, an antibody targeting cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), is considered a milestone in cancer treatment and has led to substantial patient outcomes unresectable or metastatic melanoma. Subsequently, pembrolizumab and nivolumab, antibodies that block programmed cell death protein-1 (PD- 1), were effectively used to treat a variety of tumors, such as renal cell carcinoma (RCC), advanced melanoma, non-small-cell lung cancer (NSCLC), breast cancer, and advanced ATTORNEY DOCKET NO.10110-461WO1 hepatocellular carcinoma. Furthermore, the anti-PD-1 drugs demonstrated better survival rates than conventional therapies when used for cancer immunotherapy. However, a substantial number of cancer patients either failed to respond to these single-agent immune checkpoint inhibition therapeutics or demonstrated an initial response followed by acquired resistance. Effector T cells exhibit the exhaust phenotype and malfunction within the tumor microenvironment (TME). Dysfunctional T cells have increased numbers of co-inhibitory receptors, such as CTLA-4 and PD-1, on their surfaces. Based on these facts, identifying new immune checkpoint pathways might improve the immunotherapy response rates and broaden the efficacy of the treatment provided. Given the limitations described above, there is a need to explore new therapies that target other co-inhibitory receptor pathways. SUMMARY Disclosed here are isolated receptors of CD112 and CD112 binding molecules and methods of their use in the treatment, inhibition, reduction, amelioration, and/or prevention of cancer. Disclosed herein are isolated receptors or target binding molecules that binds CD112, wherein the isolated receptor or target binding molecule (such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells) comprises a CD112R extracellular domain variant. Also disclosed herein are isolated receptors or target binding molecules of any preceding aspect, wherein the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N-terminal end of SEQ ID NO: 1. In one aspect, disclosed herein are isolated receptors or target binding molecules of any preceding aspect, wherein the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 50 (such as, for example, a methionine to lysine substitution (M50K)), 92 (such as, for example, a histidine to glutamine substitution (H92Q)), 138 (such as, for example, a serine to threonine substitution (S138T)), 69 (such as, for example a serine to asparagine (S69N), serine to tyrosine (S69Y), serine to proline (S69P), serine to histidine (S69H), serine to threonine (S69T), serine to aspartic acid (S69D) or serine to alanine (S69A) substitution), 82 (such as, for example, a glycine to histidine (G82H), glycine to arginine (G82R), glycine to asparagine (G82N), glycine to serine (G82S), glycine to aspartic acid (G82D), glycine to proline (G82P), glycine to threonine (G82T) or glycine to alanine (G82A) substitution), 83 (such as, for example, an alanine to glycine (A83G), alanine to ATTORNEY DOCKET NO.10110-461WO1 threonine (A83T), alanine to asparagine (A83N), alanine to aspartic acid (A83D) or alanine to serine (A83S) substitution), 90 (such as, for example, a valine to leucine (V90L), valine to alanine (V90A), valine to isoleucine (V90I), valine to proline (V90P), valine to threonine (V90T), valine to histidine (V90H), valine to asparagine (V90N) or valine to aspartic acid (V90D) substitution), and/or 143 (such as, for example, a serine to threonine (S143T), serine to histidine (S143H), serine to asparagine (S143N), serine to arginine (S143R), serine to proline (S143P), serine to isoleucine (S143I) or serine to leucine (S143L) substitution) of SEQ ID NO: 1. For example the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 2); or a M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 5). Also disclosed herein are isolated receptors or target binding molecules of any preceding aspect wherein the isolated receptor binds an epitope on tumor cells comprising CD112. In one aspect, disclosed herein are isolated receptors or target binding molecules of any preceding aspect, wherein the CD112R is a human CD112R. Also disclosed herein are pharmaceutical compositions comprising the isolated receptor or target binding molecule of any preceding aspect, and pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition can comprise at least one additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB. Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis (such as for example, breast cancer including, but not limited to triple negative breast cancer) in a subject in need thereof, comprising administering to a subject an effective amount of the isolated receptor, target binding molecules, or pharmaceutical compositions of any preceding aspect. For example, disclosed herein are methods of treating cancer in a subject in need thereof, comprising administering to a subject an isolated receptor or target binding molecule that binds CD112, wherein the isolated receptor or target binding molecule (such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells) comprises a CD112R extracellular domain variant or administering to the subject a pharmaceutical composition comprising said isolated receptor or target binding molecule. In some aspects, the pharmaceutical composition can further comprise at least one ATTORNEY DOCKET NO.10110-461WO1 additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB. In one aspect, disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect, wherein the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N-terminal end of SEQ ID NO: 1. Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect, wherein the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 50 (such as, for example, a methionine to lysine substitution (M50K)), 92 (such as, for example, a histidine to glutamine substitution (H92Q)), 138 (such as, for example, a serine to threonine substitution (S138T)), 69 (such as, for example a serine to asparagine (S69N), serine to tyrosine (S69Y), serine to proline (S69P), serine to histidine (S69H), serine to threonine (S69T), serine to aspartic acid (S69D) or serine to alanine (S69A) substitution), 82 (such as, for example, a glycine to histidine (G82H), glycine to arginine (G82R), glycine to asparagine (G82N), glycine to serine (G82S), glycine to aspartic acid (G82D), glycine to proline (G82P), glycine to threonine (G82T) or glycine to alanine (G82A) substitution), 83 (such as, for example, an alanine to glycine (A83G), alanine to threonine (A83T), alanine to asparagine (A83N), alanine to aspartic acid (A83D) or alanine to serine (A83S) substitution), 90 (such as, for example, a valine to leucine (V90L), valine to alanine (V90A), valine to isoleucine (V90I), valine to proline (V90P), valine to threonine (V90T), valine to histidine (V90H), valine to asparagine (V90N) or valine to aspartic acid (V90D) substitution), and/or 143 (such as, for example, a serine to threonine (S143T), serine to histidine (S143H), serine to asparagine (S143N), serine to arginine (S143R), serine to proline (S143P), serine to isoleucine (S143I) or serine to leucine (S143L) substitution) of SEQ ID NO: 1. For example the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 2); or a M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 5). Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect, wherein the isolated receptor binds an epitope on tumor cells comprising of CD112. ATTORNEY DOCKET NO.10110-461WO1 In one aspect, disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect, wherein the CD112R is a human CD112R. BRIEF DESCRIPTION OF FIGURES The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. FIG. 1 shows CD112R and CD112 expression in health and tumor microenvironment. In normal human peripheral blood cell subsets, CD112R is predominantly expressed in NK cells and CD8+ T cells, but not in CD4+ T cells, DCs, neutrophils, monocytes and B cells. CD112R+CD8+ T cells are mainly detected in effector memory T (CD45RA−CCR7−) cells in phenotype, and rarely in naïve T (CD45RA+CCR7+) cells. In the tumor microenvironment, CD112R is expressed abundantly in NK cells, CD4+ T cells and CD8+ T cells. Meanwhile, CD112 was detectable primarily in DCs, tumor-associated macrophages (TAMs) or monocytes, and tumors cells. FIG. 2 represents TCGA cancer types with corresponding GTEx normal tissues. The GTEx normal and TCGA normal are combined. The gene expression unit is log2(TPM+1). FIG. 3 shows a molecule of CD112R and 1 molecule of CD112 D1 crystallographic unit. CD112 D1 and CD112R have 9 antiparallel β-strands. FIG.4 shows complex structure design of Interface remodeling and Velcro engineering libraries to isolate a CD112R mutant with high affinity for CD112 ligand. FIG. 5 represents a new variant as CD112RIV which is a result of combining all the mutations from Interface and Velcro library. FIG. 6 represents staining in MDA-MB-468 triple negative breast cancer cell line. A binding test is performed where cell lines are stained with CD112RWT, CD112RIV and TIGIT extracellular domain recombinant proteins. CD112R is the dominant receptor of CD112 ligand and TIGIT (second receptor) also binds to CD112 but with very low affinity. CD112RIV binds specifically with CD112 ligands only. FIG.7 depicts affinity maturation of the CD112RIV mutant to enhance affinity. Random mutagenesis resulted in a panel of CD112R mutants with different affinities. FIG.8 represents CD112RIV and CD112RIVE as the two best binders out of the 9 clones. FIG.9 depicts a combined mutation table from CD112RIV affinity maturation. FIG.10 shows a flowchart of engineering the CD112R mutants. ATTORNEY DOCKET NO.10110-461WO1 FIG. 11 shows CD112 is a ligand in the PVR-like protein network. (A) Schematic depicting known interactions between PVR-like proteins. (B) CD112 is overexpressed in several cancers (TCGA RNAseq database) compared to corresponding normal tissues (GTEx and TCGA RNAseq databases combined). FIG. 12 shows purification of CD112R, TIGIT, and CD112 ECDs. Representative chromatograms show that all three proteins elute from the SEC column as monodisperse peaks, which is indicative of favorable biochemical behavior. Protein purity was evaluated by SDS PAGE (insets). FIG. 13 depicts CD112R binding disrupting CD112 homodimerization. CD112 and CD112R were purified individually or in complex on a SEC column. CD112 eluted as a dimer, CD112R eluted as a monomer, and CD112:CD112R eluted as a 1:1 complex. Stoichiometries were estimated based on MW standards. FIG. 14 represents a comparison of experimentally determined CD112-CD112R structure to published CD112-TIGIT structure. (A) Crystal structure of CD112R (purple) bound to CD112 D1 (beige). (B) Crystal structure of TIGIT (pink) bound to CD112 D1 (beige, PDB 5V52). In both (A) and (B), inset zoom windows depict the lock and key motif at the binding interface. (C) Table comparing the binding interfaces of CD112-CD112R and CD112- TIGIT. FIG. 15 shows the structure-guided engineering of CD112R mutant libraries. (A) The interface library was generated by mutating residues at the CD112-CD112R binding interface (red) as indicated in the table. (B) The velcro library was generated by introducing a randomized 5 amino acid peptide insertion (red) at the N-terminus of CD112R. FIG. 16 depicts CD112RIV consensus variant binds to CD112 with high affinity. Mutations (red spheres) from the interface and velcro libraries were combined to generate CD112RIV. The yeast expressing CD112RWT or CD112R variants were stained with a fixed concentration (250 nM) of fluorescently labeled CD112 and binding was detected by flow cytometry. FIG.17 depicts specific binding of CD112RIV to CD112+ TNBC cells. CD112RIV binds to the CD112-expressing TNBC cell line MDA-MB-468, but not to cells lacking CD112. Knockout of CD155 did not affect CD112RIV binding. In both the parent cell line and the CD155 knockout line, CD112RIV bound more robustly than CD112RWT. Cells were stained with 1uM biotinylated CD112RWT or CD112RIV and fluorescently labeled streptavidin (SA- AF647), and binding was detected by flow cytometry. ATTORNEY DOCKET NO.10110-461WO1 FIG. 18 represents CD112 blockade increases CAR-T cell cytotoxicity. (A) CAR-T cells were co-cultured with CD112+ MDA-MB-468 cells in the presence of IgG1 isotype control or Fc-CD112RIV at a 1:1 E: T ratio. After 24 hours, killing was measured by luciferase assay. (B) CAR-T cells showed surface expression of CD112R detected by flow cytometry in resting TnMUC1.28z CAR-T cells. FIG.19 shows CD112R variants as soluble inhibitors construct design. FIG. 20 shows CD112R library design and selection strategy. (A) Flow chart and structural representation of the CD112R affinity-maturation scheme. The interface library was generated by mutating hotspot residues at the CD112-CD112R interface (red spheres), the velcro library was generated by introducing a randomized 5 amino acid peptide insertion at the N-terminus of CD112R (orange spheres), and the unbiased library was generated using error- prone PCR (cyan spheres). Mutations that were isolated from each library are indicated on the structures. (B) Yeast expressing CD112 D1 were stained with a fixed concentration (100 nM) of biotinylated CD112R or CD112R variants and binding was analyzed by flow cytometry. FIG. 21 shows comparison of CD112-CD112R and CD112-TIGIT structures. (A) TIGIT (pink) and CD112R (light purple) ectodomains from CD112-bound structures were superimposed and shown in cartoon representation to compare their binding modes. D1 of CD112 is shown in surface representation (beige). The right panels indicate the CD112R- and TIGIT-binding interfaces. The table below compares various structural parameters for each interface. (B) A sequence alignment of the CD112-binding regions of CD112R and TIGIT. Interface residues (purple for CD112R, pink for TIGIT) and lock/key motifs are indicated on the alignments. FIG. 22 shows molecular interactions at THE CD112R/CD112 interface. Cartoon representation of the CD112R-CD112 structure (left) and a table indicating contact residues between the two proteins (right). The zoom panels highlight various H-bonds, salt bridges, and van der Waals interactions listed in the table. H-bonds and salt bridges are shown as dotted lines, and H-bond cutoffs were defined as contact distances of less than 3.5 Å. FIG. 23 shows selection strategy for CD112R libraries. (A) Flow charts depict the selection strategies used to isolate high-affinity CD112 variants from the interface, velcro, and unbiased libraries. Red arrows and text indicate negative selections. (B) The gating strategy used to isolate yeast singlets is shown (bottom). The gate in the SSC/FSC plot (left) was drawn to select for the bulk yeast population, and these yeast were again gated in the FSCH/FSCA plot (middle) to isolate singlets. Yeast singlets were then analyzed based on surface expression ATTORNEY DOCKET NO.10110-461WO1 (FL-1, staining with myc-488 antibody) and CD112 (FL-4, staining with biotinylated CD112 bound to SA-647). FIG.24 shows expression yield of CD112R and CD112R variants. SDS-PAGE analysis of CD112R or CD112R variants purified from 1 mL of insect cell supernatant following infection with recombinant Baculovirus (left). The table (right) indicates the yield of CD112R, CD112RIV, or CD112RIVE from 1L of infected insect cell cultures. FIG. 25 shows AlphaFold3 model of CD112RIVE bound to D1 of CD112. The Alphafold3 model of CD112RIVE (light purple, cartoon representation) bound to D1 of CD112 (beige surface representation with positive/negative charges colored). Zoom windows highlight mutations from the interface (red), velcro (orange), and unbiased (cyan) libraries. FIG. 26 shows CD112R variants have increased binding to recombinant CD112 and CD112-expressing cells. (A) SPR was used to determine the binding affinities of CD112R, CD112RIV, and CD112RIVE. Site-specifically biotinylated CD112R proteins were immobilized on a Streptavidin (SA)-coated sensor chip and 3-fold dilutions of the full-length CD112 ECD were injected over the surface, starting at a concentration of 20,000 nM. Representative SPR sensograms from a single experiment are shown, and the KD and s.d. were determined by averaging the values from two-independent experiments. (B) Wild-type, CD112 knockout, CD155 knockout, and double CD112/CD155 knockout MDA-MB-468 cells were stained with CD112R variants to assess binding and specificity. The cells were stained with 1 µM biotinylated CD112R, CD112RIV, or CD112RIVE or SA-647 alone (Control) followed by staining with Alexa Fluor 647-labeled SA (SA-647), and binding was detected by flow cytometry. The graph represents the median fluorescent intensity (MFI) and s.d. of n=3 replicates of one independent experiments. FIG. 27 shows MDA-MB-468 TNBC cell line variants utilized for functional assays. (A) Histograms depict flow cytometry characterization of CD112 (left) and CD155 (right) surface expression on the parental and variant cells generated by Crispr-Cas9 KO of CD112 and CD155 (B) Gating strategy used to analyze MDA-MB-468 cell line staining with soluble CD112R variants. FIG.28 shows CD112R variants are effective CD112 traps and CD112-targeting T cell engagers. (A) A competition assay was used to measure the blockade of CD112-CD112R interactions by CD112RIV and CD112RIVE. Yeast expressing CD112 were stained with a fixed 100nM tetramer concentration of fluorescently labeled CD112R in the presence of increasing concentrations of CD112RIV or CD112RIVEFc fusion proteins, and CD112-CD112R binding was monitored by flow cytometry. (B) Illustration of TCEs incorporating CD112R variants in ATTORNEY DOCKET NO.10110-461WO1 co-culture with MDA-MB-468 cells and T cells. (C) Tumor cell killing post 48-hour treatment with CD112R variant TCEs added to co-cultures of donor T cells and MDA-MB-468 cells (E:T= 2.5:1). (D) IFNγ production was measured after 48-hour TCE treatment co-culture (E:T=2.5:1). In A, C, and D, graphs are representative of one of three independent experiments. FIG.29 shows TCE purification and analysis of TCE antigen specificity. (A) Overlaid size exclusion chromatograms show the purification of CD112R, CD112RIV and CD112RIVE TCEs from a size-exclusion column. (B) IFNγ production following 10 nM TCE treatment of donor T cells and MDA-MB-468 (CD112- CD155+ and CD112+ CD155+) co-cultures (E:T=2.5:1). This graph represents n=3 replicates representative of one independent experiment. DETAILED DESCRIPTION The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof. Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Terminology Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, ATTORNEY DOCKET NO.10110-461WO1 the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings: “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation ATTORNEY DOCKET NO.10110-461WO1 "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient. “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. The term “comprising”, and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. Compositions CD112 is part of the PVR-like protein co-signaling network. CD112 (also known as Nectin-2 or PVRL2) is a ligand in the Polio Virus Receptor-like (PVR-like) network of immune co-signaling molecules (FIG. 11A). The transmembrane CD112 protein is predominantly localized to the adherens junction in epithelial cells, but it is also expressed in endothelial cells, neurons, and fibroblasts. Several human tumors overexpress CD112, including breast cancer, ovarian cancer, and pancreatic cancer, making it an intriguing target for immunomodulatory drugs (FIG.11B). Interface remodeling and Velcro engineering libraries were used to isolate a CD112R mutant with high affinity for CD112 ligand. Use of the “Interface library” introduced mutations at interface hotspots to improve surface complementarity to CD112 and have the binding energetics of CD112R. To decipher protein-protein interaction and affinity computational alanine scanning, free energy decomposition and molecular dynamic are used as methods for the determination of individual residue contributions to binding energy. “Interface remodeling” is used to investigate protein interactions between CD112R and CD112 by changing amino acids in the ATTORNEY DOCKET NO.10110-461WO1 CD112R extracellular domain. The resulting variant is a high affinity variant of CD112R which is CD112RIV and CD112RIVE. Design of tailored protein interfaces wherein practical structural and functional modifications are achieved for higher affinity towards CD112 antigen binding. Disclosed herein are isolated receptors or target binding molecules that binds CD112, wherein the isolated receptor or target binding molecule (such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells) comprises a CD112R extracellular domain variant. In some embodiments “Velcro engineered” libraries are assessed for high affinity binding between CD112 variants and CD112 ligand expressed by cancer cells. The Velcro library is designed by introducing a randomized nine-residue peptide extending from the N- terminus of CD112R. Accordingly, also disclosed herein are isolated receptors or target binding molecules, wherein the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N-terminal end of SEQ ID NO: 1. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly under- stood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY 35 AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure. The terms CD112R polypeptide", "CD112R protein" and "CD112R" are used interchangeably herein and refer to polypeptide sequences including homologues and embodiments thereof, capable of binding to a CD112 polypeptide. The CD112R described herein may be isolated from a variety of sources, such as from human or from a nonhuman organism, or prepared by recombinant or synthetic methods. CD112R is a polypeptide capable of binding to CD112 and having at least 90% sequence identity to at least a 10 amino acid consecutive sequence to SEQ ID NO: 1. In embodiments CD112R is a polypeptide capable of binding to CD112 and having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least a 10 amino acid consecutive sequence of SEQ ID NO: 1. All disclosures in this specification which refer to the "CD112R" refer to each of the polypeptides individually as 6 well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the ATTORNEY DOCKET NO.10110-461WO1 disclosure individually. The terms "CD112R polypeptide", "CD112R protein" or "CD112R" also include variants of the CD112R disclosed herein or known in the art. In one aspect, disclosed herein are isolated receptors or target binding molecules, wherein the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 50 (such as, for example, a methionine to lysine substitution (M50K)), 92 (such as, for example, a histidine to glutamine substitution (H92Q)), 138 (such as, for example, a serine to threonine substitution (S138T)), 69 (such as, for example a serine to asparagine (S69N), serine to tyrosine (S69Y), serine to proline (S69P), serine to histidine (S69H), serine to threonine (S69T), serine to aspartic acid (S69D) or serine to alanine (S69A) substitution), 82 (such as, for example, a glycine to histidine (G82H), glycine to arginine (G82R), glycine to asparagine (G82N), glycine to serine (G82S), glycine to aspartic acid (G82D), glycine to proline (G82P), glycine to threonine (G82T) or glycine to alanine (G82A) substitution), 83 (such as, for example, an alanine to glycine (A83G), alanine to threonine (A83T), alanine to asparagine (A83N), alanine to aspartic acid (A83D) or alanine to serine (A83S) substitution), 90 (such as, for example, a valine to leucine (V90L), valine to alanine (V90A), valine to isoleucine (V90I), valine to proline (V90P), valine to threonine (V90T), valine to histidine (V90H), valine to asparagine (V90N) or valine to aspartic acid (V90D) substitution), or 143 (such as, for example, a serine to threonine (S143T), serine to histidine (S143H), serine to asparagine (S143N), serine to arginine (S143R), serine to proline (S143P), serine to isoleucine (S143I) or serine to leucine (S143L) substitution) of SEQ ID NO: 1. For example the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 2); or a M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 5). In an aspect the present disclosure provides a CD112R protein, or a mutant CD112R protein. In embodiments, mutant CD112R is a polypeptide capable of binding to CD112 and having at least 90% sequence identity to at least a 10 amino acid consecutive sequence to SEQ ID NO: 2 or SEQ ID. NO: 5. In embodiments, mutant CD112R is a polypeptide capable of binding to CD112 and having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence. "CD112RWT" refers to naturally occurring receptor molecules with single pass membrane structure. The structure includes one transmembrane spanning region, a long intracellular domain, and a single extracellular immunoglobulin variable-like (IgV) domain encoded inside the CD112R. ATTORNEY DOCKET NO.10110-461WO1 The terms “CD112R” refers to human CD112R present on T cells and NK cells, unless otherwise specifically indicated (e.g. mouse CD112R, cynomolgus CD112R, etc.). The term includes full -length, unprocessed CD112R as well as any form of CD112R that results from processing in the cell. The term encompasses naturally occurring variants of human CD112R, e.g., splice variants allelic variants. External ID's for CD112R gene includes Entrez Gene: 79037, Ensembl: ENSG00000213413, OMIM; 617012, and UniProtKB: Q6DKI7. "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid 50 sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. "Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a target). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., receptor and target). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kn). Affinity can be measured by common methods known in art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following. An "affinity matured" receptor refers to a receptor with one or more alterations in extracellular domain, compared to a parent receptor which does not possess such alterations, such alterations optionally resulting in an improvement in the affinity of the receptor for target. An "Extracellular immunoglobulin variable-like (IgV) domain" refers to the extracellular domain of the 36kD single-pass transmembrane protein CD112R. Also disclosed herein are isolated receptors or target binding molecules, wherein the isolated receptor binds an epitope on tumor cells comprising CD112. In one aspect, disclosed ATTORNEY DOCKET NO.10110-461WO1 herein are isolated receptors or target binding molecules wherein the CD112R is a human CD112R. Also disclosed herein are pharmaceutical compositions comprising the isolated receptor or target binding molecule of any preceding aspect, and pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition can comprise at least one additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB. The current disclosure provides, inter alia, compositions and methods for modulating T-cell mediated immune response in a subject in need thereof. The method includes administering to the subject an effective amount of a CD112R protein, or a mutant CD112R protein of the present disclosure, which are collectively referred to herein as “CD112 targeting molecules ” or “ anti-CD112 binding molecules ” or “ high affinity CD112R mutants” . Methods of Use The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphomas such as B cell lymphoma and T cell lymphoma; mycosis fungoides; Hodgkin’s Disease; myeloid leukemia (including, but not limited to acute myeloid leukemia (AML) and/or chronic myeloid leukemia (CML)); bladder cancer; brain cancer (including, but not limited to brain lower grade glioma); nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer (including, but not limited to kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, and kidney chomophobe); lung cancers such as small cell lung cancer, non- small cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LUAD); neuroblastoma/glioblastoma; adrenocortical carcinoma, ovarian cancer; pancreatic cancer; prostate cancer; skin cancer (including, but not limited to skin cutaneous melanoma); hepatic cancer; melanoma; squamous cell carcinomas of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma (including, but not limited to cecial squamous cell carcinoma and endocervical adenocarcinoma); breast cancer (including, but not limited to triple negative breast cancer and breast invasive carcinoma); genitourinary cancer; pulmonary cancer; esophageal carcinoma; glioblastoma; lymphoid neoplasm diffuse large B- cell lymphoma; head and neck carcinoma; large bowel cancer; hematopoietic cancers; uterine cancer (including, but not limited to uterine corpus endometrial carcinoma); testicular cancer (including, but not limited to testicular germ cell tumors); and colon cancer, and rectal cancers. ATTORNEY DOCKET NO.10110-461WO1 In one aspect, disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis (such as for example, breast cancer including, but not limited to triple negative breast cancer) in a subject in need thereof, comprising administering to a subject an effective amount of the isolated receptor, target binding molecules, or pharmaceutical compositions of any preceding aspect. For example, disclosed herein are methods of treating cancer in a subject in need thereof, comprising administering to a subject an isolated receptor or target binding molecule that binds CD112, wherein the isolated receptor or target binding molecule (such as, for example, a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells) comprises a CD112R extracellular domain variant or administering to the subject a pharmaceutical composition comprising said isolated receptor or target binding molecule. In some aspects, the pharmaceutical composition can further comprise at least one additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB. In one aspect, disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis, wherein the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N-terminal end of SEQ ID NO: 1. Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis, wherein the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 50 (such as, for example, a methionine to lysine substitution (M50K)), 92 (such as, for example, a histidine to glutamine substitution (H92Q)), 138 (such as, for example, a serine to threonine substitution (S138T)), 69 (such as, for example a serine to asparagine (S69N), serine to tyrosine (S69Y), serine to proline (S69P), serine to histidine (S69H), serine to threonine (S69T), serine to aspartic acid (S69D) or serine to alanine (S69A) substitution), 82 (such as, for example, a glycine to histidine (G82H), glycine to arginine (G82R), glycine to asparagine (G82N), glycine to serine (G82S), glycine to aspartic acid (G82D), glycine to proline (G82P), glycine to threonine (G82T) or glycine to alanine (G82A) substitution), 83 (such as, for example, an alanine to glycine (A83G), alanine to threonine (A83T), alanine to asparagine (A83N), alanine to aspartic acid (A83D) or alanine to serine (A83S) substitution), 90 (such as, for example, a valine to leucine (V90L), valine to alanine (V90A), valine to isoleucine (V90I), valine to proline (V90P), valine to threonine (V90T), valine to histidine (V90H), valine to asparagine ATTORNEY DOCKET NO.10110-461WO1 (V90N) or valine to aspartic acid (V90D) substitution), or 143 (such as, for example, a serine to threonine (S143T), serine to histidine (S143H), serine to asparagine (S143N), serine to arginine (S143R), serine to proline (S143P), serine to isoleucine (S143I) or serine to leucine (S143L) substitution) of SEQ ID NO: 1. For example the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 2); or a M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution (including, but not limited to SEQ ID NO: 5). Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect, wherein the isolated receptor binds an epitope on tumor cells comprising of CD112. In one aspect, disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect, wherein the CD112R is a human CD112R. An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant. A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. ATTORNEY DOCKET NO.10110-461WO1 By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control. By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific ATTORNEY DOCKET NO.10110-461WO1 therapy directed toward the improvement of the associated disease, pathological condition, or disorder. "Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject. A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration. "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions ATTORNEY DOCKET NO.10110-461WO1 (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein. “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. ATTORNEY DOCKET NO.10110-461WO1 The term "block," in the context of an interaction between two or more molecules, is used herein to refer to inhibition or prevention of said interaction between the two or more molecules, wherein the inhibition or prevention of said interaction between the two or more molecules is complete or nearly complete under at least one condition. A "nearly complete" inhibition is a percent inhibition of about 70-99.9%, and a "complete" inhibition is 100%. For example, a molecule is said to "block" an interaction between two or more other molecules if it completely or nearly completely inhibits such interaction at certain concentrations in a dose dependent manner. The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. A cancer may be benign (also referred to as a benign tumor), pre-malignant, or malignant. Cancer cells may be solid cancer cells or leukemic cancer cells. The term "tumor" is used herein to refer to a cell or cells that comprise a cancer. The term “tumor growth” is used herein to refer to proliferation or growth by a cell or cells that comprise a cancer that leads to a corresponding increase in the size or extent of the cancer. The term " chimeric” receptor refers to a receptor in which a portion of the extracellular immunoglobulin variable-like (IgV) domain is mutated via random mutations derived from Interface library and Velcro library. The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, ' E, y, and µ, respectively. Administration "in combination with" one or more further therapeutic agents include simultaneous (concurrent) and consecutive (sequential) administration in any order. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, 112s, y9o, Re1s6, R Sm1s3, Bi212, p32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anti-cancer agents disclosed below. ATTORNEY DOCKET NO.10110-461WO1 "Effector functions" refer to those biological activities attributable to the Fe region of an antibody, which vary with the antibody isotype. Examples of receptor effector functions include: Clq binding and complement dependent cyto- toxicity (CDC); Fe receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor/ T cell surface receptor); and T cell activation. An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fe region as defined herein. The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell and may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of human antibody specifically excludes a humanized antibody comprising non- human antigen-binding residues. The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent. The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression ATTORNEY DOCKET NO.10110-461WO1 of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In some embodiments “immune checkpoint receptors” are checkpoint blockade inhibitors used in cancer therapies that target the extracellular domains (ECDs) of immune inhibitory receptors. These successful drugs reverse T cell exhaustion by blocking ligand- mediated activation of receptor signaling. Current invention focuses on novel immunotherapies targeting the next-generation checkpoint protein CD112. In some embodiments, high affinity mutant CD112R are provided. In certain embodiments, the high affinity mutant CD112R shares certain structural and/or functional features. In some embodiments, the high affinity mutant CD112R includes a parent receptor with random mutation induced via Interface or Velcro methods in the extracellular domain and affinity matured thereof. In some embodiments, the affinity matured variants comprise variants CD112IVA3 and CD112IVA5. In some embodiments, the affinity matured variants comprise receptors with random mutations as compared to wild type receptor. In some embodiments, a pharmaceutical composition comprising the variant receptor of CD112R and a pharmaceutically acceptable carrier, wherein the composition optionally comprises an opsonizing agent, a regulatory T cell depleting agent, chemotherapy, and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB. In one embodiment, the variant receptor of CD112R or pharmaceutical composition is used for treating cancer in a subject, wherein the cancer is optionally carcinoma, lymphoma, blastoma, sarcoma, or leukemia, or wherein the cancer is optionally squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulva, cancer, thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer, testis cancer, cholangiocarcinoma, gallbladder carcinoma, gastric cancer, melanoma, or various types of head and neck cancer (including squamous cell carcinoma of the head and neck). In one embodiment, a pharmaceutical composition comprising the variant receptor of CD112R for use in treating cancer, wherein the use further comprises administering a second ATTORNEY DOCKET NO.10110-461WO1 therapy, wherein the second therapy is optionally radiotherapy, surgery or administration of a second agent, wherein the second agent is optionally an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3 or TIM-3, or an antagonist of TIGIT or CD96, or an antagonist of PVRLl, PVRL2, PVRL3, PVRL4, and CD155, or is an antagonist of CD47, or is an antagonist of CD39, or is an antagonist of IL-27, or is STING agonist, wherein the second agent is optionally an antagonist receptor. In some aspects, the second agent can be an agonist for a stimulatory receptor on a T cell including, but not limited to OX40L (such as, for example, OX40), CD40L (such as, for example, CD40), CD28 (such as, for example, CD80/CD86), Inducible costimulatory (ICOS)(such as, for example, ICOS ligand), lymphocyte function associated antigen 1 (LFA-1) (such as, for example, intracellular adhesion molecule 1 (ICAM-1)), and T cell immunoglobulin and mucin domain 1 (TIM-1) (such as, for example, TIM-4), 4-1BB (such as, for example, 4-1BBL). A method of producing the variant receptor of CD112 comprises culturing the yeast cells of under conditions wherein the receptor is expressed, optionally further comprising purifying the receptor. In some embodiments, the additional therapeutic agent or second agent is a chemotherapeutic agent, an opsonizing agent, a regulatory T cell ("Treg") depleting agent, an antagonist of a target other than CD112R, or an agonist of a target other than CD112R. In certain embodiments, the second agent is a chemotherapeutic agent described herein or any known chemotherapeutic agent. In some embodiments, the second agent is an opsonizing agent, wherein the opsonizing agent is an antibody other than an anti-CD112R antibody that targets cancer or tumor cells. In some embodiments, the second agent is a Treg depleting agent described herein or any known Treg depleting agent. In some embodiments, the second agent is an antagonist of a target other than CD112R. In some embodiments, the second agent is an agonist of a target other than CD112R. In certain embodiments, a variant of CD112R provided herein is a multispecific T cell engager, e.g. a bispecific T cell engager. In certain embodiments, one of the binding specificities is for CD112 and the other is for any other target. In certain embodiments, one of the binding specificities is for CD112 and the other is for selected independently from one (in the case of bispecific) or more (in the case of multispecific) of OX40, CD28, CD40L, LFA-1, ICOS, 4-1BB, PD-1, PD-Ll, CTLA-4, Lag- 3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, STING, CD47, CD39, and IL-27. In certain embodiments, bispecific antibodies may bind to two different epitopes of ATTORNEY DOCKET NO.10110-461WO1 CD112R. Bispecific T cell engagers (BiTEs) may also be used to localize cytotoxic agents to cells which express CD112. BiTEs can be prepared as full-length receptor or fragments thereof. In certain embodiments, amino acid sequence variants of CD112R provided herein are contemplated. For example, it is desirable to improve the binding affinity and/or other biological properties of the receptor. Amino acid sequence variants of the receptor may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the receptor, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the receptor. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., target binding. In certain embodiments, antibody variants having one or more amino acid substitutions are provided. In embodiments, one or more bispecific T-cell engagers of the disclosure are used in combination with one or more anti-CD112 binding antibodies, one or more cancer vaccines, Freund's Adjuvant, one or more CAR-T cell therapies, one or more stem cell therapies, and/or one or more scFv therapies. It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane),ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, ATTORNEY DOCKET NO.10110-461WO1 BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil--Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clofarabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR-U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil--Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EVACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil--Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® ATTORNEY DOCKET NO.10110-461WO1 (Fluorouracil--Topical), Fluorouracil Injection, Fluorouracil--Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE- OXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL- 2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado- Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, ATTORNEY DOCKET NO.10110-461WO1 MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride) , MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R- EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R- ATTORNEY DOCKET NO.10110-461WO1 ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil--Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate) , UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VeIP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XTANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron ATTORNEY DOCKET NO.10110-461WO1 Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and/or ZYTIGA® (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD- 1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT-011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR- 1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep). EXAMPLES The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that are apparent to one skilled in the art. Example 1: CD112 is part of the PVR-like protein co-signaling network. CD112 is part of the PVR-like protein co-signaling network. CD112 (also known as Nectin-2 or PVRL2) is a ligand in the Polio Virus Receptor-like (PVR-like) network of immune co-signaling molecules (FIG. 11A). The transmembrane CD112 protein is predominantly localized to the adherens junction in epithelial cells, but it is also expressed in endothelial cells, neurons, and fibroblasts. Several human tumors overexpress CD112, including breast cancer, ovarian cancer, and pancreatic cancer, making it an intriguing target for immunomodulatory drugs (FIG. 11B). CD112 was first characterized as a ligand for the immunostimulatory receptor CD226 and the immunosuppressive receptor TIGIT. However, the protein CD112R (formerly known as PVRIG) was recently determined to be the dominant receptor for CD112 ATTORNEY DOCKET NO.10110-461WO1 (FIG.11A). This assertion was supported by surface plasmon resonance (SPR) binding studies that revealed that CD112R-CD112 interactions are > 100-fold higher affinity than CD112 interactions with CD226 or TIGIT. Mechanistically, CD112 engagement of CD112R induces the phosphorylation of ITIM motifs in the CD112R intracellular domain (ICD), initiating a signaling cascade that inhibits T cell function by suppressing expression of the transcriptional activator NFAT. In addition to CD112, CD112R, TIGIT, and CD226, the PVR-like protein network includes the inhibitory receptor CD96, and the TIGIT/CD96-activating ligand CD155 (FIG.11A). Example 2: Determine structures of CD112 bound to inhibitory (CD112R) and activating receptors (CD226), and quantitatively measure the binding affinities between all PVR- like proteins. Establish the molecular basis for CD112 interactions with activating and inhibitory receptors. Several proteins in the PVR-like network are promiscuous and form cis or trans interactions to regulate immune signaling. For example, structural studies have revealed that TIGIT dimerizes upon engagement of CD155 homodimers to form 2:2 heterotetramers, whereas TIGIT binding disrupts CD112 dimerization and forms a 1:1 complex. TIGIT has also been shown to inhibit CD226 signaling through the formation of cis TIGIT-CD226 complexes on the cell surface. Compared to TIGIT, little information about the CD112R interactions is known in the larger context of the PVR-like network, and there are no structures available of CD112R alone or in complex with CD112 to indicate how CD112R becomes activated. More broadly, understanding how the molecular assemblies of PVR-like proteins activate or inhibit signaling illuminates new opportunities for the development of highly selective inhibitors. In this Aim, it is determined as to how CD112 binds and activates the inhibitory CD112R receptor as well as the activating CD226 receptor. In parallel, a PVR-like interactome binding study was performed to quantitatively measure the affinities between all possible protein-protein interactions. Preliminary Data Purification of recombinant receptor and ligand ECDs. The ECDs of CD112, CD112R, and TIGIT were purified to facilitate the structural and biophysical studies. These domains contain disulfide bonds and N-linked glycans, so they are not amenable to expression in prokaryotic systems such as E. coli. To ensure the proteins were properly folded, the constructs were expressed in insect (T. Ni) cells using recombinant Baculovirus. Each protein was purified ATTORNEY DOCKET NO.10110-461WO1 from cell culture supernatants using nickel affinity and size-exclusion chromatography (SEC). CD112, CD112R, and TIGIT all eluted from the size-exclusion column as monodisperse peaks, which is indicative of favorable biochemical behavior (FIG. 12). To reconstitute a CD112- CD112R complex, an excess of CD112R was added to CD112 and injected the mixture onto an SEC column. The two proteins co-eluted from the column, indicating that they form a stable interaction. Previous structural studies have shown that CD112 forms homodimers, and that TIGIT binding to CD112 breaks this homodimer to form a 1:1 complex. To test whether CD112R similarly disrupts CD112 dimerization, analytical SEC was utilized to estimate the molecular weight (MW) of CD112 and in complex with CD112R. CD112 homodimers (76.8 kD) have a larger predicted MW than the CD112-CD112R complex (54 kD), a rightward shift corresponding to a decrease in MW was observed if CD112R binding disrupts homodimerization. A right shift in the peak was observed when CD112 was co-purified with CD112R, indicating that both TIGIT and CD112R disrupt CD112 dimerization upon binding (FIG.13). Crystal structure of CD112 D1 bound to CD112R. It was determined that the initiating step in CD112R signaling was to visualize the crystal structure of a CD112-CD112R complex. To probe for crystallization conditions, the ECD of CD112R was co-purified, and the receptor-binding D1 domain of CD112 and subjected the proteins to sparse matrix screening. Optimized crystals of the complex diffracted to 2.2 Å resolution and the structure was solved by molecular replacement. Following multiple rounds of building and refinement, the Rwork/Rfree were 0.20/0.24 (FIG. 14A), which is within the publishable range for a 2.2 Å resolution structure. Analysis of the structure revealed that the CD112R ECD is a prototypical Ig-like fold that interacts with CD112 D1 (also an Ig domain) in an antiparallel orientation (FIG. 14A). As opposed to Ig-antigen interactions, which are dominated by CDR loops, it was found that CD112R interacts with the core beta-sheet of CD112 through a lock-and-key motif that has been characterized in CD155-TIGIT and CD112- TIGIT complexes (FIG. 14A and 14B). The binding mode of CD112-CD112R resembles that of a previously determined CD112-TIGIT structure. However, the CD112- CD112R complex is more extensive both concerning buried surface area (884 Å2 vs 790 Å2) and polar contacts (8 H-bonds, 2 salt bridges vs 9 H-bonds, 0 salt bridge) (FIG. 14B and 14C). This analysis provides a structural explanation for the much higher reported binding affinity of CD112 for CD112R (~88 nM KD) compared to TIGIT (>5 μM KD). ATTORNEY DOCKET NO.10110-461WO1 The 2.2 Å-resolution structure of CD112R bound to D1 of CD112 was determined to establish the molecular determinants of CD112R-CD112 interactions (FIG. 14A and SEQ ID NOS: 15-29). The crystal unit cell contained one copy of CD112R and one copy of CD112D1, which is consistent with the 1:1 stoichiometry estimated from SEC purification of the complex (FIG. 14A). The antiparallel binding mode of CD112R-CD112 resembles those adopted by other PVR-like receptor-ligand pairs, including TIGIT-CD112 (FIG. 21A) and a recently published structure of CD112R-CD112. The interacting domains of PVR-like proteins contain complementary lock (T/S, F/Y, P) and key (AX6G) motifs that stabilize the binding interface (FIG.21B). In the SFP key motif of CD112R, the aromatic residue, F139, inserts into a concave hydrophobic pocket formed by the lock motif (AAFHPKMG (SEQ ID NO: 30), residues 83 to 90) of CD112 (FIG.14A). Likewise, residue F145 of the CD112 “TFP” key motif also inserts into the hydrophobic lock motif of CD112R (AVLHPERG (SEQ ID NO: 13), residues 89 to 96) (FIGS. 14A and 21B). Apart from the lock-and-key binding regions, CD112R interacts with CD112 via a broad interface centered around the β-sheet formed by the C, D, G, and H strands (FIG.22). Structure-guided engineering of high-affinity CD112R variants. Soluble receptor ECDs compete with endogenous receptors for ligand binding, making them appealing low-molecular weight alternatives to monoclonal antibodies. However, the moderate affinity (~500nM) of the CD112R ECD limits its utility for biomedical applications. To address this problem, we engineered high-affinity CD112R variants using structure-guided directed evolution. Based on our analysis of the CD112R-CD112 complex, we utilized two distinct affinity-maturation strategies: interface remodeling and “velcro” engineering. For the first strategy, we designed an “interface library” by conservatively mutating CD112R hotspot residues that contact CD112. The second “velcro library” was generated by inserting a randomized, 5-residue peptide at the N-terminus of CD112R. This approach is feasible when the N- or C-terminus of one protein is adjacent to its binding partner and has previously been used to strengthen CD47-SIRPα and TCR-MHC interactions. In the case of CD112R-CD112, the velcro peptide is predicted to enhance binding by creating new contacts with a patch of CD112 surface residues in proximity to the CD112R N-terminus (FIG.14A). To design the CD112R interface library we introduced conservative mutations at nine hotspot residues (S69, L72, G82, A83, T87, V90, A137, F139 and S143) that contact CD112 (FIG.15A). The library was generated using assembly PCR with degenerate primers and has a theoretical diversity of 6x107 unique clones. Purified library DNA was then transformed into ATTORNEY DOCKET NO.10110-461WO1 yeast such that the number of transformants was > 6x107 to ensure full coverage of the sequence space. We performed five rounds of selections using progressively decreasing concentrations of CD112 to isolate high-affinity CD112R variants (FIG. 23A). Sequencing of the enriched library revealed recurring S69N, G82H, A83G, V90L and S143T mutations in multiple clones, and the CD112R ‘interface variant’ containing these mutations was named CD112RI (FIGS. 22A and 8). To generate the velcro library, we introduced a 5-residue peptide at the N-terminus of CD112R using degenerate NNK codons that encode for all possible amino acids (FIG. 15A). We then transformed the library into yeast and performed and six rounds of selections against CD112 (FIG. 21A). Sequencing revealed two distinct velcro peptide sequences: LNVRQ and WDPSM. Because that the “LNVRQ” sequence was more abundant, we selected this CD112R velcro variant (CD112RV) for further characterization (FIGS.22A and 8). After completing the selections above, we performed two additional steps to improve binding affinity. First, we engrafted the LNVRQ peptide onto the CD112RI scaffold to generate a consensus variant containing interface and velcro mutations (CD112RIV). The gene encoding CD112RIV was then subjected to error-prone PCR to generate an unbiased mutant library. We hypothesized that this approach could indirectly enhance affinity by stabilizing the global protein fold or the β-sheet comprising the binding interface. After four rounds of yeast selections, we identified three additional affinity-enhancing mutations (M50K, H92Q and S138T) from the unbiased mutant library. The variant containing the interface, velcro, and error-prone PCR mutations was named CD112RIVE (FIGS.22A, 25 and 8). To purify the recombinant CD112R ECDs, we expressed CD112R and each variant in insect cells using Baculovirus. Although the yield of each protein was sufficient for downstream experiments, we observed that the CD112IV and CD112RIVE variants had particularly high levels of expression. To quantitatively measure the expression levels, we purified CD112R, CD112RIV, and CD112RIVE from 1L cultures of Baculovirus-infected insect cells. Following the purification, the yields of CD112R, CD112RIV, and CD112RIVE were 3 mg/L, 8 mg/L, and 13 mg/L, respectively (FIG.24). These data suggest that the CD112RIV and CD112RIVE mutations are biochemically well-tolerated and may have serendipitously improved protein folding or secretion. Yeast expressing CD112 with recombinant CD112R, CD112RI, CD112RV, CD112RIV or CD112RIVE was stained to directly compare the binding affinities of each variant (FIG.20B). It was found that the binding level was highest for CD112RIVE, followed by CD112RIV, CD112RI, CD112RV, and CD112R. These data indicate that (i) the interface and velcro ATTORNEY DOCKET NO.10110-461WO1 mutations had an additive effect on binding, (ii) the interface mutations had a greater effect on binding than the velcro peptide, and (iii) mutations isolated from the unbiased library further increased CD112-binding affinity. An AlphaFold 3 model of the CD112RIVE-CD112 complex was generated to investigate how various mutations increase CD112RIVE binding affinity (FIG.25). The predicted interface buried surface area of the model was increased by ~70 Å2 compared to the CD112R-CD112 complex structure, and mutated residues contribute both hydrophobic and hydrophilic contacts. Three mutations, V90L, S138T, and S143T, are predicted to create new van der Waals contacts at the interface through their gain of an additional side chain carbon atom (FIG. 25). On the other hand, S69N, G82H, and H92Q gained bulky or charged side chains that may engage in polar contacts with opposing CD112 residues (FIG. 25). The M50K and A83G mutations do not directly affect the interface but may improve protein solubility or favor the receptor-bound conformation, respectively. Lastly, the velcro peptide was predicted to make only glancing contacts with CD112, which is consistent with its relatively minor effect on binding affinity (FIGS.22B and 25). Determine the structure of a CD112-CD226 complex. CD112 transduces inhibitory or activating signals depending on the receptor subtype it engages. Here, the crystal structure of CD112 bound to the activating receptor CD226 was determined to directly compare the structural features of each class of receptor. In contrast to the ECD CD112R, which contains only a single Ig domain, CD226 contains two tandem Ig domains that have each been implicated in ligand binding. Therefore, the D1-D2 region of CD226 was co-purified with the full ECD (D1-D2-D3) of CD112 for use in the structural studies. Co-crystallization and structure determination were carried out as described above for CD112-CD112R. The structure was analyzed to assess how multidomain interactions, receptor assembly, and ligand-induced conformational changes contribute to signal transduction. Perform a comparative structural analysis of CD112 signaling complexes. The structures of CD112-CD112R and CD112-CD226 were determined and a detailed computational analysis was performed to identify conserved and receptor-specific features of the binding interfaces. This data ultimately led to improved selectivity in drugs targeting conserved epitopes of this receptor family, as there is a high degree of conservation among CD112-receptor binding interfaces. Given that there is also a CD112-TIGIT structure available, this therapeutically important checkpoint receptor complex was included in the analysis. ATTORNEY DOCKET NO.10110-461WO1 Previous structural studies suggest that the lock-and-key motif in D1 is the principal determinant of receptor-ligand selectivity. However, the contribution of D2 of CD226 in ligand interactions suggests that additional structural features are important for signaling through activating receptors. The investigation focuses on how specific signatures in the key motif can confer receptor-ligand compatibility. The results of these analyses were then coupled with the biophysical studies to define binding and signaling hierarchies among the full CD112-CD155- CD96-CD112R-TIGIT-CD226 axis. Biophysically characterize interactions among members of the CD112-CD155-CD96- CD112R- TIGIT-CD226 axis. CD112 and CD155 interact with multiple receptors to induce activating and inhibitory signals. The binding affinities of these interactions have been individually measured using various techniques, but the full interactome has not been systematically characterized using a single standard methodology. As CD112R was only recently discovered, it was important to understand whether binding partners outside of CD112 regulate its function. There is also growing evidence that both cis- and trans-interactions can influence immune checkpoint signaling. Therefore, surface plasmon resonance (SPR) was used to determine the kinetics and affinity of interactions among CD112, CD155, CD96, CD112R, TIGIT, and CD226. In the SPR experiment, individual proteins were immobilized on sensor chips and then the full gamut of recombinant proteins flowed over the surface. The curves were analyzed to determine the dissociation constants as well as kinetic parameters such as kon, koff, and binding half-life (t1/2). This study revealed important information about crosstalk between ligands and checkpoint receptors, which can in turn be coupled with the structural analysis to understand how conserved and divergent features confer selectivity. Biophysical characterization of CD112R variants. Surface plasmon resonance (SPR) was used to quantitatively measure the binding affinities of CD112 and CD112R, CD112RIV and CD112RIVE (FIG.26). Recombinant CD112R ECDs were injected over a sensor chip coated with CD112, and the curves were fitted to calculate the dissociation constant (KD) of each interaction. Wild-type CD112R, CD112RIV, and CD112RIVE bound CD112 with KD values of 526.0 nM, 77.0 nM and 36.9 nM, respectively. Compared to CD112R, the affinity was enhanced by ~7-fold for CD112RIV and ~14-fold for CD112IVE. The rapid dissociation of wild-type CD112R from CD112 precluded kinetic fitting. However, inspection of the SPR sensograms suggest that the affinity ATTORNEY DOCKET NO.10110-461WO1 improvement in each variant is largely due to a decrease in off-rate (FIG. 26). Our measured CD112R-CD112 KD (526 nM) differs from that of a previous binding study measuring CD112 interactions with a CD112R-Fc fusion protein (88nM) .We attribute this difference to avidity effects from the dimeric Fc tag, especially since our measurements are in agreement with a more recent study reporting a KD of 500nM between monomeric CD112R and CD112. Although CD112R variants have increased affinity for CD112, it is unclear whether their mutations affect binding to the CD112 paralog CD155. To address this question, we used a cell-based binding assay to evaluate the CD112RIV and CD112RIVE binding specificity. We selected the triple negative breast cancer (TNBC) cell line MDA-MB-468 for this study, as it expresses high levels of both CD112 and CD155 (FIG.27A). We then generated three variants of this cell line through CRISPR/Cas9 knockout of CD112, CD155, or both ligands (CD112- CD155+, CD112+ CD155- and CD112- CD155-, respectively). We then compared the binding of biotinylated CD112R, CD112RIV and CD112RIVE to each variant. We found that CD112RIVE and CD112RIV bound more robustly to cells expressing CD112 (CD112+ CD155+ and CD112+ CD155-) compared to CD112R, which is consistent with their higher affinities. On the other hand, none of the variants bound appreciably to cells that did not express CD112 (CD112- CD155+ and CD112-CD155-) (FIGS. 28B and 29B). Collectively, these data suggest that CD112RIV and CD112RIVE binding is highly selective for CD112. Soluble CD112R variants inhibit CD112-CD112R interactions. A competition assay was performed to test the variants’ ability to inhibit CD112- CD112R interactions. Yeast expressing CD112 were stained with a fixed concentration (100nM) of fluorescently labeled CD112R tetramers in the presence of increasing concentrations of CD112RIV and CD112RIVE Fc-fusion proteins, and binding was detected by flow cytometry. In this format, tetramers were used to enhance CD112R avidity because monomeric CD112R binding to CD112 was undetectably weak. We observed a dose- dependent inhibition for both variants, with IC50 of 41.9 and 24.5 nM for CD112RIV-Fc, and CD112RIVE-Fc respectively. Notably, the dimeric Fc-fusion proteins blocked CD112 interactions with higher-avidity CD112R tetramers, suggesting that they may have even greater inhibitory potency than could be measured in this assay (FIG.28A). Incorporation of CD112R variants into CD112-targeting TCEs. In contrast to checkpoint inhibitors, which block T cells from receiving immunosuppressive signals, TCEs function by tethering T cells to tumor antigens while ATTORNEY DOCKET NO.10110-461WO1 activating their T cell receptors (TCRs). We hypothesized that the enhanced affinity of CD112RIV and CD112RIVE would enable them to function as effective binding modules in CD112-targeting TCEs. We generated three TCEs by fusing CD112R, CD112RIV, or CD112RIVE to an anti-CD3 single-chain antibody variable fragment (scFv) using a flexible (GGGGS)4 linker (SEQ ID NO: 31) (FIGS.30B and 31A). To characterize the activity of these TCEs, human T cells isolated from healthy donor peripheral blood mononuclear cells (PBMCs) were co-cultured with CD112-expressing MDA-MB-468 at a 2.5:1 ratio in the presence of each TCE. After 48hs, tumor cell killing (FIG.28C) and interferon gamma (IFNγ) levels (FIG.28D) were evaluated using a Luciferase-based assay and ELISA, respectively. We found that the wild-type CD112R TCE mediated tumor cell killing only at high concentrations with minimal IFNγ secretion. On the other hand, the TCEs incorporating the CD112RIV and CD112RIVE variants each triggered a dose-dependent activation of T cells and both had greatly increased potency compared to the CD112R TCE. The highest-affinity CD112RIVE variant exhibited the highest potency, both in terms of tumor cell killing and IFNγ secretion. When the co-cultures were performed with MDA-MB-468 cells that did not express CD112, the TCEs did not trigger IFNγ production, highlighting the specificity of the response (FIG.28B). These results indicate that high-affinity CD112R variants can function as effective TCE binding modules and outperform the wild-type CD112R ECD in both potency and efficacy. Example 3: To engineer high-affinity CD112R decoys and assess their ability to enhance T cell function via blockade of CD112 signaling. Clinical development of TIGIT and CD112R inhibitors. TIGIT and CD112R are the most potent immunosuppressive receptors in the PVR-like protein network, and both TIGIT and CD112R inhibitors are under development as cancer immunotherapies. Pre-clinically, it has been shown that co-administration of anti-TIGIT and anti-CD112R antibodies boosts NK and CD8⁺ T cell function. In clinical trials, TIGIT or CD112R inhibitors are most commonly co-administered or given in combination with PD-1 inhibitors or chemoradiotherapy. Several TIGIT antagonist antibodies are now in phase 3 trials for the treatment of non-small cell lung cancer (NSCLC), esophageal cancer, and squamous cell carcinoma, and a CD112R inhibitor recently entered phase 2 trials for the treatment of a variety of solid tumors. Thus far, these agents have been associated with minimal toxicity and the success rate of several trials is pending. By contrast, no antibodies targeting CD112 are under clinical development despite its role as a dual TIGIT/CD112R ligand, and the lack of a viable CD112 inhibitor is a notable void in the toolkit of PVR-like protein-modulating drugs. ATTORNEY DOCKET NO.10110-461WO1 Evolve high-affinity CD112R decoys and assess their ability to enhance T-cell responses. Although monoclonal antibodies (MW ~150 kD) are the most prevalent class of biologic drug, a variety of lower molecular weight proteins have emerged as clinically viable alternatives. Camelid-derived nanobodies and soluble receptor ECDs (also known as ligand traps) have become increasingly popular due to their favorable biochemical properties. The smaller size of these proteins is associated with increased tissue penetrance and thermostability, and their single-chain structure allows for more straightforward genetic manipulation. Nanobodies and ligand traps have already been approved by the FDA for the treatment of thrombotic thrombocytopenic purpura and myelodysplastic syndrome, and several nanobodies and ligand traps are under active clinical development. In this Aim, structure-guided engineering was utilized to generate high-affinity CD112R variants that function as potent CD112 traps. The ability of these lower molecular weight inhibitors was tested to enhance the antitumor activity of CAR-T cells in TNBC. Blockade of CD112 was especially beneficial given that CD112 signals through both major inhibitory receptors (CD112R and TIGIT) in the PVR-like family, and it was anticipated that CD112 traps boost the efficacy of T cell therapy against a wide range of CD112-expressing tumors. Preliminary Data Development of yeast display libraries targeting the CD112-CD112R binding interface. The yeast display was used to evolve CD112R variants with enhanced binding to CD112. The yeast display affinity-maturation process begins with the generation of a mutant library of the target protein (CD112R). The library is then stained with a fluorescently labeled ligand (CD112) and subjected to several rounds of MACS or FACS to enrich for high-affinity binders. The high-resolution CD112-CD112R structure allowed us to take a structure-guided approach to CD112R library generation. For the first library (the “interface library”), conservative mutations were introduced at interface hotspots to improve surface complementarity and bind energetics (FIG. 15A). For the second library (the “velcro library”), a randomized 5-residue peptide insertion extending from the N-terminus of CD112R (FIG. 15B) was introduced. Structural analysis revealed that the N-terminus of CD112R was adjacent to the CD112 binding interface, so the introduction of this velcro-like peptide is predicted to strengthen interactions by creating additional interface contacts. Following yeast transformation, the interface and peptide libraries each contained >10⁷ unique variants were determined. ATTORNEY DOCKET NO.10110-461WO1 Selection of high-affinity CD112R variants from yeast display libraries. To isolate high-affinity CD112R variants, the libraries above were subjected to several rounds of MACS or FACS selection against the recombinant CD112 ECD. Decreasing concentrations of CD112 were used in subsequent rounds to increase stringency and retain only the highest affinity binders. Once selections were completed, individual clones were sequenced to identify affinity-enhancing mutations. Sequencing of individual clones revealed that the most abundant interface library variant (CD112RI) contained S69N, G82H, A83G, V90L, and S143T mutations and that the most abundant velcro library variant (CD112RV) contained an “LNVRQ” insertion at the N-terminus. These two mutants were selected for further characterization. Design and purification of a high-affinity CD112R consensus variant. A high-affinity CD112R interface/velcro consensus was generated variant (CD112RIV) by combining the mutations from the interface and peptide libraries onto a single CD112R sequence. The previous structural analysis did not predict any clashes between the peptide extension and native binding interface, so it was hypothesized that combining mutations from the two libraries should further increase binding affinity. The binding of CD112R, CD112RV, CD112RI, and CD112RIV in a fluorescence-based yeast staining assay (FIG.16) was compared. It was found that CD112RIV bound to CD112 more robustly than CD112R, CD112RV, or CD112RI, suggesting that combining the interface and velcro mutations had an additive effect on binding affinity (FIG.16). CD112RIV was then cloned into a Baculovirus expression vector as a fusion to human IgG1 Fc (Fc- CD112RIV) and purified from insect cells as described herein. The yield from the purification was approximately 8mg/L, which is sufficient for downstream in vivo experiments. Characterization of the binding specificity of CD112RIV on a TNBC cell line. To generate a model to study the antigen specificity of the novel CD112R variants, MDA-MB-468 was selected as a representative TNBC cell line that expresses high levels of CD112 and CD155. Different MDA-MB-468 variants were generated through CRISPR/Cas9 knock-out of CD112 and CD155 genes, resulting in a bulk cell population with around 95% loss of protein surface expression as detected by flow cytometry. The binding of wild-type CD112R (CD112RWT) and CD112RIV to these cells was compared. CD112RIV showed higher binding to CD112+ CD155+ cells than CD112RWT, and the binding was abrogated in the ATTORNEY DOCKET NO.10110-461WO1 absence of CD112 (CD112- CD155+) (FIG.17). In contrast, the binding was not affected when cells lacked CD155 (CD112+ CD155-), indicating high specificity of CD112RIV for CD112. Affinity-mature CD112R variants using an unbiased mutagenesis strategy. Receptor-ligand affinity was altered directly through modifications to interface residues, or indirectly through allosteric effects or global stabilization. To identify affinity- enhancing mutations that are not easily predicted through structural analysis, an unbiased mutant library of CD112RIV using error-prone PCR was generated. The library was selected against decreasing concentrations of fluorescently labeled CD112 to isolate additional affinity- enhancing mutations. Clones isolated from the library were purified and dissociation constants were determined by SPR as described herein. Together with the previous variants, these ultra- potent binders were contributed to a diverse panel of CD112R variants that can “affinity-tune” CD112-targeted inhibitors and CSRs for maximal efficacy. Assess the ability of CD112R variants to inhibit CD112 interactions with CD112R. SPR- and cell-based competition assays were utilized to evaluate the efficacy of the CD112R variants as CD112 inhibitors. In the SPR format, CD112R was immobilized on the surface of a sensor chip and then flowed over a fixed concentration of CD112 (approximately 100 nM) to obtain a baseline binding curve. Increasing concentrations of CD112RV, CD112RI, or CD112RIV were titrated. The decrease in binding signal was monitored as concentrations of the variants increased, and the maximum signal at each inhibitor concentration was plotted and fitted to obtain an IC50 for each variant. In the cell-based format, the CD112ʰ^ TNBC cell line MDA-MB-468 (FIG.17) was used to evaluate the CD112R decoys. The cells were stained with a fixed 50 nM concentration of fluorescently CD112R tetramers alone or in the presence of increasing concentrations of inhibitor, and then the binding was detected by flow cytometry. The mean fluorescence intensity (MFI) of binding at each inhibitor concentration was plotted and fitted to obtain IC50 values. CONCLUSION In the present study, it is shown that CD112RIV and CD112RIVE function as soluble CD112 traps and TCEs. Different binding affinities are associated with distinct functional properties. Several studies have now demonstrated that CAR single-chain variable fragments (scFvs) with moderate affinities have optimal therapeutic efficacy. In part, this is because this reduced affinity helps to minimize off-target toxicity and T cell exhaustion from sustained ATTORNEY DOCKET NO.10110-461WO1 receptor activation. It was also recently shown that IFNγ signaling is important for CAR T cell killing of solid, but not liquid, tumors, likely due to the increased adhesion of CAR T cells to tumors following IFN-γ stimulation. In CD112R-based TCEs, it is found that wild-type CD112R induced very low levels of tumor killing. This finding agrees with previous studies showing that higher affinity target-binding arms are associated with improved TCE efficacy. On the other hand, the increased affinity of CD112RIV and CD112RIVE TCEs drove high levels of IFN-γ. Therefore, affinity-optimization should be considered for the development of TCEs and other isolated receptors incorporating engineered receptor ECDs. In addition to their enhanced affinity, we found that both CD112RIV and CD112RIVE had greatly increased expression yields compared to the wild-type CD112R ECD. Yeast display selections isolate clones with the highest levels of bound ligand, irrespective of whether this binding is due to an increase in affinity, or the number of receptors displayed on the yeast. Therefore, we speculate that increased purification yields are a serendipitous byproduct of our selections enriching for both CD112 binding and surface expression. Additionally, the inclusion of a velcro peptide at the N-terminus of the CD112R protein may facilitate more efficient cleavage of the signal peptide, leading to increased levels of secretion. Regardless, these data suggest that yeast display affinity-tuning may have unexpected manufacturing benefits beyond their increased binding potency. Among immune checkpoint proteins, the Nectin and PVR-like families have been under intense scrutiny due to the large number of clinical studies of TIGIT and CD112R inhibitors. Previous studies have shown that inhibitory CD112R signaling mediated by CD112 does not overlap with inhibitory CD155-TIGIT signaling, so combination therapies focused on the dual or triple blockade of CD112R, TIGIT, and PD-1 may be the most promising approaches. Despite the dual role of CD112 as a ligand for both CD112R and TIGIT, there are still no CD112-targeting therapies in clinical studies. This study outlines the development of affinity- tuned CD112-targeting biologics to fill this void in our pharmacological toolkit. CD112 expression profile indicates that it is often overexpressed in cancer, including ovarian, breast, pancreatic and colorectal cancers, compared to normal tissues. However, lower levels of CD112 are also expressed on healthy cells, so careful evaluation of toxicity due to CD112 inhibition, or T cell killing of CD112+ cells will be important for optimal therapeutic development. Affinity-tuned biologics can provide one possible approach to mitigating such toxicities in future studies. MATERIALS AND METHODS ATTORNEY DOCKET NO.10110-461WO1 Protein expression and purification. Human CD112 D1 (residues 32-160) [uniprot: Q92692] was cloned into a pET26b(+) vector containing a N-terminal pelB periplasmic signal peptide and a C-terminal 6xHis tag. The recombinant protein was expressed in Escherichia coli strain BL21 (DE3). Cells were grown to optical density 600 nm of 0.6 to 0.8 and expression was induced through the addition of 0.4 mM isopropyl β-D thiogalactoside (IPTG), followed by an overnight incubation at 16 ºC. Following induction, the cells were harvested by centrifugation and the pellet was stored at -20 ºC for one hour. The frozen pellet was then thawed, and the pellet was resuspended in SET buffer (0.5 M sucrose, 0.5 mM EDTA and 0.2 M Tris pH 8.0), stirred for 30 minutes, then an equal volume of buffer containing 150 nM NaCl, and 20 mM MgCl2 and 2µL benzonase, was added and the mixture was stirred for an additional 45 minutes. The lysate was then centrifuged at 20,000g for 20 mins and the protein was purified from the supernatant using nickel affinity (Nickel-NTA resin, Qiagen) chromatography followed by size-exclusion chromatography (SEC) on an Superdex 75 Increase 10/300 GL column (GE). Prior to purification on the size-exclusion column, the protein was treated with bovine carboxypeptidase A (1:100) and bovine carboxypeptidase B (1:200) to remove the C- terminal 6xHis tag. The full-length CD112 ECD (D1 to D3), the CD112R ECD, CD112R variants, and CD112R-based TCEs were all expressed in insect cells using Baculovirus. All proteins were cloned into the pAcGp67A Baculovirus transfer vector and containing a N-terminal gp67 signal peptide, however, the C-terminal tags varied based on the individual protein purified. The ECD of CD112 (D1 to D3, residues 32 to 350) and the ECD of human CD112R (residues 41-172) [Uniprot ID: Q6DKI7] contained a C-terminal 8xHis tag. The ECDs of CD112R, CD112RI, CD112RV, CD112RIV, and CD112RIVE were modified to include a C-terminal biotin acceptor peptide (BAP) tag upstream of their C-terminal 6xHis tag for site-specific labeling with biotin. CD112RIV and CD112RIVE Fc fusion proteins contained C-terminal human IgG1 Fc domains followed by 6xHis tags. The CD112R-based TCEs were generated by fusing CD112R, CD112RIV, or CD112RIVE to the N-terminus of a single-chain antibody variable fragment (scFv) derived from the anti-CD3 antibody OKT3 via a (GGGGS)4 linker. The TCEs contained a C-terminal Myc tag and 8xHis tag. All of the above CD112R proteins, CD112R variants, or TCEs were expressed by infecting Trichoplusia ni cells (Expression Systems) at a density of 2 × 106 cells/mL with Baculovirus followed by a 72-hour incubation at 27 °C. The proteins were then purified from culture supernatants using nickel and size-exclusion chromatography. ATTORNEY DOCKET NO.10110-461WO1 Protein purity was analyzed by SDS–PAGE using TGX 12% Precast gels (Bio-Rad). All proteins were flash-frozen in liquid nitrogen and stored at −80 °C for later use. For CD112R protein preps that would be used for crystallography, 5 µM kifunensine was also added to sensitize the protein for deglycosylation. The purified CD112R ECD were incubated overnight at 4 °C with 1:200 (w/w) endoglycosidase F1, 1:100 (w/w) bovine carboxypeptidase A (Sigma-Aldrich) and 1:200 (w/w) bovine carboxypeptidase B (Sigma- Aldrich) to remove N-linked glycans and disordered residues at the C terminus of the protein, respectively. The enzymatically processed CD112R was mixed with CD112 D1 in 1:1 ratio and applied to a Superdex 200 Increase 10/300 GL column (GE) and fractions corresponding to the co-eluted complex were pooled and concentrated to 14.7 mg/mL. Crystallization and structure determination. Crystals of the concentrated, deglycosylated CD112 D1-CD112R complex (described above) were grown by sitting drop vapor diffusion. Drops containing 0.1µL of protein were combined with 0.1µL of mother liquor from JCSG1-4 sparse matrix screens (Qiagen). Diffracting crystals were grown in 0.1M CHES pH 9.5, 20% w/v PEG 8000. Crystals were cryoprotected by adding ethylene glycol to the condition to a final concentration of 25% before plunge freezing in liquid nitrogen. Diffraction data of the crystals were collected at Advanced Photon Source beamline 22- BM. The data sets were collected at the detector at a crystal-to-detector distance ranging from 300 mm with a 1° oscillation angle and an exposure time of 1 second per image. A total of 360 images were collected. The data was indexed, integrated, scaled and merged with XDS. The complex structure was solved by Molecular replacement (MR) in Phenix. The MR model for CD112R was generated with AlphaFold2 using the Colabfold server and we used the previously determined structure of CD112 D1 (PDB ID: 4DFH) as the model for CD112. Following molecular replacement, the structure was subjected to rigid body refinement using Phenix.refine followed by iterative rounds of building and refinement in COOT and Phenix.refine, respectively. The final 2.2 Å-resolution structure of CD112 D1-CD112R has an Rwork of 20.8% and Rfree of 24.5%. The complex structure has unmodeled gaps from Ser121 to Ser125 and Ser154 to Leu172 in CD112R. Yeast display of CD112R. The extracellular domain of CD112R was cloned into a modified pCT vector as an N-terminal fusion to a c-Myc epitope tag (EQKLISEEDL, (SEQ ID NO: 32) and the yeast cell wall protein Aga2. The plasmid was transformed into EBY100 cells by electroporation and recovered in SD-CAA growth medium at 30 °C. Yeast cultures were allowed to grow overnight and then induced in SG-CAA medium at 20°C for 24-48 hours. ATTORNEY DOCKET NO.10110-461WO1 After a 48-hour induction, expression of CD112R was detected by flow cytometer with Alexafluor-488-conjugated Myc antibody (Cell Signaling) or Alexafluor-647-conjugated Myc antibody (Cell Signaling). Mutant library generation. To generate all three libraries (the interface library, velcro library, and unbiased library), we transformed 50 µg of insert DNA and 10 µg of linearized pCT vector DNA into electrocompetent yeast as previously described. The inserts each contained 40-50 bp overlaps with the ends of the vector DNA to allow for in vivo recombination of the vector and insert. To generate the CD112R interface library insert, 9 interface residues were selected for mutagenesis, and then 12 overlapping primers with degenerate codons at the desired positions were used to perform assembly PCR (SEQ ID NOS: 15-29). Flanking primers (CD112Rlib_F1 and CD112Rlib_R6, SEQ ID NOS: 15-29) were used to introduce the 40-50 bp overhangs between the library insert and the linearized vector. The theoretical diversity of the interface library is 6x107, and we obtained 1x108 transformants following electroporation. To generate the velcro library, we used a primer containing five NNK codons upstream of the mature CD112R N-terminus to amplify the CD112R ECD gene (SEQ ID NOS: 15-29). The Velcro peptide-containing insert was subjected to a second round of PCR amplification using the flanking primers described above. The theoretical diversity of the velcro library is 3.2x106 and we obtained 5x107 transformants. The unbiased CD112RIV mutant library was generated through PCR amplification of the CD112RIV gene from the pCT vector. The PCR was performed using an error-prone polymerase (Genemorph II kit, Agilent) and the CD112RIV and modified pCT vector overhang flanking primers (SEQ ID NOS: 15-29). We obtained 1x108 transformants following co- electroporation of the vector + insert. Yeast display library selections. For each round, a number of yeasts representing >5- to 10-fold excess of the library diversity were pelleted and used for selections. Yeast were pre- washed with HBM buffer (20mM HEPES+ 150 mM NaCl+0.1% bovine serum albumin plus 5mM maltose to prevent yeast flocculation). The selections were then done in two steps: (1) a negative selection to remove non-specific binders to SA-coated MACS beads, and (2) a positive selection to isolate binders to CD112. For all round 1 negative selections, yeast were washed with HBM buffer and mixed with 250 µL SA-coated magnetic microbeads (Miltenyi Biotech), incubated for 30 minutes, and then flowed over a magnetic-activated cell sorting (MACS) LS column (Miltenyi Biotech) to remove non-specific binders. For negative selections in subsequent rounds, yeast were incubated with 100 nM SA-647 for 30 min, washed, incubated ATTORNEY DOCKET NO.10110-461WO1 for 30 min with 50 µM of anti-647 microbeads (Miltenyi), washed, and then flowed through an LS column to remove non-specific binders. Interface library. For the round 1 positive selection, 250 µL SA-coated microbeads were coated with biotinylated CD112 to a final concentration of 450 nM. The beads were then mixed with the negatively selected yeast in a 10 ml volume of HBM incubated for 2 hours at 4°C. After 2 hours, the yeasts were washed with HBM buffer and passed through the MACS LS column. The column-bound yeasts were then recovered in SD-CAA media for 24 hours and then induced in SG-CAA media for 24-48 hours prior to the round 2 selection. The second selection was performed using a 100nM concentration of CD112 tetramers. Tetramers were formed by mixing biotinylated CD112 with SA-647 in a 4.5: 1 ratio followed by a 10-minute incubation on ice. After a 2-hour incubation at 4 Cº, yeast were washed and then incubated with 50 µL anti-647 microbeads (Miltenyi Biotech) for 30 minutes. Finally, the yeast were washed and CD112 binders were captured by flowing the mixture over a magnetic LS column. This selection process was then repeated for 3 additional rounds using CD112 protein concentrations of 250 nM, 50 nM and 5nM, respectively. Velcro library. The positive selection for round 1 was performed as described above for the interface library. In round 2, yeast were incubated with 1 µM biotinylated CD112 for two hours, washed and incubated with SA-647 (1:50 dilution) for 30 minutes. After 30 minutes the yeast was washed and incubated with 50 µL anti-647 microbeads (Miltenyi Biotech) for 30 minutes. After incubation, the yeast were washed and flowed over the LS column, and the column-bound yeast were collected. The selection process was repeated for four additional rounds using 1 µM, 200nM, 50 nM and 5 nM concentrations of CD112, respectively. Unbiased CD112RIV mutant library. The round 1 positive selection was performed by incubating yeast with 100nM biotinylated CD112 for 2 hours, followed by a wash with HBM, followed by a 30-minute incubation with SA-647. Yeast were then washed and incubated with 50 µL anti-647 microbeads (Miltenyi Biotech) for 30 minutes. After this incubation, the yeast were washed and flowed over the LS column to isolate binders. This selection process was repeated for 3 rounds at 20nM, 15nM and 5nM concentrations of CD112, respectively. Sequencing of yeast clones following selections. Upon completion of selections, yeast isolated from the final round of selection from each library were plated on SD-CAA plates. Individual colonies from the libraries were picked, cultured in SD-CAA, and induced in SG- ATTORNEY DOCKET NO.10110-461WO1 CAA. Plasmids from the selected clones were extracted using the Zymoprep Yeast Plasmid Miniprep II Kit (Zymo Research) and sequenced to identify affinity-enhancing mutations. SPR binding studies. The SPR experiment was performed on a BiacoreT200 instrument (GE Healthcare). Approximately 150 resonance units (RUs) of biotinylated CD112RWT, CD112RIV and CD112RIVE were immobilized on streptavidin-coated sensor chips (series S SA-coated sensor chip; GE healthcare). An increasing concentration of recombinant His-tagged CD112 was flowed over the chip in HBS buffer (HEPES) supplemented with 0.005% P20 surfactant at 20°C. Binding and dissociation phases were performed at 45µL/min for 120s and 120s, respectively. The chip was regenerated after every injection with 30 second washes of 0.5 MgCl2+10% Ethylene glycol. The curves were reference-subtracted from the flow cell containing negative control protein (extracellular domain of human carbonic anhydrase IX). The maximum RU for each experiment was normalized to 100 RU and plotted as a function of concentration using PRISM 9 (GraphPad). Steady-state binding curves were fitted using T200 BIAcore evaluation software (Cytiva) to a 1:1 Langmuir model to determine KD. Competition assay measuring CD112R variant inhibition of CD112-CD112R interactions. The extracellular domain of CD112 (D1) was cloned into a modified pCT vector as an N-terminal fusion to myc epitope tag and the yeast cell wall protein Aga2. The plasmid was transformed into EBY100 cells by electroporation, recovered in SD-CAA and induced in SG-CAA. CD112R tetramers (200 nM) were formed by mixing biotinylated CD112R with SA- 647 at a 4.5:1 ratio on ice for 10 minutes. During the incubation, CD112RIV-Fc and CD112RIVE-Fc proteins were serially diluted from 1µM to 0.5nM. After the 10-minute incubation, the CD112R tetramers were mixed 1:1 with the serially diluted CD112RIV-Fc CD112RIVE-Fc proteins, resulting in final concentration of CD112R 100nM tetramer plus 500nM to the CD112R variants. CD112R tetramer binding to the yeast was then detected by flow cytometry. Flow cytometry acquisitions were performed on a BD Accuri analyzer unless specified otherwise, and analyzed with FlowJo software (BD Biosciences). Cell Line and Crispr-Cas9 gene targeting. MDA-MB-468 (ATCC HTB-132) cell line was acquired from ATCC, authenticated by short tandem repeat (STR), and regularly tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection kit (Lonza). Cells were grown at 37°C, 5% CO2 with RPMI-1640 Medium supplemented with 2mM L- glutamine, 1X MEM Non-Essential Amino Acids, 1X Sodium Pyruvate, 55μM 2- Mercaptoethanol, 1X Penicillin- Streptomycin (from Gibco), and 10% fetal bovine serum ATTORNEY DOCKET NO.10110-461WO1 (FBS, GeminiBio). Cells were transduced to express green fluorescent protein (GFP)-firefly luciferase (FFLuc). For CRISPR/Cas9-mediated deletion of CD112 and CD155, we used two Predesigned Alt-R CRISPR-Cas9 crRNA guides per gene were used (SEQ ID NOS: 33-36). The crRNAs were combined with Alt-R CRISPR-Cas9 tracrRNA and Alt-R S.p. Cas9 Nuclease V3 to form CRISPR-Cas9 ribonucleoproteins and with Alt-R Cas9 Electroporation Enhancer (all from IDT). MDA-MD-468 cells were transfected with the 10μl Neon transfection system (Invitrogen) using 2 pulses at 1400 V and 20ms width. The efficiency of the gene deletion was at least 95% as measured by flow cytometry after surface staining with PE anti-human CD112 (clone TX31, Biolegend) or PE/Dazzle 594 anti-human CD155 (clone SKII.4, Biolegend) (FIG. S7A). Flow cytometry was performed on a LSRII flow cytometer using FACSDiva software (BD Biosciences) at the Moffitt Cancer Center Flow Cytometry Core. MDA-MB-468 cell line staining. For this assay, 0.25x106 MDA-MB-468 cells from each cell variant (CD112+ CD155+; CD112- CD155+; CD112+ CD155-; CD112- CD155-) were stained with 1µM biotinylated recombinant CD112R, CD112RIV and CD112RIVE for 60min at 4°C, washed 2 times, followed by staining with SA-647. Binding was detected by flow cytometry. Peripheral blood mononuclear cell and T cell isolation. De-identified healthy donor Buffy Coats were obtained from Lifesouth Community Blood Centers. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation using Lymphoprep (StemCell) and cryopreserved in FBS plus 10% dimethyl sulfoxide (DMSO). After thawing, T cells were isolated from PBMC using EasySep Human T Cell Isolation Kit (Stemcell) following the manufacturer’s instructions and cultured in RPMI-1640 Medium supplemented with 2mM L-glutamine, 1X Penicillin- Streptomycin, and 10% FBS (cRPMI) at 37°C, 5% CO2. Three different healthy donors were used for the functional experiments. T cell engager functional evaluation. To evaluate the TCE activity, 2x105 T cells were co-cultured with 8x104 MDA-MB-468 cells (2.5:1 E:T ratio) in 200ul cRPMI, and the different TCE were added to the media in the concentrations indicated in the FIG.. After 48hr, supernatant was collected and kept at -80°C for cytokine measurement. Cells were incubated with Bright-Glo Luciferase Assay System (Promega) for 5min to allow complete cell lysis and the lysed mixture was then transferred into a plate compatible with luminescence measurements. Relative luminescence intensity (RLI) was measured in a GloMax Discover ATTORNEY DOCKET NO.10110-461WO1 microplate reader (Promega). The percentage of cytotoxicity was calculated as: 100- (RLI sample/ RLI control* 100), where the control was target cells without effector cells. Enzyme-linked immunosorbent assay (ELISA). 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ATTORNEY DOCKET NO.10110-461WO1 SEQUENCES SEQ ID. NO: 1 > CD112RWT TPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIR QWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDP GLSAPPTPAPILRADL SEQ ID. NO: 2 >CD112RIV LNVRQTPEVWVQVRMEATELSSFTIRCGFLGSGNISLVTVSWGGPNHGGGTTLALLH PERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGTWEACGSL PPSSDPGLSAPPTPAPILRADL SEQ ID. NO: 3 >CD112RIV.A3 LNVRQTPEVWVQVRKEATELSSFTIRCGFLGSGNISLVTVSWGGPNHGGGTTLALLH PERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFATFPEGTWEACGSL PPSSDPGLSAPPTPAPILRADL SEQ ID. NO: 4 >CD112RIV.A5 LNVRQTPKVWVHVRKEATELSSFTIRCGFLGSGNISLVTVSWGGPNHGGGTTLALLQ PERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGTWEACGSL PPSSDPGLSAPPTPAPILRADL SEQ ID. NO: 5 PERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFATFPEGTWEACGSL PPSSDPGLSAPPTPAPILRADL SEQ ID. NO: 6 ATTORNEY DOCKET NO.10110-461WO1 >hIgG1 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPG K SEQ ID. NO: 7 > CD112RWTFc TPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPE RGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGS LPPSSDPGLSAPPTPAPILRADLGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHN HYTQKSLSLSPGK CD112R-Bold GSG linker- Underline hIgG1- Italics SEQ ID. NO: 8 >CD112RIVFc LNVRQTPEVWVQVRMEATELSSFTIRCGFLGSGNISLVTVSWGGPNHGGGTTL ALLHPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGT WEACGSLPPSSDPGLSAPPTPAPILRADLGSGDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEGLHNHYTQKSLSLSPGK CD112RIV-Bold GSG linker- Underline hIgG1- Italics ATTORNEY DOCKET NO.10110-461WO1 SEQ ID. NO: 9 >CD112RIVEFc LNVRQTPEVWVQVRKEATELSSFTIRCGFLGSGNISLVTVSWGGPNHGGGTTLA LLQPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFATFPEGTW EACGSLPPSSDPGLSAPPTPAPILRADLGSGDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEGLHNHYTQKSLSLSPGK CD112RIVE-Bold GSG linker- Underline hIgG1- Italics SEQ ID. NO: 10 >five amino acid anchor at N-terminus of CD112R LNVRQ SEQ ID. NO: 11 >TIGIT amino acid sequence TIETTGNISAEKGGSIILQCHL-SSTTAQVTQVNWEQQDQL----LAICNADLGWH- ISPSFKDRVAPGPGLGLTLQS---LTVNDTGEYFCIYHTYPDGTYT SEQ ID. NO: 12 >TIGIT Lock motif AICNADLG SEQ ID. NO: 13 >CD112R lock motif residues 89 to 96 AVLHPERG SEQ ID. NO: 14 ATTORNEY DOCKET NO.10110-461WO1 TPEVWVQVRMEATELSSFTIRCGFLGSGNISLVTVSWGGPNHGGGTTLALLHPERGIR QWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGTWEACGSLPPSSDP GLSAPPTPAPILRADL SEQ ID. NO: 15 >CD112Rlib_F1 Interface library overlap primers CATACATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATT SEQ ID. NO: 16 >CD112Rlib_F2 Interface library overlap primers TGTTTTTCAATATTTTCTGTTATTGCTAGCGTTTTAGCAGGATCCACCCCGGAGGT G SEQ ID. NO: 17 >CD112Rlib_F3 Interface library overlap primers GGATGGAGGCCACCGAGCTCTCGTCCTTCACCATCCGTTGTGGGTTCCTG SEQ ID. NO: 18 >CD112Rlib_F4 Interface library overlap primers ACTGTGAGCTGGGGGGGCCCCAACVVTRVTGGGGGGACCVMGCTGGCTVHCTTG CACCCAGAACGTGGCA SEQ ID. NO: 19 >CD112Rlib_F5 Interface library overlap primers CTGCTCGCCAGGCCCGCTGGGAAACCCAGAGCAGCATCTCTCTCATCCTGGAAGG CTCTG SEQ ID. NO: 20 >CD112Rlib_F6 Interface library overlap primers CACCACCTTCTGCTGCAAGTTTVHTTCCYHTCCTGAGGGAMNCTGGGAGGCTTGT GGTAGTGAACAAA SEQ ID. NO: 21 ATTORNEY DOCKET NO.10110-461WO1 >CD112Rlib_R1 Interface library overlap primers GAGCTCGGTGGCCTCCATCCGAACTTGAACCCACACCTCCGGGGTGGATCCTGCT AA SEQ ID. NO: 22 >CD112Rlib_R2 Interface library overlap primers GCCCCCCCAGCTCACAGTCACANKGGAGATAKNGCCAGACCCCAGGAACCCACA ACGGATGG SEQ ID. NO: 23 >CD112Rlib_R3 Interface library overlap primers CCAGCGGGCCTGGCGAGCAGGGGCCCATTGCCGGATGCCACGTTCTGGGTGC SEQ ID. NO: 24 >CD112Rlib_R4 Interface library overlap primers TTGCAGCAGAAGGTGGTGTTGGCGCAGGGGCTGCTGGCCCCAGAGCCTTCCAGG ATGAGA SEQ ID. NO: 25 >CD112Rlib_R5 Interface library overlap primers ACCCAAGTCTTCTTCGGAGATAAGCTTTTGTTCACTACCACAAGC SEQ ID. NO: 26 >CD112Rlib_R6 Interface library overlap primers GAGAACCACCACCACCAGAACCACCACCACCAGATCCACCACCACCCAAGTCTT CTTCGGAGATAAG SEQ ID. NO: 27 >CD112RVelcro_F Velcro library primer CATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTA GCGTTTTAGCANNKNNKNNKNNKNNKACCCCG SEQ ID. NO: 28 >CD112RIV EP-PCR_F Unbiased library primer ATTORNEY DOCKET NO.10110-461WO1 CCTCTATACTTTAACGTCAAGGAG SEQ ID. NO: 29 >CD112RIV EP-PCR_R Unbiased library primer GGGATTTGCTCGCATATAGTTG SEQ ID. NO: 30 >CD112R lock motif residues 83 to 90 AAFHPKMG SEQ ID. NO: 31 >Linker GGGGSGGGGSGGGGSGGGGS SEQ ID. NO: 32 >c-Myc epitope tag EQKLISEEDL SEQ ID. NO: 33 Hs.Cas9.NECTIN2.1.AA: crRNA GCGAGTTCAAGTGCTACCCG SEQ ID. NO: 34 Hs.Cas9.NECTIN2.1.AB: crRNA ACACCATCTGCTCGGCCCGA SEQ ID. NO: 35 Hs.Cas9.PVR.1. AB: crRNA CTATTCGGAGTCCAAACGGC SEQ ID. NO: 36 Hs.Cas9.PVR.1.AC: crRNA CACGGAGTCGCCCAAGAAGC ATTORNEY DOCKET NO.10110-461WO1 TABLES Table 1: X-ray data and refinement statistics* CD112R-CD112 complex (PDB ID: 9E6Y) 2) 2 38 90 3) 2) 2) 3) 9) 71 9) 4) 8) 5) 6) 1) 8) 5) 4) 0) 3) ATTORNEY DOCKET NO.10110-461WO1 Number of non-hydrogen atoms 1973 10 58 05 38 02 56 55 45 00 51 30 69 16 45 70

Claims

ATTORNEY DOCKET NO.10110-461WO1 CLAIMS What is claimed is: 1. An isolated receptor or target binding molecule that binds CD112, wherein the isolated receptor or target binding molecule comprises a CD112R extracellular domain variant. 2. The isolated receptor or target binding molecule of claim 1, wherein the isolated receptor or target binding molecule comprises of a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells. 3. The isolated receptor or target binding molecule of claim 1 or 2, wherein the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N- terminal end of SEQ ID NO: 1. 4. The isolated receptor or target binding molecule of claim 1 to 3, wherein the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 69, 82, 83, 90, 143, 50, 92, or 138 of SEQ ID NO: 1. 5. The isolated receptor or target binding molecule of claim 4, wherein the substitution at residue 50 comprises a methionine to lysine substitution (M50K). 6. The isolated receptor or target binding molecule of claim 4 or 5, wherein the substitution at residue 92 comprises a histidine to glutamine substitution (H92Q). 7. The isolated receptor or target binding molecule of any of claims 4-6, wherein the substitution at residue 138 comprises a serine to threonine substitution (S138T). 8. The isolated receptor or target binding molecule of any of claims 4-7, wherein the substitution at residue 69 comprises a serine to asparagine substitution (S69N). 9. The isolated receptor or target binding molecule of any of claims 4-8, wherein the substitution at residue 82 comprises a glycine to histidine substitution (G82H). 10. The isolated receptor or target binding molecule of any of claims 4-9, wherein the substitution at residue 83 comprises an alanine to glycine substitution (A83G). 11. The isolated receptor or target binding molecule of any of claims 4-10, wherein the substitution at residue 90 comprises a valine to leucine substitution (V90L). ATTORNEY DOCKET NO.10110-461WO1 12. The isolated receptor or target binding molecule of any of claims 4-11, wherein the substitution at residue 143 comprises a serine to threonine substitution (S143T). 13. The isolated receptor or target binding molecule of any of claims 4-12, wherein the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution. 14. The isolated receptor or target binding molecule of claim 13, wherein the CD112R extracellular domain variant comprises of SEQ ID NO: 2. 15. The isolated receptor or target binding molecule of any of claims 4-12, wherein the isolated receptor or target binding molecule comprises an M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution. 16. The isolated receptor or target binding molecule of claim 15, wherein the CD112R extracellular domain variant comprises of SEQ ID NO: 5. 17. The isolated receptor of any of claims 1-16, wherein the isolated receptor binds an epitope on tumor cells comprising of CD112. 18. The isolated receptor or target binding fragment of any of claims 1-17, wherein the CD112R is a human CD112R. 19. A pharmaceutical composition comprising the isolated receptor or target binding molecule of any of claims 1-18, and pharmaceutically acceptable carrier. 20. The pharmaceutical composition of claim 19, further comprising at least one additional therapeutic agent. 21. The pharmaceutical composition of claim 19, further comprising: an antagonist of PD-1, PD- Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB. 22. The pharmaceutical composition of claim 19, further comprising an antagonist of TIGIT. 23. A method of treating cancer in a subject in need thereof, comprising administering to a subject an effective amount of the isolated receptor of any of claims 1-18 or pharmaceutical composition of any of claims 19-22. ATTORNEY DOCKET NO.10110-461WO1 24. A method of treating cancer in a subject in need thereof, comprising administering to a subject an isolated receptor or target binding molecule that binds CD112, wherein the isolated receptor or target binding molecule comprises a CD112R extracellular domain variant or administering to the subject a pharmaceutical composition comprising said isolated receptor or target binding molecule. 25. The method of claim 24, wherein the isolated receptor or target binding molecule comprises of a bispecific T cell engagers (BiTEs), diabody, nanobody, single chain Fv (scFv), or antibody (Ab) that binds to CD112 on tumor cells. 26. The method of claim 23 or 24, wherein the isolated receptor or target binding molecule comprises the amino acid sequence LNVRQ added to the N-terminal end of SEQ ID NO: 1. 27. The method of any of claims 23-26, wherein the isolated receptor or target binding molecule comprises one or more amino acid substitutions at residues 69, 82, 83, 90, 143, 50, 92, or 138 of SEQ ID NO: 1. 28. The method of claim 27, wherein the substitution at residue 50 comprises a methionine to lysine substitution (M50K). 29. The method of claim 27 or 28, wherein the substitution at residue 92 comprises a histidine to glutamine substitution (H92Q). 30. The method of any of claims 27-29, wherein the substitution at residue 138 comprises a serine to threonine substitution (S138T). 31. The method of any of claims 27-30, wherein the substitution at residue 69 comprises a serine to asparagine substitution (S69N). 32. The method of any of claims 27-31, wherein the substitution at residue 82 comprises a glycine to histidine substitution (G82H). 33. The method of any of claims 27-32, wherein the substitution at residue 83 comprises an alanine to glycine substitution (A83G). 34. The method of any of claims 27-33, wherein the substitution at residue 90 comprises a valine to leucine substitution (V90L). ATTORNEY DOCKET NO.10110-461WO1 35. The method of any of claims 27-34, wherein the substitution at residue 143 comprises a serine to threonine substitution (S143T). 36. The method of any of claims 27-35, wherein the isolated receptor or target binding molecule comprises an S69N, G82H, A83G, V90L, and S143T substitution. 37. The method of claim 36, wherein the CD112R extracellular domain variant comprises of SEQ ID NO: 2. 38. The method of any of claims 27-35, wherein the isolated receptor or target binding molecule comprises an M50K, H92Q, S138T, S69N, G82H, A83G, V90L, and S143T substitution. 39. The method of claim 38, wherein the CD112R extracellular domain variant comprises of SEQ ID NO: 5. 40. The method of any of claims 23-39, wherein the isolated receptor binds an epitope on tumor cells comprising CD112. 41. The method of any of claims 23-39, wherein the CD112R is a human CD112R. 42. The method of any of claims 23-41, wherein the pharmaceutical composition further comprises at least one additional therapeutic agent and/or an antagonist of PD-1, PD-Ll, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRLl, PVRL2, PVRL3, PVRL4, CD155, CD47, CD39 and/or IL-27 and/or an agonist of OX40, CD28, CD40L, LFA-1, ICOS, and/or 4-1BB. 43. The method of claim 42, wherein the pharmaceutical composition further comprises an antagonist of TIGIT. 44. The method of any of claims 23-43, wherein the cancer comprises triple negative breast cancer.
PCT/US2025/014297 2024-02-01 2025-02-03 Reprogramming the cd112-cd112r axis for optimized cancer immunotherapy Pending WO2025166339A1 (en)

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Citations (4)

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US20170240613A1 (en) * 2015-09-02 2017-08-24 The Regents Of The University Of Colorado Compositions and methods for modulating t-cell mediated immune response
US20230241234A1 (en) * 2017-08-23 2023-08-03 Cygenica Limited Cell membrane penetrating conjugates
US20240025973A1 (en) * 2015-02-19 2024-01-25 Compugen Ltd. Pvrig polypeptides and methods of treatment

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140056890A1 (en) * 2012-06-06 2014-02-27 Oncomed Pharmaceuticals, Inc. Binding Agents That Modulate the Hippo Pathway and Uses Thereof
US20240025973A1 (en) * 2015-02-19 2024-01-25 Compugen Ltd. Pvrig polypeptides and methods of treatment
US20170240613A1 (en) * 2015-09-02 2017-08-24 The Regents Of The University Of Colorado Compositions and methods for modulating t-cell mediated immune response
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