MACROPHAGE-MEDIATED ANTIBODY GUIDED CANCER CELL

OR TUMOR ERADICATION

RELATED APPLICATIONS

This Patent Convention Treaty (PCT) International Application claims the benefit of priority to U.S. Provisional Application Serial No. (USSN) 62/532,661 filed July 14, 2017. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under National Institutes of

Health (NIH) grant nos. HL098472, HL 109142, HL121365 and National Science Foundation (NSF) grant nos. CBET1360341, DMS1361421, and NSFC 11428207. The government has certain rights in the invention.

TECHNICAL FIELD

This invention generally relates to cell biology, cellular genetic engineering and directed cancer immunotherapeutics. In alternative embodiments, provided is a Shp2-and Spleen tyrosine kinase (Syk)-integrated sensing and actuating protein (iSNAP) (Shp2- and Syk-iSNAP) chimeric protein comprising: a bi-phosphorylatable peptide, optionally a bisphosphoryl tyrosine-based activation (BTAM) motif; a Fluorescent Protein (FP) Forster Resonance Energy Transfer (FRET) (or FP FRET) pair or pair of motifs, optionally an enhanced cyan fluorescent protein (ECFP) motif and a YPet fluorochrome; a truncated Shp2 domain comprising an N-Src Homology 2 (N-SH2) domain and a C-Src Homology 2 (C-SH2) domain; and, a phosphatase (PTP) domain or a kinase domain. Also provided are engineered cells and methods for cancer cell or tumor eradication, or for the treatment or amelioration of a cancer, tumor or dysfunctional cell, or for promoting an anti-cancer, anti-tumor or anti- dysfunctional cell inflammatory response, including enhancing macrophage-, monocyte-, dendritic cell-, microglia-, osteoclast- or Kupffer cell- mediated antibody- or monoclonal antibody (mAb)-guided cancer or dysfunctional cell or tumor eradication, amelioration, or treatment. BACKGROUND

Immunotherapy has emerged as a promising novel direction for cancer therapeutics. Monoclonal antibody (mAb) therapies directly targeting cancer cells have also been widely used. Macrophages play a prominent role in mAb-based cancer immunotherapy by phagocytosis of tumor cells (Weiskopf and Weissman 2015). Macrophage phagocytosis results from a balance between the pro-phagocytic ("eat me") and anti -phagocytic ("don't eat me") signals. Antigen-targeting antibody can ligate FcyRs on macrophages and trigger pro-phagocytic signaling, while the antiphagocytic signaling is mediated by CD47 (also known as integrin associated protein (IAP)) on the target cell engaging the corresponding receptor SIRPa on macrophages (Barclay and Van den Berg 2014). In fact, anti-CD47 antibody has been applied to prevent anti -phagocytic signaling and to promote tumor eradication in various cancer types (Chao, Alizadeh et al. 2010; Jaiswal, Chao et al. 2010; Tseng, Volkmer et al. 2013). CD47 is, however, also expressed at high levels in red blood cells (RBCs), and undesirable anemia may complicate anti-CD47 cancer therapy (Barros, Yamamoto et al. 2009; Liu, Wang et al. 2015).

Current monoclonal antibody (mAb) mediated cancer immunotherapy is now one of the most successful and important strategies, however its efficacy is compromised by the "don't eat me" signal CD47 highly expressed on cancer cells (Weiskopf and Weissman 2015). Developing anti-CD47 cancer therapy utilizing antibodies shows high potential for cancer immunotherapy, however undesirable anemia may complicate this approach (Barros, Yamamoto et al. 2009; Liu, Wang et al. 2015).

SUMMARY

In alternative embodiments, provided is a Src homology region 2 domain- containing phosphatase-2 (Shp2)- and Spleen tyrosine kinase (Syk)-integrated sensing and actuating protein (iSNAP) (Shp2- and Syk-iSNAP, or Shp2-iSNAP) chimeric protein comprising:

(a) a phosphorylatable peptide, optionally a bi-phosphorylatable peptide, wherein optionally the bi-phosphorylatable peptide comprises a bisphosphoryl tyrosine-based activation (BTAM) motif,

(b) a Fluorescent Protein (FP) Forster Resonance Energy Transfer (FRET) (FP FRET) pair or pair of motifs, wherein optionally the FP FRET pair or pair of motifs is or comprises an enhanced cyan fluorescent protein (ECFP) motif and a YPet fluorochrome,

(c) a truncated Shp2 domain comprising an N-Src Homology 2 (N-SH2) domain and a C-Src Homology 2 (C-SH2) domain; and,

(d) a phosphatase (PTP) domain or a kinase domain,

wherein optionally the kinase comprises a tyrosine kinase (Syk),

wherein the unphosphorylated N-SH2 domain binds or quenches the PTP domain or the kinase domain,

and when phosphorylated (or bi-phosphorylated), the phosphorylated (or bi- phosphorylatable) peptide or BTAM motif binds the N-SH2 domain and the C-SH2 domain and liberates or unquenches (activates) the PTP domain or the kinase domain, and unquenching or activating of the PTP domain or the kinase domain causes (or results in the) emission of a detectable signal by the FP FRET pair or pair of motifs, and optionally, if the FP FRET pair or pair of motifs is or comprises an enhanced cyan fluorescent protein (ECFP) motif and a YPet fluorochrome, when the YPet fluorochrome comes into physical proximity to the enhanced cyan fluorescent protein (ECFP) motif, the physical proximity causes the YPet fluorochrome to emit a 535 nm signal.

In alternative embodiments, the Shp2-iSNAP chimeric protein as provided herein further comprises:

(a) a transmembrane domain;

(b) an extracellular domain capable of binding to a ligand; or

(c) a transmembrane domain and an extracellular domain capable of binding to a ligand,

wherein optionally the transmembrane domain is joined or fused to the phosphorylatable peptide, or bi-phosphorylatable peptide or BTAM motif,

and when the extracellular domain binds to its ligand the phosphorylatable peptide are phosphorylated (or the bi-phosphorylatable peptide or BTAM motif are biphosphorylated), and when phosphorylated (or bi-phosphorylated), the

phosphorylated (or bi-phosphorylated peptide or BTAM motif) binds the N-SH2 domain and the C-SH2 domain and liberates or unquenches (activates) the PTP domain or the kinase domain, and the unquenching or activating of the PTP domain or the kinase domain causes emission of a detectable signal by the FP FRET pair or pair of motifs, and optionally, if the FP FRET pair or pair of motifs is or comprises an enhanced cyan fluorescent protein (ECFP) motif and a YPet fluorochrome, the YPet fluorochrome comes into physical proximity to the enhanced cyan fluorescent protein (ECFP) motif, thereby causing the YPet fluorochrome to emit a 535 nm signal,

wherein optionally the extracellular domain capable of binding to a ligand comprises a Signal Regulatory Protein a (SIRPa), or a human SIRPa, domain and the ligand comprises a Cluster of Differentiation 47 (CD47) protein, optionally a CD47- coated particle or a CD47-expressing liposome or cell.

In alternative embodiments, provided are recombinant nucleic acids encoding a Shp2-iSNAP chimeric protein as provided herein.

In alternative embodiments, provided are expression vehicles or cassettes, vectors, viruses (e.g., recombinant viruses), and plasmids comprising or having contained therein a nucleic acid as provided herein, wherein optionally the virus is or is derived from a lentivirus, a poliovirus, or an adenovirus.

In alternative embodiments, provided are engineered (or recombinant) cells comprising or having contained therein a nucleic acid as provided herein, or an expression vehicle or cassette, vector or plasmid as provided herein, wherein optionally the cell is a bacterial cell, a yeast cell, a mammalian cell or a human cell, and optionally the cell is a macrophage, a microglial cell, an osteoclast, a Kupffer cell or a monocyte.

In alternative embodiments, provided are engineered eukaryotic cells comprising or expressing a Shp2-iSAP chimeric protein as provided herein, or comprising or having contained therein a nucleic acid as provided herein, or an expression vehicle or cassette, vector or plasmid as provided herein, wherein optionally the cell is a mammalian cell or a human cell, or an immune cell, or a macrophage, a monocyte, a microglial cell, an osteoclast, a Kupffer cell or a dendritic cell, wherein optionally the immune cell, or the macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell is a human macrophage, microglial cell, osteoclast, Kupffer cell monocyte or dendritic cell.

In alternative embodiments, provided are methods for:

- enhancing a macrophage-, monocyte-, microglia-, osteoclast- or Kupffer cell- or dendritic cell- mediated antibody (Ab)- or monoclonal antibody (mAb)-guided cancer cell, dysfunctional cell or tumor treatment or eradication,

- promoting development of an Ml macrophage phenotype,

- treating or ameliorating a cancer, a tumor or a condition caused by a dysfunctional cell, or

- for enhancing or initiating a macrophage-, monocyte-, microglia-, osteoclast- or Kupffer cell- or dendritic cell- mediated anti-cancer, antitumor or anti -dysfunctional cell inflammatory response,

the method comprising:

(a) providing or having provided an engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell as provided herein, wherein the macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell expresses a Shp2-iSNAP chimeric protein comprising a transmembrane domain and an extracellular domain capable of binding to a ligand (wherein optionally the ligand is a CD47 ligand) expressed on the cancer or dysfunctional cell or tumor, wherein the transmembrane domain is joined or fused to the phosphorylatable peptide (optionally a bi-phosphorylatable peptide or BTAM motif),

and when the extracellular domain binds to its ligand (optionally a CD47 ligand) the phosphorylatable peptide is phosphorylated (or the bi-phosphorylatable peptide or BTAM motif are biphosphorylated), and when phosphorylated (or bi- phosphorylated), the phosphorylated (or bi-phosphorylated peptide or BTAM motif) binds the N-SH2 domain and the C-SH2 domain and liberates or unquenches (activates) the PTP domain or the kinase domain, and the unquenching or activating of the PTP domain or the kinase domain causes emission of a detectable signal by the FP FRET pair or pair of motifs, and optionally, if the FP FRET pair or pair of motifs is or comprises an enhanced cyan fluorescent protein (ECFP) motif and a YPet fluorochrome, the YPet fluorochrome comes into physical proximity to the enhanced cyan fluorescent protein (ECFP) motif, thereby causing the YPet fluorochrome to emit a 535 nm signal, and liberating or unquenching (activating) the PTP domain or the kinase domain bypassing a CD47 mediated phagocytosis-block signal,

wherein optionally the fusion of Shp2-iSNAP to the C-tail of SIRPa (SIRPa Shp2- or Syk-iSNAP) rewires the anti -phagocytic "don't eat me" CD47/SIRPa/Shpl negative signaling (the native CD47-SIRPa pathway in myeloid cells is mediated by the negative regulator Shpl) into a positive Shp2 or Syk action upon the engagement of CD47 (or binding of the extracellular domain to its ligand CD47), thus facilitating phagocytosis of opsonized tumor cells, wherein the opsonization is initiated by IgG- FcyR interactions;

(b) providing or having provided an anti-tumor, anti-cancer or anti- dysfunctional cell Fc domain-comprising antibody or mAb capable of specifically binding to the cancer or dysfunctional cell or tumor, and via its Fc domain the antibody or mAb is capable of binding to or interacting with a macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell FcyR receptor capable of initiating phagocytosis of the cancer or dysfunctional cell or tumor; and

(c) exposing or contacting, or having exposed or having contacted, the cancer or dysfunctional cell or tumor to the antibody or mAb, wherein when the exposure is in vivo and the exposure comprises administering or having administered to an individual in need thereof the antibody or mAb, wherein optionally the antibody or mAb is administered in situ in or near the cancer or tumor or dysfunctional cell, or by injection (optionally intravenously (IV), intraperitoneally (IP), or intramuscularly (EVI)) to the individual in need thereof, wherein optionally the antibody or mAb administration in situ in or near the cancer or tumor or dysfunctional cell is by injection or by implanting a device (an implant) comprising the antibody or mAb, and optionally the dosage of the antibody or mAb is between about 0.1 to 1000 μg/ml, and optionally when the exposure is in vivo or to an individual in need thereof the method comprises:

(i) preparing or having prepared a Shp2-iSNAP chimeric protein-expressing engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell ex vivo and administering or having administered a Shp2-iSNAP chimeric protein-expressing engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell in vivo or to the individual in need thereof, wherein optionally between about 10 ³ to 10 ⁴ to about 5xl0 ⁶ to 5xl0 ⁸ engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cells are administered per unit dosage, optionally administered intravenously (IV), intraperitoneally (IP), intrathecally, or intramuscularly (EVI), optionally the administration is by in situ injection or deposition (optionally by or via an implant), wherein optionally the Shp2-iSNAP chimeric protein-expressing engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell is administered in situ in or near the cancer or tumor or dysfunctional cell, wherein optionally the Shp2-iSNAP chimeric protein-expressing engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell administration in situ in or near the cancer or tumor or dysfunctional cell is by injection or by implanting a device (an implant) comprising the Shp2-iSNAP chimeric protein- expressing engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell, or

(ii) engineering or having engineered a macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell in vivo by administering or having administered to the individual in need thereof a nucleic acid encoding the Shp2- iSNAP chimeric protein, wherein optionally the nucleic acid is contained in a gene therapy vector or virus (or the expression vehicle or cassette, a vector, a virus, or a plasmid as provided herein, which optionally is designed to target a macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell), wherein the nucleic acid is expressed in the macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell thereby causing it to express the Shp2-iSNAP chimeric protein.

In alternative embodiments, provided are methods for:

- cancer cell or tumor eradication in an individual in need thereof,

- for treating or ameliorating a cancer or a tumor or a condition caused by a dysfunctional cell,

- promoting development of an Ml macrophage phenotype, or for promoting an anti -cancer, anti-tumor or anti-dysfunctional cell inflammatory response,

in an individual in need thereof,

the method comprising:

(a) administering or having administered to the individual in need thereof an antibody or mAb capable of specifically binding to the cancer cell, tumor or dysfunctional cell, the administration as set forth in any method or embodiment as provided herein, e.g., intravenously (IV), intrathecally, intraperitoneally (IP), or intramuscularly (EVI), and (b) (i) preparing or having prepared a Shp2-iSNAP chimeric protein- expressing engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell as provided herein ex vivo and administering or having administered the Shp2-iSNAP chimeric protein-expressing engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell in vivo or to the individual in need thereof, or

(ii) engineering or having engineered a macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell in vivo by administering or having administered to the individual in need thereof a nucleic acid encoding the Shp2- iSNAP chimeric protein, wherein optionally the nucleic acid is contained in a gene therapy vector or virus (or the expression vehicle or cassette, a vector, a virus, or a plasmid of claim 4, which optionally is designed to target a macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell), wherein the nucleic acid is expressed in the macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell thereby causing it to express (to express extracellularly an extracellular domain of) the Shp2-iSNAP chimeric protein.

In alternative embodiments, provided are Uses of a Shp2-iSNAP chimeric protein-expressing engineered macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell for:

- a cancer cell or a tumor eradication,

promoting the development of an Ml macrophage phenotype, for the treatment or amelioration of a cancer, a tumor or a condition or disease caused by a dysfunctional cell, or

for promoting an anti-cancer, anti-tumor or anti-dysfunctional cell inflammatory response,

in an individual in need thereof,

wherein the Shp2-iSNAP chimeric protein-expressing engineered

macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell is an engineered cell of any of the preceding claims, and the cancer, tumor or dysfunctional cell is eradicated, treated or ameliorated in the individual in need thereof by practicing a method of any of the preceding claims. In alternative embodiments, provided are Shp2-iSNAP chimeric protein- expressing engineered macrophages, monocytes, microglial cells, osteoclasts, Kupffer cells or dendritic cells for use in:

a cancer cell or a tumor eradication,

- for the treatment or amelioration of a cancer, a tumor or a condition or disease caused by a dysfunctional cell, or

for promoting an anti-cancer, anti-tumor or anti-dysfunctional cell inflammatory response,

in an individual in need thereof,

wherein the Shp2-iSNAP chimeric protein-expressing engineered macrophage or dendritic cell is an engineered cell of any of the preceding claims, and the cancer cell or tumor is eradicated in the individual in need thereof by practicing a method of any of the preceding claims.

In alternative embodiments, provided are kits comprising an engineered eukaryotic cell as provided herein (e.g., a macrophage, monocyte, microglial cell, osteoclast, Kupffer cell or dendritic cell), where the kit optionally comprising instructions for practicing a method of any of the preceding claims, or the kit optionally further comprises an antibody or mAb capable of specifically binding to the cancer cell, tumor or dysfunctional cell, or the kit optionally further comprises any component for practicing a method of any of the preceding claims.

In alternative embodiments, provided are products (or articles) of manufacture comprising an engineered eukaryotic cell as provided herein, wherein optionally the product of manufacture is: a device; an implant; a vial, a carpule or storage container; or a catheter.

In alternative embodiments, in practicing methods as provided herein the cancer or tumor can be: a lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma,

rhabdomyosarcoma, leukemia or lymphoma, Hodgkin's lymphoma or childhood acute lymphoblastic leukemia, a mastocytoma or a mast cell tumor, an ovarian cancer or carcinoma, pancreatic cancer, a non-small cell lung cancer, small cell lung cancer, hepatocarcinoma, melanoma, retinoblastoma, breast tumor, colorectal carcinoma, leukemia, lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphoid leukemia, acute myeloid leukemia (AML), a histiocytic sarcoma, a brain tumor, an astrocytoma, a glioblastoma, a neuroma, a colon carcinoma, cervical carcinoma, sarcoma, bladder tumor, tumor of the reticuloendothelial tissues, Wilm's tumor, a bone cancer, an osteosarcoma, a renal cancer, or head and neck cancer, oral cancer, a laryngeal cancer, or an oropharyngeal cancer.

In alternative embodiments, in practicing methods as provided herein the antibody or the mAb is selected from the group consisting of (or, can be) an anti-IGF- 1R (e.g., hRl) (), an anti-mucin (e.g., hPAM4 or KC4), an anti-CD20 (e.g., rituximab, tositumomab, ibritumomab tiuxetan, GA101 or hA20 ), an anti-CD19 (e.g., hA19 ), an anti-AFP (e.g., hEVIMl l ), an anti-CD74 (e.g., hLLl ), an anti-CD22 (e.g., hLL2 or RFB4), an anti-CSAp (e.g., hMu-9 ), an anti-HLA-DR (e.g., hL243 ), an anti- CEACAM-5 (e.g., hMN-14 ), an anti-CEACAM-6 (e.g., hMN-15 or hMN-3 (), an anti-TROP-2 (e.g., hRS7 ), an anti-TAG-72 (e.g., CC49 ), an anti-PSMA (e.g., J591 or D2/B ), an anti-carbonic anhydrase IX (e.g., G250 ), an anti-T F-α (e.g., infliximab or certolizumab pegol, or adalimumab), anti-CD52 (e.g., alemtuzumab ), anti-VEGF (e.g., bevacizumab ), anti-EGFR (e.g., panitumumab or cetuximab ), anti- CD33 (e.g., gemtuzumab ), anti-HER2/neu (e.g., trastuzumab ), anti-IL-6 receptor (e.g., tocilizumab , anti-CD25 (e.g., basiliximab or daclizumab), anti-CDl la (e.g., efalizumab ), anti-CD3 receptor (e.g., muromonab-CD3), anti-a4 integrin (e.g., natalizumab ), anti-histone H2A/H4 (e.g., BWA-3 ), anti-histone H3 (e.g., LG2-1 ), an anti-histone HI (e.g., MRA12 ), an anti-histone H2B (e.g., PRl-1, LG11-2, LG2-2).

In alternative embodiments, in practicing methods as provided herein the antibody or the mAb specifically targets an antigen selected from the group consisting of a-fetoprotein (AFP), a-actinin-4, A3, antigen specific for A33 antibody, ART -4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m, CCL19, CCL21, CD1, CDla, CD2, CD3, CD4, CD5, CD8, CD11 A,

CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-Ια, colon-specific antigen-p (CSAp), CEA (CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gplOO, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, la, IGF-IR, IFN-γ, IFN-a, IFN-β, IFN- λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL- 17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1- antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MTF), MAGE, MAGE-3, MART-1, MART -2, NY-ESO-1, TRAG-3, mCRP, MCP-1, ΜΠ>- 1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM- 1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-R, L-6, IL-25, RS5, RANTES, T101, SAGE, SI 00, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-a, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1 A-antigen, complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl-6, and Kras. The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1 A-D illustrate the designation, activation mechanism and functionality of the iSNAP, SIRPa Shp2-iSNAP and SIRPa Syk-iSNAP engineered proteins and constructs as provided herein:

FIG. 1 A illustrates a schematic drawing of the Shp2-iSNAP and its putative activation mechanism;

FIG. IB illustrates a schematic drawing of the SIRPa Shp2-iSNAP and its putative activation mechanism upon engagement of CD47; FIG. 1C illustrates a schematic of SIRPa Syk-iSNAP, where the activated kinase domain is labeled "Kin" (and colored in red);

FIG. ID illustrates a schematic of engineering macrophages for mAbs-guided cancer cell eradication;

as described in detail, below.

FIG. 2A-G illustrate the characterization of the Shp2-iSNAP in vitro and in cells:

FIG. 2A graphically illustrates the emission spectrum change of the Shp2- iSNAP before (black) and after (red) Src kinase incubation in vitro;

FIG. 2B-C graphically illustrate the emission ratio time courses (FIG. 2B) and phosphatase activities (FIG. 2C) of Shp2-iSNAP and its FF mutant before and after Src incubation in vitro as indicated;

FIG. 2D illustrates ratiometric images of a MEF cell transfected with the Shp2-iSNAP before and after PDGF stimulation at indicated time points;

FIG. 2E graphically illustrates the ratio time courses (mean ± S.E.M) of MEFs expressing the Shp2-iSNAP or its FF mutants;

FIG. 2F illustrates a schematic drawing of the inhibitory effect of the non- fluorescent Shp2-iSNAP upon activation on its target FAK monitored by a membrane-bound FAK biosensor;

FIG. 2G illustrates bar graphs of FAK biosensor emission ratios (mean ±

S.E.M) in PDGF-stimulated MEFs expressing control vector, the Shp2-iSNAP, or its FF mutant; *, P < 0.05 (n=3),

as described in detail, below.

FIG. 3 A-F illustrate the activation and phagocytic functions of SIRPa Shp2- iSNAP in macrophages:

FIG. 3 A schematically illustrates the SIRPa Shp2-iSNAP and its putative activation mechanism upon engagement of CD47;

FIG. 3B illustrates ratiometric, YPet, and Differential Interference

Contrast (DIC) images of RAW264.7 macrophages expressing SIRPa Shp2-iSNAP or FF mutant after incubation with beads coated by CD47 plus IgG, CD47 only, or IgG only, respectively;

FIG. 3C graphically illustrates quantification of local FRET response in macrophages stimulated by beads with different coatings as indicated in FIG. 3B; FIG. 3D graphically illustrates the time course of FRET ratio of SIRPa Shp2- iSNAP in response to CD47-coated beads and 10 μΜ SFKs inhibitor PPl treatment;

FIG. 3E illustrates Ratiometric, YPet, and DIC images of phagocytosis of opsonized RBCs by a representative RAW264.7 macrophage expressing SIRPa Shp2-iSNAP at indicated time points;

FIG. 3F graphically illustrates a Bar graph (mean ± S.E.M.) of normalized phagocytic rate of macrophages expressing SIRPa Shp2-iSNAP or its controls against the opsonized RBCs at 37 °C for 30 min, FF mutations in the BTAM peptide of iSNAP; "ΔΡΤΡ", SIRPa Shp2-iSNAP without PTP domain; "SIRP", full length SIRPa fused with YPet; "SIRP-no ΠΤΜ", ITFM-truncated SIRPa fused with YPet, as described in detail, below.

FIG. 4A-E illustrate the activation and function of exemplary chimeric SIRPa Shp2-iSNAP proteins on phagocytosis of cancer cells:

FIG. 4A and FIG. 4D illustrate Ratiometric, YPet, and DIC images of a representative BMDM expressing exemplary chimeric SIRPa Shp2-iSNAP proteins before and after incubation with 10μg/ml rituximab-opsonized Toledo cells (FIG. 4A) or 2μg/ml cetuximab-opsonized DLD1 (FIG. 4D) at indicated time points;

FIG. 4B graphically illustrates the time course of FRET ratio of SIRPa Shp2- iSNAP in the BMDM at the region around the engaging Toledo cell in (FIG. 4 A);

FIG. 4C and FIG. 4E graphically illustrates a Bar graph (mean ± S.E.M.) of normalized phagocytic rate of macrophages expressing different constructs as described in Figure 3 against rituximab-opsonized Toledo (C) or cetuximab- opsonized DLD1 cells (E).

FIG. 5A-E illustrate the activation and function of exemplary chimeric SIRPa Syk-iSNAP proteins in macrophages:

FIG. 5 A schematically illustrates a SIRPa Syk-iSNAP and its putative activation mechanism (activation of kinase, or "Kim") upon CD47 engagement by phosphorylation of the BTAM2 motif;

FIG. 5B illustrates Ratiometric, YPet, and DIC images of RAW264.7 macrophages expressing SIRPa Syk-iSNAP before and after incubation with CD47- coated beads; FIG. 5C illustrates Ratiometric and DIC images of RAW264.7 macrophages expressing dark SIRPa Syk-iSNAP together with Ca ²⁺ or Erk FRET biosensors before and after the incubation with CD47-coated beads;

FIG. 5D illustrates Ratiometric and DIC images of phagocytic processes of opsonized RBCs by a representative RAW264.7 macrophage expressing SIRPa Syk- iSNAP;

FIG. 5E graphically illustrates a Bar graph (mean ± S.E.M.) of normalized phagocytic rate of RAW264.7 macrophages expressing SIRPa Syk-iSNAP or control constructs against the rabbit anti-human RBC IgG-opsonized RBCs at 37 °C for 30 min; FF, FF mutations in the BTAM peptide of iSNAP; K402R, a kinase dead mutant K402R in Syk kinase domain of iSNAP; other constructs are the same as described for FIG. 3,

as described in detail, below.

FIG. 6A-D illustrate stable expression of exemplary chimeric iSNAP proteins in engineered THPl monocyte cell lines:

FIG. 6A: illustrates a schematic drawing of an activation mechanism for phagocytosis in the iSNAP-engineered monocytes;

FIG. 6B illustrates fluorescence images of YFP signals in the iSNAP proteins stably expressed in the engineered THPl monocytes;

FIG. 6C illustrates flow cytometry results of YFP signals in the iSNAP proteins stably expressed in the exemplary engineered THPl monocytes to quantify the expression of iSNAPs;

FIG. 6D graphically illustrates quantification of mRNA levels of endogenous SIRPa receptors and exemplary engineered-iSNAP in THPl cells,

as described in detail, below.

FIG. 7A-C illustrate the expression of iSNAP as provided herein affected the phenotypes of engineered THPl monocytes:

FIG. 7A graphically illustrates the expression of lineage markers in the control and exemplary iSNAP-engineered THPl monocytes. CD31 : monocyte marker;

CD1 lb and CD 14: macrophage markers;

FIG. 7B graphically illustrates the expression of macrophage polarization markers in the control and exemplary iSNAP-engineered THPl monocytes. CD68, CD80, CD86: Ml macrophage polarization markers; CD163, CD206: M2

macrophage polarization markers;

FIG. 7C graphically illustrates the expression of macrophage proinflammatory cytokines in the control and exemplary iSNAP-engineered THP1 monocytes, IL-6 and TNFa: pro-inflammatory cytokine markers; IL-10: antiinflammatory cytokine marker,

as described in detail, below.

FIG. 8A-B illustrate that exemplary iSNAP-engineered THP1 monocytes as provided herein are better than the naive THP1 cells in eliminating B-lymphoma (Toledo cells) tumors in mice, guided by the Rituximab recognizing the B-lymphoma Toledo antigen CD20:

FIG. 8 A schematically illustrates the treatment of human B-lymphoma (Toledo) tumors in nude mice models by exemplary iSNAP-engineered macrophages as provided herein and control THP1 cells, guided by Rituximab recognizing Toledo antigen CD20;

FIG. 8B graphically illustrates the time course quantification of B-lymphoma tumor sizes after the treatment by exemplary iSNAP-engineered and control THP1 cells, guided by Rituximab recognizing Toledo antigen CD20,

as described in detail, below. Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In alternative embodiments, provided are engineered immune cells, including macrophages, monocytes, dendritic cells, microglia, osteoclasts and Kupffer cells comprising chimeric Signal Regulatory Protein a (SIRPa) integrated sensing and actuating proteins (iSNAPs), and methods for making and using them. In alternative embodiments, provided is a Shp2-and Syk-integrated sensing and actuating protein (iSNAP) (Src homology region 2 domain-containing phosphatase-2 (Shp-2)- and Syk- iSNAP) chimeric protein comprising: a phosphorylatable peptide (e.g., a bi- phosphorylatable peptide, optionally a bisphosphoryl tyrosine-based activation (BTAM) motif); a Fluorescent Protein (FP) Forster Resonance Energy Transfer (FRET) (or FP FRET) pair or pair of motifs, and optionally an enhanced cyan fluorescent protein (ECFP) motif and a YPet fluorochrome; a truncated Shp2 domain comprising an N-SH2 domain and a C-SH2 domain; and, a phosphatase (PTP) domain or a kinase domain. Also provided are methods and uses for enhancing macrophage-, monocyte-, dendritic cell-, microglia-, osteoclast- or Kupffer cell- mediated antibody- or monoclonal antibody (mAb)-mediated inflammatory responses, including enhancing guided cancer cell, dysfunctional cell or tumor eradication.

In alternative embodiments, engineered macrophages, monocytes, dendritic cells, microglia, osteoclasts and Kupffer cells with (expressing) chimeric Signal Regulatory Protein a (SIRPa) integrated sensing and actuating proteins (iSNAPs) as provided herein are used to target different types of cancers, tumors or dysfunctional cells, and in alternative embodiments, this tumor, cancer or dysfunctional cell targeting is integrated with monoclonal antibody (mAb)- mediated immunotherapy, including e.g., cancer immunotherapy, or immunotherapy for any condition caused by a dysfunctional cell.

In alternative embodiments, the Signal Regulatory Protein a (SIRPa) integrated sensing and actuating proteins (iSNAPs) and engineered cells provided herein use are used platforms for the high throughput screening of potential SIRPa inhibitors for cancer, tumor or dysfunctional cell therapy, e.g., to enhance the efficiency of therapeutic mAbs (e.g. anti-CD 19, anti-CD20, anti-PSMA mAbs) against any cell target, including tumor or cancer cells, or any dysfunctional cell, including infected cells, e.g., viral or bacterium infected cells.

In alternative embodiments, chimeric SIRPa iSNAPs as provided herein are used as biosensors to study the activation and regulation of SIRPa and related molecular mechanism.

In alternative embodiments, provided herein are fluorescent protein-based biosensors and controllable protein actuators that are engineered to visualize and manipulate, respectively, signaling events in live cells as separate, parallel

approaches. Here we integrate protein modules with sensing and actuating functions to engineer integrated sensing and actuating proteins (iSNAPs) capable of detecting tyrosine phosphorylation events and activating desired enzymatic domains to reprogram the cell. An iSNAP utilizing Shp2 phosphatase as the actuator (Shp2- iSNAP) was characterized and connected to the macrophage SIRPa receptor, whose native form transduces a "don't eat me" signal from CD47 ligand during

phagocytosis. The macrophages engineered with this SIRPa Shp2-iSNAP not only allowed visualization of spatiotemporal dynamics of SIRPa phosphorylation upon CD47 engagement, but also rewired the detected input into the activation of positive Shp2 signaling, leading to enhanced phagocytosis of opsonized tumor cells.

Since the design of iSNAPs is highly modular, we further replaced the sensor and actuator domains in Shp2-iSNAP to create a Syk-iSNAP, which likewise rewired the CD47/SIRPa axis to the pro-phagocytic Syk kinase activation when fused to SIRPa. Thus, our approach can be extended to execute a broad range of sensing and automated reprogramming actions for macrophage engineering and directed cell, e.g., tumor, cancer or dysfunctional cell, immunotherapeutics.

In alternative embodiments, the dysfunctional cell is an infected cell, e.g., a viral or bacterium infected cell, or a cell made dysfunctional by a genetic or inherited condition.

The novel engineered integrated sensing and actuating proteins (iSNAPs) provided herein, and the engineering of macrophages by iSNAPs as provided herein, provide new immunotherapies. Previously, numerous genetically encoded biosensors based on fluorescent proteins (FPs) and FRET have been generated to visualize dynamics of signal transduction in live cells at subcellular levels (Wang, Shyy et al. 2008). In parallel, synthetic proteins that can undergo dimerization or allosteric conformational change upon stimulation by radio wave, light, chemical compounds, or cell-cell interactions have been designed to actuate signal cascades and control cellular behavior for therapeutic purposes (Toettcher, Voigt et al. 2011; Toettcher, Weiner et al. 2013; Chakravarti and Wong 2015; Wu, Roybal et al. 2015; Wu, Rupp et al. 2015; Roybal, Rupp et al. 2016). However, these two separate approaches have not been integrated into one platform to engineer proteins with both sensing and actuating functions. The new designs as provided herein combine the advantage of those approaches using integrated sensing and actuating proteins (iSNAPs) to survey the intracellular space, and to trigger designed molecular actions upon detection and visualization of specific signals, with the consequences of modulating the downstream signaling cascades and cellular (e.g., macrophage, monocyte, microglia, osteoclast, Kupffer cell or dendritic cell) functions.

In alternative embodiments, compositions and methods provided herein solve the problem of anti-CD47-based therapies, where CD47, being expressed at high levels in red blood cells (RBCs), may cause undesirable anemia may complicate anti-CD47 cancer therapy. By using engineering macrophages, monocytes, microglia, osteoclasts and Kupffer cells or dendritic cells by iSNAP with rewired CD47 signaling, as provided herein, together with monoclonal antibodies (mAbs) to specifically target a tumor, a cancer or any dysfunctional cell, provided herein is a revolutionary next-generation immunotherapy.

In alternative embodiments, provided herein is an engineered class of iSNAPs that can sense specific biochemical events and consequently activate the reprogramming of cellular (e.g., macrophage, monocyte, microglia, osteoclast, Kupffer cell or dendritic cell) functions. We first engineered a Shp2-based iSNAP (Shp2-iSNAP) for the sensing of intracellular tyrosine phosphorylation and the consequent activation of an important phosphatase (PTP) Shp2 (Tonks 2006). Structurally, the enzymatic PTP domain of Shp2 is masked by the auto-inhibitory N-SH2, which can be released upon binding of a phosphorylated peptide (Tonks 2006). We have hence fused a phosphorylatable peptide (bisphosphoryl tyrosine-based activation motif, BTAM) with a FRET pair (ECFP and YPet) and truncated Shp2 to eliminate the potential interfering binding of the truncated C-terminal tyrosine-containing tail toward the N- SH2 within the iSNAP (Fig. 1 A). As such, the peptide upon kinase phosphorylation can bind to the N-SH2 domain to cause FRET changes and subsequently relieve the inhibitory effect of N-SH2 on the PTP domain, thus activating Shp2. Hence, FRET signals of the sensor module within iSNAPs can provide immediate readings and serve as "digital multi-meters" for functional calibration and optimization of engineered iSNAPs. When Shp2-iSNAP was fused to the C-tail truncated SIRPa (SIRPa and Shp2-iSNAP), it could sense CD47 induced phosphorylation of SIRPa and initialize positive Shp2 action (Fig. IB).

In one embodiment, this modular approach is used to engineer a new type of iSNAPs employing spleen tyrosine kinase (Syk) (Syk-iSNAP) (Fig. 1C).

We further applied those SIRPa iSNAPs to engineer macrophages, and this engineered macrophage was activated by CD47 expressed on tumor cells and promoted the antibody-mediated tumor cell phagocytosis comparing to native macrophage (Fig. ID).

Figure 1 schematically illustrates a diagram describing the designation, activation mechanism and functionality of exemplary Shp2-iSNAP, SIRPa Shp2- iSNAP, SIRPa Syk-iSNAP chimeric proteins as provided herein; this drawing schematically illustrates the designation, activation mechanism and functionality of the iSNAP, SIRPa Shp2-iSNAP and SIRPa Syk-iSNAP engineered proteins and constructs as provided herein:

(A) Schematic drawing of the Shp2-iSNAP and its putative activation mechanism. Shp2-iSNAP consists of a phosphorylatable peptide, a FP FRET pair, a truncated Shp2 consisting of two SH2 domains (N-SH2 and C-SH2) and a PTP domain. Two sensing tyrosine sites are indicated as Y (without phosphorylation), P indicates phosphorylation, activated PTP domain is colored in red.

(B) Schematic drawing of the SIRPa Shp2-iSNAP and its putative activation mechanism upon engagement of CD47. Structurally, SIRPa Shp2-iSNAP contains a human SIRPa without its ΠΤΜ-containing C-tail, fused to the Shp2-iSNAP. At rest, PTP domain is quenched by its SH2 domains. Upon CD47 stimulation, tyrosines in BTAM motif will be phosphorylated to bind SH2 domains and result in the release and activation of the PTP domain.

(C) SIRPa Syk-iSNAP has similar design with BTAM2 instead of BTAM,

Syk instead of Shp2. Activated kinase domain is colored in red.

(D) Engineering macrophage for mAbs-guided cancer cell eradication. In native macrophages, pro-phagocytic activities mediated by the antigen-recognizing antibody and FcyR is inhibited by CD47-SIRPa signal pathway via the recruitment of negative regulator Shpl . In engineered macrophages, the anti -phagocytic signals of CD47-SIRPa axis are rewired by SIRPa iSNAPs to promote phagocytic activities for the tumor cell eradication.

Characterize cytosolic Shp2-iSNAP in vitro and in mammalian cells

To characterize the exemplary cytosolic Shp2-iSNAP, purified Shp2-iSNAP was incubated with recombinant Src, a kinase known to phosphorylate SIRPa in response to various mitogens (Tsuda, Matozaki et al. 1998). As we designed, the responsiveness of this iSNAP depended upon phosphorylation of tyrosines in the BTAM peptide, since mutation of these tyrosines (called an "FF" mutation) abolished phosphorylation, FRET response and PTP activation (Fig. 2A-C). When Shp2- iSNAP was expressed in mouse embryonic fibroblasts (MEFs), PDGF caused a FRET increase (Fig. 2D), which is dependent on the phosphorylatable tyrosines in the BTAM peptide (Fig. 2E). Therefore, the exemplary Shp2-iSNAP as provided herein can sense and report PDGFR activation-induced tyrosine phosphorylation events in live cells.

We further evaluated whether the Shp2-iSNAP can rewire and activate designed downstream signaling events in mouse embryonic fibroblasts (MEFs) after detecting input signals. To quantify the activation effect of the Shp2-iSNAP, we utilized a FRET-based Lyn-focal adhesion kinase (FAK) biosensor to monitor the membrane activity of FAK (Fig. 2F). As a downstream target of Shp2, FAK can be dephosphorylated at Y397 to inhibit its kinase activity (Seong, Ouyang et al. 2011). To avoid interference between two pairs of enhanced cyan fluorescent protein (ECFP) and YPet in the same cell, we mutated the ECFP (W65 A/W66 A) and YPet (G66 A) in the Shp2-iSNAP to eliminate its fluorescence while leaving the other domains and functions intact (Fig. 2F). Shp2-iSNAP significantly reduced PDGF-induced FAK activity compared to empty vector or the FF mutant (Fig. 2G). These results confirmed the designed functionality of Shp2-iSNAP in vitro and in live cells.

Figure 2 illustrates the characterization of the Shp2-iSNAP in vitro and in cells:

(A) graphically illustrates the emission spectrum change of the Shp2-iSNAP before (black) and after (red) Src kinase incubation in vitro;

(B, C) graphically illustrate the emission ratio time courses (B) and phosphatase activities (C) of Shp2-iSNAP and its FF mutant before and after Src incubation in vitro as indicated;

(D) illustrate ratiometric images of a MEF cell transfected with the Shp2- iSNAP before and after PDGF stimulation at indicated time points. Color scale bar represents the FRET/CFP emission ratio, with cold (the shading or blue coloring closer to the indicated 0.9 on the FRET/CFP scale) and hot (the shading or orange/red coloring closer to the indicated 1.5 on the FRET/CFP scale) colors representing low and high FRET efficiency of iSNAP, respectively; Size scale bar, 20 μπι;

(E) graphically illustrates the ratio time courses (mean ± S.E.M) of MEFs expressing the Shp2-iSNAP or its FF mutants;

(F) illustrates a schematic drawing of the inhibitory effect of the non- fluorescent Shp2-iSNAP upon activation on its target FAK monitored by a membrane-bound FAK biosensor; the activated iSNAP and FAK are colored in orange and red, respectively; (G) illustrates Bar graphs of FAK biosensor emission ratios (mean ± S.E.M) in PDGF-stimulated MEFs expressing control vector, the Shp2-iSNAP, or its FF mutant; *, P < 0.05 (n=3).

SIRPa Shp2-iSNAP activated by CD47 and promote phagocytic functions in macrophages

While the native CD47-SIRPa pathway in myeloid cells is mediated by the negative regulator Shpl (Tsai and Discher 2008), Shp2 can act as a positive regulator in macrophages (Neel, Gu et al. 2003; Matozaki, Murata et al. 2009). The fusion of Shp2-iSNAP to the C-tail of SIRPa (SIRPa Shp2-iSNAP) may rewire the anti- phagocytic "don't eat me" CD47/SIRPa/Shpl negative signaling into a positive Shp2 action upon the engagement of CD47, thus facilitating phagocytosis of opsonized tumor cells initiated by IgG-FcyR interactions (Fig. 3 A). When CD47-coated beads engaged the RAW264.7 macrophages expressing SIRPa Shp2-iSNAP, a local FRET increase of the iSNAP was observed (Fig. 3B). This activation was specific to CD47 and depended on tyrosine phosphorylation, since it was abolished in groups using beads coated with IgG only or with iSNAP FF mutant (Fig. 3B-C). Consistent with the potent capability of Src family kinases (SFKs) in phosphorylating SIRPa (Tsuda, Matozaki et al. 1998; Johansen and Brown 2007), the SFKs inhibitor PP1 markedly suppressed the FRET response of SIRPa Shp2-iSNAP (Fig. 3D).

We next examined macrophages engineered with SIRPa Shp2-iSNAP for their phagocytic activity against the human RBCs that endogenously express high levels of CD47 (Oldenborg, Zheleznyak et al. 2000). Indeed, comparing to control groups, RAW264.7 macrophages expressing SIRPa Shp2-iSNAP exhibited a significantly increased capability in engulfing opsonized-RBCs, accompanied by a transient FRET response of SIRPa Shp2-iSNAP, which returned to the basal level once the RBCs were completely engulfed by the macrophages (Fig. 3E-F). These results indicate that the rewiring of CD47 signaling by SIRPa Shp2-iSNAP can promote the phagocytic ability of the engineered macrophages toward RBCs.

Figure 3 illustrates the activation and phagocytic functions of SIRPa Shp2- iSNAP in macrophages:

(A) schematically illustrates the SIRPa Shp2-iSNAP and its putative activation mechanism upon engagement of CD47; (B) illustrates ratiometric, YPet, and DIC images of RAW264.7 macrophages expressing SIRPa Shp2-iSNAP or FF mutant after incubation with beads coated by CD47 plus IgG, CD47 only, or IgG only, respectively;

(C) graphically illustrates quantification of local FRET response in

macrophages stimulated by beads with different coatings as indicated in (B);

(D) graphically illustrates the time course of FRET ratio of SIRPa Shp2- iSNAP in response to CD47-coated beads and 10 μΜ SFKs inhibitor PP1 treatment;

(E) illustrates Ratiometric, YPet, and DIC images of phagocytosis of opsonized RBCs by a representative RAW264.7 macrophage expressing SIRPa Shp2-iSNAP at indicated time points; RBCs were opsonized with anti -human RBC IgG; Arrows point to the positions of ingested RBCs;

(F) graphically illustrates a Bar graph (mean ± S.E.M.) of normalized phagocytic rate of macrophages expressing SIRPa Shp2-iSNAP or its controls against the opsonized RBCs at 37 °C for 30 min, FF mutations (tyrosine mutations) in the BTAM peptide of iSNAP; "ΔΡΤΡ", SIRPa Shp2-iSNAP without PTP domain;

"SIRP", full length SIRPa fused with YPet; "SIRP-no ΠΤΜ", ITFM-truncated SIRPa fused with YPet. * : P<0.05; Color and size scale bars are the same as Figure 2.

SIRPa Shp2-iSNAP activated by CD47 and promote phagocytic functions in macrophages.

We then examined whether an exemplary chimeric SIRPa Shp2-iSNAP protein can rewire the "don't eat me" signal of CD47/SIRPa to produce engineered macrophages with high phagocytic capabilities against tumor cells expressing CD47. We then targeted two types of cancer cells expressing high levels of CD47 and tumor- specific antigens (TSAs), viz., Toledo, a human non-Hodgkin's lymphoma (NHL) cell line representing hematologic cancer that expresses CD20, and DLDl, a human colon cancer cell line representing nonhematopoietic solid tumor that expresses EGFR. The engineered bone marrow-derived macrophages (BMDMs) led to a rapid engulfment of engaged Toledo cells opsonized by anti-CD20 antibody, accompanied by a locally activated and transient FRET signals of SIRPa Shp2-iSNAP which returned to the basal level after the completion of Toledo phagocytosis (Fig. 4A-B). The mutation of sensing tyrosines or PTP domain in SIRPa Shp2-iSNAP caused a significant reduction of the phagocytic activity of the engineered BMDMs (Fig. 4C). Similar results can be observed in engineered BMDMs when they engaged with DLD1 (ATCC ^® CCL-221 ^™ ) cells opsonized by anti-EGFR antibody (Fig. 4D-E). These results indicate that the rewiring of CD47 signaling in macrophages by an exemplary chimeric SIRPa Shp2-iSNAP can promote the antibody-mediated tumor cell phagocytosis.

Figure 4 illustrates the activation and function of SIRPa Shp2-iSNAP on phagocytosis of cancer cells:

Figures (A) and (D) illustrate Ratiometric, YPet, and DIC images of a representative BMDM expressing exemplary chimeric SIRPa Shp2-iSNAP proteins before and after incubation with 10 μg/ml rituximab-opsonized Toledo cells (A) or 2μg/ml cetuximab-opsonized DLD1 (D) at indicated time points; the arrows point to the positions where opsonized tumor cells engage the engineered macrophages; the colors of the images represent the ratiometric signals of biosensors within iSNAPs, with cold and hot colors represent the low and high levels of iSNAP activations.

(B) graphically illustrates the time course of FRET ratio of SIRPa Shp2- iSNAP in the BMDM at the region around the engaging Toledo cell in (A);

(C) and (E) graphically illustrates a Bar graph (mean ± S.E.M.) of normalized phagocytic rate of macrophages expressing different constructs as described in Figure 3 against rituximab-opsonized Toledo (C) or cetuximab-opsonized DLD1 cells (E). SIRPa Syk-iSNAP activated by CD47 and promote phagocytic functions in macrophages.

We further extended our modular approach to engineer an exemplary chimeric protein, which is new type of iSNAP employing spleen tyrosine kinase (Syk) (Syk- iSNAP) (Mocsai, Ruland et al. 2010), with the sensing peptide derived from the IT AM (immunoreceptor tyrosine-based activation) motif of FcyRIIA followed by the same FRET pair and the full-length human Syk. Syk-iSNAP was also fused to SIRPa to develop SIRPa Syk-iSNAP (Fig. 5 A). CD47-coated beads induced the FRET response of the SIRPa Syk-iSNAP (Fig. 5B). Intracellular Ca ²⁺ and Erk activation was detected by the Ca ²⁺ and Erk FRET biosensors in RAW264.7 macrophages engineered with the dark SIRPa Syk-iSNAP (Fig. 5C). SIRPa Syk-iSNAP in engineered RAW264.7 macrophages could also be activated by opsonized RBCs to cause enhanced phagocytic activity, mediated by the tyrosine phosphorylation and the kinase domain of the iSNAP (Fig. 5C-E). Therefore, our modular design and strategies can be readily extended to engineer kinase-based iSNAPs capable of sensing, rewiring, and reprogramming cells.

Figure 5 illustrates the activation and function of SIRPa Syk-iSNAP in macrophages:

(A) schematically illustrates a SIRPa Syk-iSNAP and its putative activation mechanism upon CD47 engagement;

(B) illustrates Ratiometric, YPet, and DIC images of RAW264.7 macrophages expressing SIRPa Syk-iSNAP before and after incubation with CD47-coated beads;

(C) illustrates Ratiometric and DIC images of RAW264.7 macrophages expressing dark SIRPa Syk-iSNAP together with Ca ²⁺ or Erk FRET biosensors before and after the incubation with CD47-coated beads;

(D) illustrates Ratiometric and DIC images of phagocytic processes of opsonized RBCs by a representative RAW264.7 macrophage expressing SIRPa Syk- iSNAP;

(E) illustrates a Bar graph (mean ± S.E.M.) of normalized phagocytic rate of RAW264.7 macrophages expressing SIRPa Syk-iSNAP or control constructs against the rabbit anti-human RBC IgG-opsonized RBCs at 37 °C for 30 min (FF, FF mutations in the BTAM peptide of iSNAP; K402R, a kinase dead mutant K402R in Syk kinase domain of iSNAP; other constructs are the same as described in Figure 3).

We further engineered THPl monocyte cell lines with an exemplary iSNAP protein and examined their capability in eradicating tumors in vivo with nude mice models. As shown in Figure 6A, the intracellular domain of SIRPa receptor was replaced by Fcy-IA IT AM (Immunoreceptor tyrosine-based activation motif) and fused with SYK kinase, and a Ypet (YFP) reporter to generate the iSNAP construct. In the iSNAP-engineered THPl cells, the exemplary engineered iSNAP proteins are expected to rewire the endogenous SIRPa signaling pathway and activate

phagocytosis when the "don't eat me" SIRPa receptor is engaged. We have introduced the exemplary engineered iSNAP construct by lentivirus infection into THPl monocyte cell lines. The results clearly indicate that the exemplary engineered iSNAP proteins can be stably expressed in THPl cells, measured by both

fluorescence imaging and flow cytometry (Fig. 6B-C). Quantitative PCR to measure mRNA levels of iSNAP in Fig. 6D also verified the protein expression results. We then examined the molecular profiles of the exemplary iSNAP-engineered THP1 monocytes. The results revealed that the introduced iSNAP can significantly affect the phenotypes of engineered THP1 monocytes (Fig. 7). While iSNAP did not affect the monocyte marker expression CD31, it significantly promoted the expression of CD1 lb and CD14 macrophage markers (Fig. 7A). Further results indicate that iSNAP expression can induce polarization phenotypes of engineered TFIP1 monocytes, altering the profiles of Ml and M2 polarization markers, optionally promoting the Ml phenotype (Fig. 7B). The engineered iSNAP TFIP1 cells also had significantly higher production in pro-inflammatory cytokines (Fig. 7C). Taken together, these results indicate that iSNAP engineering as provided can affect the marker profiles of host TFIP1 monocytes and specifically promote their inflammatory phenotypes.

We further examined these engineered TFIP1 cells in eradicating B- Lymphoma (Toledo) tumors in vivo. Human B-lymphoma tumor model was established by subcutaneous injection of Toledo cells in nude mice (Fig. 8 A). The tumors were then treated by local injection of parental control and iSNAP-engineered TFIP cells, guided by rituximab, an anti-CD20 monoclonal antibody, in recognizing the tumor antigen CD20. The tumor sizes were followed and measured weekly (Fig. 8B). The results showed that the iSNAP-engineered TFIP1 cells had significantly enhanced capability in eliminating B-lymphoma tumors comparing to parental control TFIP1 cells, possibly due to their increased inflammatory phenotypes.

In summary, these results demonstrate that iSNAP-engineered monocytes (or macrophages) as provided herein can significantly enhance the immunotherapeutic efficacy of monocytes (and macrophages) in eradicating target cancers or tumors, e.g., as the B-lymphoma tumors used here, in vivo, as demonstrated using a mouse animal model.

Materials and Methods

Reagents and cell culture

Fetal bovine serum was obtained from Atlanta Biologicals (Lawrenceville, USA). Rat recombinant platelet-derived growth factor BB (PDGF) and PP1 were from

Sigma Aldrich (Milwaukee, USA). Rabbit anti-human RBC antibody (ab34858) was from Abeam (Cambridge, USA). The plasmid encoding the human SIRPa was a generous gift from Dr. Umemori at Department of Biological Chemistry, University of Michigan Medical School. FDA-approved therapeutic antibodies rituximab (anti- CD20, human IgGl) and cetuximab (anti-EGFR, human IgGl) were obtained from Dr. Dahl at VA Medical Center, San Diego. Cell line HEK293T (human embryonic kidney 293T cell line), MEF (mouse embryonic fibroblast), RAW264.7 (mouse macrophage cell line), DLD1 (human colon cancer cell line), Toledo (human non- Hodgkin's B cell lymphoma cell line) and L929 cell line (murine aneuploid fibrosarcoma cell line) were from American Tissue Culture Collection (Manassas, VA), with the authentication and the verification of the absence of mycoplasma contamination. These cells were cultured in ATCC recommended conditions in a humidified incubator of 95% 0 ₂ and 5% C0 ₂ at 37°C.

Human samples

Normal human peripheral blood samples were obtained from VA San Diego Medical Center, San Diego, CA with an IRB-approved protocol (VA San Diego IRB# HI 50008). Informed consent was obtained from all human participants.

Construction of plasmids

The Shp2- or Shpl-iSNAP was constructed by fusing DNA sequences encoding BTAM peptide: GGGGDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTS (SEQ ID NO: 1), derived from ITFM of SIRPa ⁴⁰ ,

ECFP (Forward primer 1 : 5'

CAACCATACCGAATATGCGAGCATTCAGACCAGCGGCGGGTCTGGCGGGA CAAT 3' (SEQ ID NO:2),

forward primer 2: 5'

CAAAAAACCGGCGCCGCAGGCGGCGGAACCGAACAACCATACCGAATAT GCGAGC 3' (SEQ ID NO:3),

forward primer 3 : 5'

ATATTACCTATGCGGATCTGAACCTGCCGAAAGGCAAAAAACCGGCGCCG CAGG 3' (SEQ ID NO:4),

forward primer 4: 5'

CGATGGATCCTGGCGGCGGCGGCGATATTACCTATGCGGATCTGAACC 3 ' (SEQ ID NO: 5),

reverse primer: 5' ACTGCATGCGGCGGCGGTCACGAACTCC 3' (SEQ ID NO: 6),

EV linker (116aa) ⁴¹ : forward primer: 5' GTAC AAGGCATGCGAGCCTGCCAGGGGTACCAG 3 ' (SEQ ID NO: 7),

reverse primer: 5' GCTTGGTCGACAGGGACATCTGGTCCGGAACC 3' (SEQ ID NO: 8),

YPet:

forward primer: 5' CTAAAGTCGACATGTCTAAAGGTGAAGAATTATTCAC 3' (SEQ ID NO: 9),

reverse primer: 5'

ACTGAGCTCCCCGCCTTTGTACAATTCATTCATACCCTCG 3' (SEQ ID NO: 10),

and the truncated human Shp2 (aa 1-532):

forward primer: 5' ACTGAGCTCATGACATCGCGGAGATGGTTTC 3' (SEQ ID NO: 11),

reverse primer: 5' ACTGAATTCTTACTACTCTTCTTC AATCCTGCGCTG 3 ' (SEQ ID NO: 12),

or Shpl (aa 1-525):

forward primer: 5' ACTGAGCTC ATGGTGAGGTGGTTTCACCGAG 3 ' (SEQ ID NO: 13),

reverse primer: 5' ACTGAATTCTTACTAGACCTCCAGCTTCTTCTTAGTGG 3' (SEQ ID NO: 14),

in a pcDNA3.1 (Invitrogen) vector using BamHI/EcoRI restriction sites with 6xHis tag in front of start codon of the BTAM coding sequence.

The Syk-iSNAPs were constructed by fusing DNA sequences encoding BTAM2 peptide GGYMTLNPRAPTDDDKNIYLTLPPN (SEQ ID NO: 15), including oligonucleotides:

5'

ATTACCTGGTCCGTCAGGTCTCGGATCCAGGCGGCTACATGACTCTGAACC CCAGGGCACCTACTGACGATGATAAAAACATCTACCTGACTCTTCCTCCC AACGGTACCGGCGGTGAGACCAGCTCACATCACCCGGG 3' (SEQ ID

NO: 16),

derived from IT AM of FcyR IIA, or GV YTGL S TRNQET YETLKHE (SEQ ID NO: 17), oligonucleotides: 5'

GAAGATTACCTGGTCCACGTCAGGTCTCGGATCCAGGTGTTTACACGGGC CTGAGCACCAGGAACCAGGAGACTTACGAGACTCTGAAGCATGAGGGTA CCGGCGGTGAGACCAGCTCACATCACCCGGGA 3' (SEQ ID NO: 18),

derived from IT AM of FcRy, or MPDYEPIRKGQRDLYSGLNQR (SEQ ID NO: 19)

oligonucleotides: 5'

CGTCAGGTCTCGGATCCACCAGACTATGAGCCCATCCGGAAAGGCCAGCG GGACCTGTATTCTGGCCTGAATCAGAGAGGCGGGTCTGGCGGGACAGGTA CCGGCGGTGAGACCAGCTCACATCACCCGGGA 3' (SEQ ID NO:20)

derived from ITAM of CD3s) ⁴⁰ ,

PCR product of ECFP:

forward primer: 5' AG ATC GGTC TC GGCGGT ATGGTGAGC A AGGGC GAGG 3' (SEQ ID NO:21),

reverse primer: 5 ' GAGTTCGTGACCGCCGCCC ATGcgGAGACC AGCTC 3 ' (SEQ ID NO:22),

EV linker, oligonucleotides: 5'

CCTGGTCCGTCAGGTCTCCATGCGAGCCTGCCAGGAGCGCAGGCGGATCA GCTGGAGGGTCTGCAGGGGGTAGTGCAGGTGGCTCAGCTGGCGGGAGCG GCTCAGCTGGGGGATCTGCTGGTGGCAGTACCTCAGCAGGCGGTAGCGCC GGAGGTTCTGCTGGTGGCTCCGCAGGAGGGTCTGCAGGCGGTTCCGGGAG TGCAGGTGGATCTGCAGGTGGGTCAACAAGTGCTGGTGGATCCGCAGGAG GTTCAGCAGGCGGGAGTGCTGGAGGCTCTGCAGGCGGTAGCGGGAGTGCC GGTGGCAGCGCAGGGGGAAGCACTAGTGCTGGAGGCAGTGCAGGTGGCA GCGCAGGAGGCTCTGCCGGGGGAAGCGCCGGGGGCTCCGGACCAGATGT CCCTGTCGACGAGACCAGCTCACATCA 3' (SEQ ID NO:23),

PCR product of YPet:

forward primer:

5' GATCGGTCTCGTCGACTCTAAAGGTGAAGAATTATTCACTG 3 ' (SEQ ID NO:24),

reverse primer:

5' AGGGTATGAATGAATTGTACAAAGAGACC AGCTC 3' (SEQ ID NO:25, and PCR product of full length human SYK: forward primer: 5'

GATCGGTCTCTACAAAGGCGGGGAgctcGCCAGCAGCGGCATGGCTGACAG CGCCAACCAC 3' (SEQ ID NO:26),

reverse primer: 5'

C AATTACTACTATGACGTGGTGAACTAAGAATTCGAGACGAGCTC 3 ' (SEQ ID NO:27),

into BamHI/ EcoRI restriction sites of pcDNA3.1 vector using Golden Gate assembly, respectively.

FF mutant of Syk-iSNAP was constructed by replacing oligonucleotides coding wild type peptide with oligonucleotides:

5'

ATTACCTGGTCCGTCAGGTCTCGGATCCAGGCGGCTTTATGACTCTGAACC CCAGGGCACCTACTGACGATGATAAAAACATCTTTCTGACTCTTCCTCCCA ACGGTACCGGCGGTGAGACCAGCTCACATCACCCGGG 3' (SEQ ID NO:28) carrying Y to F mutations.

For the SIRPa fused iSNAPs, the PCR product of gene sequence encoding human SIRPa (aa 1-425):

forward primer: 5' AGCCCAAGCTTGCCACCATGGAGCCCGCC 3 '(SEQ ID NO:29),

reverse primer: 5' TCGGGGATCCGAATTTGTGTCCTGTGTTATTTC 3' (SEQ ID NO:30),

was inserted in 5' of iSNAP coding sequences using Hindlll/BamHI restriction sites. Deletion and point mutations of iSNAP were generated using QuikChange Site- Directed Mutagenesis Kit™ (Agilent Technologies), with primer sets:

5 ' C AGGAGTGC AAACTTCTCT AC AGCC AGCGC AGGATTGAAGAAGAG 3 ' (SEQ ID NO:31), and

5' CTCTTCTTCAATCCTGCGCTGGCTGTAGAGAAGTTTGC ACTCCTG 3 ' (SEQ ID NO:32),

for SIRPa Shp2-iSNAP ΔΡΤΡ: 5'; and

GTGAAAACCGTGGCTGTGAGGATACTGAAAAACGAGGCCA 3' (SEQ ID NO:33), and

5' TGGCCTCGTTTTTCAGTATCCTCACAGCCACGGTTTTCAC 3' (SEQ ID NO:34), for SIRPa Syk-iSNAP K402R.

For SIRPa- YPet and SIRPa-no ITIM, PCR products of human SIRPa coding sequence:

forward primer: 5' AGCCCAAGCTTGCCACCATGGAGCCCGCC 3' (SEQ ID NO:35),

reverse primer: 5'

CGTGGAATTCTGTTCCGCCAGATCCGCCCTTCCTCGGGACCTG 3 ' (SEQ ID NO:36) for aa 1-504, full length; or,

reverse primer: 5'

CGTGGAATTCTGTTCCGCCAGATCCGCCATTTGTGTCCTGTGTTATTTC 3' (SEQ ID NO:37), for aa 1-425, no ITIM),

and PCR product of YPet:

forward primer: 5'

TCCGGAATTCATGTCTAAAGGTGAAGAATTATTC ACTG 3 ' (SEQ ID NO: 38), reverse primer: 5' ACAGACCTCGAGTCATTTGTACAATTCATTCATACCCT 3' (SEQ ID NO: 39),

were inserted into Hindlll/ Xho I restriction sites of pcDNA3.1.

ECFPT65A/W66A and YPet Y66A mutations were done with primer sets:

5' CGTGACCACCCTGGCCGCGGGCGTGCAGTGC 3' (SEQ ID NO:40) and, 5 ' GC ACTGC ACGCCCGCGGCC AGGGTGGTC ACG 3 ' (SEQ ID NO:41);

5' TGGCCAACCTTAGTCACTACTTTAGGTGCTGGTGTTCAATGTTTTG 3 ' (SEQ ID NO:42), and 5'

C AAAACATTGAACACC AGCACCTAAAGTAGTGACTAAGGTTGGCCA 3 '

(SEQ ID NO:43),

respectively.

The constructed plasmids were confirmed by restriction enzyme digestion and

DNA sequencing.

Protein expression and purification

HEK cells transfected with wild type iSNAPs or their mutants were washed with cold PBS and then lysed in buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl,

1 mM EDTA, 0.2 mM PMSF, 0.2% Triton X-100 and a protease inhibitor cocktail tablet (Roche). Lysates were centrifuged at 10,000 x g at 4°C for 10 min.

Supernatants were incubated with Ni-NTA agarose (Qiagen) to capture the desired protein products via their 6-His tag at the N-termini, which were then washed with 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM imidazole. iSNAP proteins were eluted in a buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, and 100 mM imidazole.

In vitro kinase assays

Purified proteins were dialyzed overnight at 4°C in kinase buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM MgCb, and 2 mM dithiothreitol). Fluorescence emission spectra of the purified iSNAP proteins (50 nM) were measured in 96-well plates with an excitation wavelength of 430 nm using a fluorescence plate reader (TECAN, Sapphire II). To detect FRET changes of the iSNAPs, emission ratios of acceptor/donor (526 nm/478 nm) were measured at 30°C before and after the addition of 1 mM ATP and 100 nM active recombinant Src (Millipore).

In vitro phosphatase activity assays

After kinase assay, phosphatase activity was measured by adding fluorogenic 6,8- difluoro-4-methylumbelliferyl phosphate (DiFMUP; Invitrogen) as the substrate. In brief, each reaction contained 50 mM Tris pH 8, 100 mM NaCl, 10 mM MgCb, 2mM dithiothreitol, ΙΟΟηΜ Src kinase, 50nM iSNAP proteins and 50 μΜ DiFMUP in a total reaction volume of 100 μΐ in a well of 96-well plates. Reactions were initiated by the addition of DiFMUP, followed by measuring the fluorescence signal of the reaction product, 6,8-difluoro-4-methylumbelliferone, at an excitation wavelength of 355 nm and an emission of 460 nm with a plate reader (TECAN, Sapphire II). The intensity slope of the fluorescent product over time was calculated and compared. Our statistics were performed with two-tailed Student t-test.

Immunoblotting

After in vitro assays, the iSNAP proteins were resolved by SDS-PAGE. The proteins were then transferred onto a nitrocellulose membrane and blocked with 5% bovine serum albumin in TTBS buffer (50 mM Tris-HCl, 145 mM NaCl, 0.05% Tween-20, pH 7.4) for 2 hours (hr) at room temperature (RT). Membranes were further incubated with primary antibodies overnight at 4°C, washed and then incubated with HRP -conjugated secondary antibodies for 2 hr at RT. Signals were detected using SuperSignal™ Western Pico or Femto ECL Kit™ (Pierce).

Monoclonal anti-phosphotyrosine pY20 antibody was from BD Transduction laboratory (61000, 1 : 1000 dilution). And polyclonal anti-GFP antibodies (sc-8334, 1 : 1000 dilution) as well as HRP -conjugated secondary antibodies (sc-2004, sc-2005, 1 :2000 dilution) were purchased from Santa Cruz Biotechnology.

Microscopy, image acquisition and analysis

Glass-bottom dishes (Cell E&G Inc.) were coated with 10 μg ml ^A -l fibronectin (Sigma) overnight at 37°C. Transfected or electroporated cells were plated onto these dishes overnight in medium containing 0.5% FBS before imaging. During imaging, cells were maintained in medium containing 0.5% FBS with 5% C02 supplement at 37°C and a few frames of images were acquired to obtain the basal level before adding stimulation. For PDGF stimulation, PDGF was added into medium to reach final concentration 10 ng ml ^A -l . For RBCs, Toledo and DLD1 cells stimulation, those cells were washed with PBS plus 0.4% BSA, and opsonized with rabbit anti-human RBC IgG (Abeam, ab34858, 5 μg ml ^A -l), rituximab (anti-CD20 mAb, 10 μg ml ^A -l), cetuximab (anti-EGFR mAb, 2 μg ml ^A -l) or trastuzumab (anti-F£ER2 mAb, 2 μg ml ^A - 1) for 60 min at RT, respectively. After washing with PBS plus 0.4% BSA, cells were re-suspended in medium with 0.5% FBS and applied into dishes. Images were obtained by a Nikon™ eclipse Ti inverted microscope equipped with a cooled charge- coupled device camera (Cascade 512B; Photometries) with 512x512 resolution using MetaFluor 6.2™ software (Universal Imaging). The following filter sets (Chroma) were used in our experiments for FRET imaging: a dichroic mirror (450 nm), an excitation filter 420/20 nm, an ECFP emission filter 475/40 nm, and a FRET emission filter 535/25 nm. The excitation filter for ECFP at 420±20 nm was specifically selected to shift toward lower wavelength away from the peak excitation spectrum of ECFP to reduce the cross-excitation of the FRET acceptor YPet, which has significantly higher brightness than ECFP. This filter selection can minimize the effect of bleed-through on the FRET channel. The fluorescence intensity of non- transfected cells was quantified as the background signal and subtracted from the ECFP and FRET signals of transfected cells. The pixel -by-pixel ratio images of FRET/ECFP were calculated based on the background-subtracted fluorescence intensity images of ECFP and FRET. These ratio images were displayed in the intensity modified display (FMD) mode in which the color and brightness of each pixel is determined by the FRET/ECFP ratio and ECFP intensity, respectively. The emission ratios were quantified by MetaFluor™ software and our statistics were performed with two-tailed Student t-test using Excel™ (Microsoft). Recombinant human CD47 production and coating on beads

Plasmid encoding the extracellular domain of human CD47 was a gift from Dr. Dennis E. Discher (Molecular and Cell Biophysics Laboratory, University of

Pennsylvania). The plasmid was transfected into HEK293T cell using Lipofectamine 2000 (Invitrogen). After 36 hr culture, medium containing secreted CD47-CD4d3 was concentrated using a 10K MWCO Amicon™ (Millipore), and CD47-CD4d3 was biotinylated at the C terminus using a biotin-protein ligase (Avidity, LLC) and dialyzed against PBS for 24 hr. The biotinylated CD47-CD4d3 was affinity purified using monomelic avidin (Promega) and dialyzed against PBS for 24 hr. Streptavidin- coated polystyrene beads of 4 μπι diameter (Spherotech) were washed in PBS plus 0.4% BSA, and then incubated with biotinylated CD47 at RT for 30 min, followed by 3X wash and re-suspension in cell culture medium with 0.5% FBS. For IgG coating, streptavidin-coated polystyrene beads were incubated with rabbit anti-streptavidin IgG (Abeam, ab6676, 1 : 3000 dilution) at RT for 30 min.

Flow cytometry

For soluble CD47 binding assay, HEK293T cells were transfected with SIRPa Shp2-iSNAP, its mutants, SIRPa-YPet, or truncated SIRPa (no LTEVI motif) fused to YPet™ by Lipofectamine 2000™ (Life technologies). After 36 hr culture, transfected HEK293T cells were detached by treatment of 10 mM EDTA and non-specific surface residues were blocked for 10 min by PBS plus 0.4% BSA. Cells were then incubated with biotinylated CD47 at room temperature (RT) for 30 min, washed and then incubated with PE conjugated streptavidin (Life technologies, SA10041™, 1 : 1000 dilution) at RT for 30 min.

For the measurement of surface CD47, CD20 and EGFR expressions, RBCs, Toledo and DLD1 (detached by treatment of Accutase™, Innovative Cell

Technologies, Inc.) cells were washed with PBS plus 0.4% BSA, and incubated with mouse anti-human CD47 IgG (Abeam, ab3283, 1 : 1000 dilution), rituximab (1 : 1000 dilution), or cetuximab (1 : 1000 dilution) at RT for 60 min, respectively. After wash with PBS plus 0.4% BSA, cells were incubated with secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse antibody for CD47, (Life technologies, A32723, 1 : 1000 dilution), PE-conjugated rabbit anti-human antibody (Abeam, ab98596, 1 :500 dilution) for CD20 and EGFR, at RT for 60 min. For RBC opsonization, RBCs were washed with PBS plus 0.4% BSA, incubated with rabbit anti-hRBC IgG (5 μg ml ^A -l) for 60 min at RT, followed by washing and incubation with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Life technologies, R37117, 1 : 1000 dilution) at RT for 60 min.

Cells were analyzed using flow cytometry after antibody staining. The acquisition configurations using a BD Accuri™ C6 (BD Immunocytometry Systems) for different fluorescent proteins and dyes are listed as following: YPet (Ex:488, Em:530/30 nm); PE (Ex:488, Em:575/26 nm); Deep red dyes (Ex:640, Em:660/20 nm) .

Differentiation of bone marrow derived macrophages

Bone marrow from wild-type C57BL/6 12-20 weeks female mice were harvested from freshly isolated femurs, tibiae, and humeri (S000227M, UCSD Animal Care Program/I ACUC). After removal of connective tissues and muscles, bone marrow cells were flushed out and single cell suspensions were made by pipetting and passing bone marrow through a sterile 70-μπι filter (BD Falcon). Remaining RBCs were lysed by ACK buffer (0.15M H ₄ C1, 10mM KHCO ₃ , O. lmM EDTA). Macrophages were differentiated by incubating bone marrow cells for 7 days with complete RPMI 1640, supplemented with 10% L929-conditioned medium (containing M-CSF).

Macrophages were harvested after 15 -minute incubation with 10 mM EDTA.

Electroporation of macrophages and phagocytosis assay

iSNAPs were introduced into RAW 264.7 macrophages and BMDMs by electroporation. Briefly, 4 x 10 ⁶ RAW 264.7 or BMDMs were re-suspended in 240 μΐ of RPMI 1640 with 20 μg plasmids, and then electroporated (250V, 950 mF,∞ Ω). For phagocytosis assays, transfected macrophages were plated in 35 mm dishes at 20% confluency. RBCs, Toledo and DLDl (detached by treatment of Accutase) cells were washed 3 times with PBS, and incubated with CellTracker™ Deep Red dye (Life technologies) in PBS (1 μΜ for RBCs labeling, 0.3 μΜ for Toledo and DLDl cells labeling) for 30 min at RT followed by 3 times of wash with PBS. RBCs were added to RAW264.7 macrophages at a ratio of 10: 1 and allowed to incubate at 37°C for 30 min in the presence of rabbit anti-hRBC IgG (5 μg ml ^A -l). Free RBCs were removed by washing with PBS plus 0.4% BSA; bounded RBCs were lysed by adding ACK buffer ( H ₄ C1 31 mM, KHC0 ₃ 2 mM, EDTA 20 μΜ) for 2 min. Toledo or DLDl cells were added to BMDMs at a ratio of 10: 1 and allowed to incubate at 37 ° C for 4 hrs in the presence of rituximab (10 μg ml ^A -l) for Toledo cells or cetuximab (2 μg ml ^A -l) for DLD1 cells, respectively. Free Toledo or DLD1 cells were removed by washing with PBS; bounded cells were dissociated by incubation with EDTA 10 mM at 37°C for 20 min and dissociation efficiency was confirmed under

microscope ^{42 43} . Macrophages were detached by scratching with plastic lifter and kept in suspension. Engineered macrophage population defined as YPet ⁺ cells were measured by flow cytometry and gated, and the signal of ingested cells (Deep Red ⁺ , Ex:640 nm, Em:660/20 nm) was determined within engineered macrophage population. Expression level of SIRPa Shp2-iSNAP or control constructs represented by the intensity of YPet signal (Ex: 488 nm, Em: 575/26 nm) were recorded simultaneously. Doublets discrimination flow cytometry was used to further distinguish internalized from externally bound tumor cells. The phagocytic potential (mean ± s.e.m. from 6-8 measurements) of different macrophage groups were normalized to the level of phagocytosis of target cells by macrophages expressing SIRPa- YPet. Each dot represents mean normalized phagocytic rate from an individual experiment, error bars represent s.e.m.

Statistical analysis

Statistical analysis was performed by two-sided Mann-Whitney U test in

MATLAB, with the p-values adjusted for comparison of multiple groups. Statistics of figure Id was done by exact randomized permutation test. All the center values are mean ± s.e.m. A significant difference was determined by P value < 0.05. All the experiments were replicated at least 3 times and represented biological replicates.

Data availability statement. The DNA sequences of iSNAP have been deposited into NCBI GenBank (Shp2-iSNAP: MF434748, SIRPa Shp2-iSNAP:

MF434749, SIRPa Shp2-iSNAP ΔΡΤΡ: MF434750, SIRPa Syk-iSNAP:

MF434751, SIRPa YPet: MF434752, SIRPa no ITFM YPet: MF434753). The data that support the findings of this study are available from the corresponding author upon request.

A number of embodiments of the invention have been described.

Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.