HK1201757B - High affinity sirp-alpha reagents - Google Patents

Description

high affinity SIRP-alpha reagents

Government authority

The invention was made with government support under contracts CA086017, HL058770 and CA139490 awarded by the national institutes of health. The government has certain rights in the invention.

Background

the process requires specific and selective removal of unwanted cells, distinguishing healthy cells from unwanted/senescent/dying cells, which exhibit markers or ligands known as "eat-me" signals, i.e., "change self," which in turn can be recognized by receptors on phagocytic cells.

CD47 is a widely expressed transmembrane glycoprotein with a single Ig-like domain and 5 transmembrane regions, which functions as a cellular ligand for SIRP α, with binding mediated by the NH2 terminal V-like domain of SIRP α is expressed primarily on myeloid cells, including macrophages, granulocytes, myeloid Dendritic Cells (DCs), mast cells and their precursors, including hematopoietic stem cells.the structural determinants on SIRP α that mediate CD47 binding are discussed by Lee et al (2007) J.Immunol.179: 7741-7750, Hatherley et al (2007) J.B.C.282: 14567-75, and the role of SIRP α dimerization in CD47 binding is discussed by Lee et al (2010) J.B.C.285: 37953-63.

Consistent with the inhibition of normal cellular phagocytosis by CD47, there is evidence that: it is transiently upregulated on Hematopoietic Stem Cells (HSCs) and progenitor cells before and during its migratory phase, and the level of CD47 on these cells determines the likelihood that they will be engulfed in vivo. CD47 is also constitutively upregulated on many cancers. Tumor cells overexpress CD47 can increase pathogenicity by allowing cells to escape phagocytosis.

Summary of The Invention

high affinity SIRP α polypeptides and analogs thereof are provided, which are referred to herein as high affinity SIRP α agents that are sequence variants of native human SIRP α proteins and have utility for blocking the interaction between native SIRP α proteins and their receptor CD47, in vivo and in vitro methods that provide increased affinity amino acid changes are localized to the d1 domain, and thus the high affinity SIRP α agents of the invention comprise the d1 domain of human SIRP α with at least one amino acid change relative to the wild-type sequence within the d1 domain.

in one embodiment, the invention provides a soluble high affinity SIRP α reagent that lacks a SIRP α transmembrane domain and that comprises at least one amino acid change within the d1 domain that increases the binding affinity of the SIRP α polypeptide to CD47 relative to wild-type human SIRP α.

the invention also includes pharmaceutical formulations of the high affinity SIRP α agents in combination with pharmaceutically acceptable excipients.

in some embodiments, methods are provided for manipulating targeted phagocytosis of cells, e.g., by macrophages or other mammalian phagocytic cells, in which methods cells expressing CD47 are contacted with a high affinity SIRP α agent of the invention in an amount effective to block the interaction between endogenous SIRP α and CD 47.

in related embodiments, phagocytic tumor cells, such as solid tumors, e.g., carcinomas, sarcomas, melanomas, and the like, leukemia, lymphomas, and the like, are targeted by contacting the tumor cells with a dose of a high affinity SIRP α polypeptide effective to block or mask CD47 on the cell surface, allowing for engulfment of targets that are not normally phagocytized, and allowing for engulfment of targets that are not normally phagocytized, it may be advantageous to administer an effective dose of the high affinity SIRP α polypeptide to the patient to prevent interaction between CD47 and SIRP α, which increases clearance of the tumor cells via phagocytosis.

such labeled reagents may be used for imaging purposes in vitro or in vivo, such as imaging of tumors.

Brief Description of Drawings

figure 1A-1e. directed evolution of high affinity SIRP α variants a. cd47 is blocked by soluble high affinity SIRP α (left) under basal conditions CD47 expression on cancer cells activates SIRP α 0 on macrophages, leading to a schematic representation of yeast surface display of high affinity SIRP α 1 protein competitively antagonizing CD47 and preventing cancer cells from phagocytosis (right) and preventing engagement with SIRP α on macrophages, inhibiting phagocytosis.b.sirpa V group Ig domain (domain 1, D1.) yeast clones (grey cells) give different variants of SIRP α (colored inflection point.) insert indicates that SIRP α is linked to yeast cell surface by fusion with Aga2 and selects with CD47a biotinylated SIRP α C. the summary of the sequence of the engineered SIRP α variants and SPR affinity measurements of SPR affinity measurements and the corresponding CD 3619℃ biotinylated SIRP α variants and SPR affinity binding sequence thereof are depicted in top indicator 1. fig. red color map of the top of the wild type allele binding residues (pd) and red color of the complex of the wild type SIRP α binding site pd +.

figure 2A-2h. high affinity sirpa variants block CD47 and stimulate phagocytosis in vitro. a. wild type sirpa allele 1(WTa1, pink), anti-CD 47 clone B6H12 Fab fragment (orange), or two high affinity sirpa 0 variants (FD6, CV1, green) on Raji lymphoma cells dose-response curves that antagonize CD47 as CD 3985, cells stained with titration concentrations of CD47 blocking agent competing with 100nM Alexa Fluor 647-conjugated sirpa 1 tetramer, phagocytosis assay with CFSE labeled Raji lymphoma cells and RFP + macrophages with vehicle phagocytic control (PBS) or fused with human IgG4, high affinity sirpa 2 variant (CV 1-hgg 4) as a high phagocytic antibody to CD 19 α phagocytic macrophage alone (CD 631) or with a high affinity to CD 19 g β -macrophage cell phagocytosis receptor, indicated by a high affinity of sirpa β -phagocytosis receptor ligand binding, or high affinity of sirpa β -IgG 12 g β -CD 19B + phagocytosis receptor, indicated by the presence of a high affinity of a CD 19 phagocytic antibody alone (WT β -phagocytic antibody alone) in a t β -phagocytosis of a t β -macrophage cell phagocytosis receptor, CD 19, indicated by a high affinity of a t β -phagocytic antibody alone, or high affinity of a t β -macrophage cell phagocytic antibody alone (CD 19, indicated by a t β -phagocytic assay with No. 2H β -phagocytic macrophage as a t β -phagocytic antibody, no-phagocytic antibody alone, No. 2 t β -phagocytic antibody, No. 2H 2, No. 2 phagocytic antibody, No. 2H 2, No. 2B phagocytic antibody, No. 2, No. 12, No. 2, No. 12, No. 2H 2, No. 12, No. 2, No. 12B, No. 12, No..

a. mice implanted with GFP-luciferase + DLD-1 cells survive treatment with vehicle control (PBS, red) or high affinity SIRP α -hIgG4 fusion protein (CV1-hIgG4, blue.) b. mice implanted with GFP-luciferase + DLD-1 cells survive a representative analysis of human Fc bound to the surface of whole blood cells from treated animals in b. blood analysis of treated animals in D.b shows the mean and standard deviation of four animals per cohort over time. dashed lines show the lower limit of normal values. GFP-639-V cancer cells grow after treatment with therapy as indicated, as assessed by bioluminescence imaging. median values of bars, points show the median values from individual implanted mice, g-g +639-V cells after implantation of GFP-g mice, g-g +639-V cancer cells after treatment with therapy, g-g + g.

figure 4A-4F. high affinity SIRP α monomer enhances the efficacy of monoclonal antibodies in vivo a. gfp-luciferase + Raji lymphoma tumors grow after daily treatment with PBS (red), CV1 monomer (orange), rituximab (green), or rituximab plus CV1 monomer (blue), as assessed by bioluminescence imaging the bars indicate median values, dots indicate values from individual mice, b. representative bioluminescence images of mice on day 29 post implantation red circles indicate sites of primary tumors, red arrows indicate sites of metastasis to axillary lymph nodes c. average tumor volume measurements from mice of a. error bars depict standard deviation d. survival from lymphoma bearing mice of a. e.gfp-luciferase + Raji lymphoma tumors stop after treatment with PBS (red), CV1 monomer (orange), alemtuzumab (green), or alemtuzumab plus 1 monomer (blue) every two times the growth of mice per week, as indicated by the black arrows indicate that the mice had stopped growth relative to the individual mice, as assessed by PBS, (g. < g > 360.g. < CV 360, no more mean tumor volume measurements relative to the growth of mice from a.

fig. 5A-5b. library design and sequences from first generation selection a. left: random positions of 'contact residue' libraries with possible amino acid variants and a table that the random positions are located within sirpa.right: location and description of random positions of non-contact 'core residue' libraries.sirpa is depicted in green, CD47 is depicted in dark red, and random positions are represented as space filling side chains.b. summary of sequences of SIRP α variants obtained after first generation selection.

FIG. 6. library design for second generation selection A table of random positions and possible amino acids and positions of variable residues within the SIRPa structure for the second generation libraries SIRPa is depicted in green, CD47 in dark red, and random positions are indicated as space filling side chains.

Fig. 7.FD 6: representative electron density plots of CD47 complexes. 2mFo-DFc electron density plots outlined at 2.0 σ. The simulated amino acid residues were depicted as rods, with FD6 residues being yellow and CD47 residues being green. Pyroglutamic acid residue 1 of CD47 is indicated as PCA1 above the corresponding residue and density.

FIGS. 8A-8C. high affinity SIRP α variants efficiently bind and block CD47.A. binding to wild type SIRP α allele 1 monomer on Jurkat leukemia cells (WTa1 monomer, pink), wild type SIRP α allele 1 tetramer (WTa1 tetramer, chestnut) or high affinity SIRP α variant (FD6, FA4, green) titration curves error bars indicate standard deviation B. CD47 blocking assay for Jurkat cells addition of CD47 antagonist competes with Alexa Fluor647 conjugated wild type SIRP α tetramer first generation SIRP α mutant as monomer (1A5 monomer, blue green), second generation SIRP α mutant as monomer (FD6 monomer, green), second generation SIRP α mutant as Fc fusion with human IgG 84 (FD6-hIgG4, blue) and anti-SIRP α mutant as anti-IgG 47 (FD 363629 monomer, green), second generation SIRP α mutant as Fc fusion with human IgG 84 (FD6-hIgG4, blue) and anti SIRP α mutant as IgG clone (FD 599-orange) clone, WTT 5826, yellow, orange, IgG 639-IgG, IgG 639-binding to wild type SIRP α variant IgG2, IgG 599-IgG, IgG 599-binding to Jurkat leukemia cells, and yellow.

figure 9 high affinity SIRP α -Fc variants limited the growth of DLD-1 colon cancer cells in vivo tumor growth curves after treatment with vehicle (PBS, red) or high affinity SIRP α -hIgG4 fusion protein (CV1-hIgG4, blue) as measured by bioluminescence imaging of the peritoneal cavity of treated mice.

fig. 10A-10d. treatment with high affinity sirpa-Fc variants caused macrophage infiltration and affected the red cell index a. an anatomically accessible subcutaneous tissue mass from CV1-hIgG4 treated mice, left white light, right GFP fluorescence, dashed ovals surrounded two superficial tumor nodules, an asterisk marked macrophage enriched stroma infiltration, 5 mm.b. hematoxylin and eosin staining of accessible subcutaneous tissue mass from CV1-hIgG4 treated mice, demonstrating the presence of infiltrated macrophages, tumor nodules visible above the left of the image, surrounding it with inflammatory infiltration, inset shows representative macrophages in the dashed box outline region, c. immunohistochemically stained macrophage cell marker F4/80 in the accessible subcutaneous tissue mass from CV1-hIgG4 treated mice, histochemical stained tumor nodules visible in the right portion of the image, stained macrophage infiltration in the left border region, black inset shows that macrophage infiltration of macrophage cell stain in the visible macrophage cell area, macrophage infiltration area, white macrophage cell-macrophage infiltration area, white cell fluorescence, white light, GFP fluorescence, dashed arrows 34, white blood cell staining of macrophage infiltration, macrophage cell line, macrophage infiltration, and yellow macrophage cell line staining after the dashed arrow 34, macrophage infiltration process, no evidence of macrophage infiltration, no macrophage cell staining of macrophage cell, no macrophage cell staining in the dashed arrows, no macrophage cell line 34, see the dashed arrows 369-GFP, no.

fig. 11A-11c. treatment with high affinity SIRP α monomer did not cause erythrotoxicity a. hematocrit measurements from mice during treatment with the indicated therapies b. hemoglobin levels from mice during treatment with the indicated therapies c. absolute red blood cell counts from mice during treatment with the indicated therapies a-c.ns is not significant black arrows indicate the start and stop of daily treatment.

figure 12. high affinity SIRP α variants show safety in non-human primates cynomolgus monkeys were treated with a single intravenous injection of high affinity SIRP α -Fc (FD6-hIgG4, red) or a series of dose escalating high affinity SIRP α monomers (FD6 monomer, blue).

fig. 13A-13c. radiolabeled high affinity SIRP α variants are effective as noninvasive imaging agents for cancer a.nsg mice were implanted with DLD-1 human colon cancer cells subcutaneously in the right upper flank.tumor-bearing mice were injected with Cu-64 labeled FD6 monomer and imaged by PET scan.red arrows indicate uptake of the tumor, black arrows indicate uptake by the kidney.the upper panel shows a back image and the lower panel shows cross sections through the tumor.m 2, M3 represent images from two independent animals.

fig. 14A-14F. fluorescence activated cell sorting of macrophages demonstrates that high affinity SIRP α variants induce phagocytosis of cancer cells a. major human macrophages and GFP + DLD-1 colon cancer cells were co-cultured in the presence of 100nM CV1-hIgG 4. phagocytosis was quantified as the percentage of CD45+ macrophages that became GFP +.

figure 15 high affinity SIRP α variants synergize with trastuzumab (trastuzumab) against Her2+ breast cancer major human macrophages were co-cultured with GFP + SK-BR-3 breast cancer cells and indicated therapies.

fig. 16A-D high affinity SIRP α variants induce maximal potency in the presence of tumor bound antibody Fc chains a shows a schematic of high affinity SIRP α variants b. analysis of gel filtration shows that high affinity SIRP α -Fc fusion proteins are purified as a single species (elution volume 13.44) with limited aggregation.c. phagocytosis assays using RFP + mouse macrophages and GFP + human lymphoma Raji cells CD47 blockade with high affinity SIRP α variant CV1 monomer or anti-CD 47 Fab fragment produced marginal increases in phagocytosis, while treatment with high affinity SIRP α -Fc or intact anti-CD 47 antibody produced increased phagocytosis.

figure 17A-17D high affinity SIRP α variants bind and block other mammalian CD47 lineages.a. high affinity SIRP α variant FD6 but not wild type allele 1 human SIRP α binds to mouse CT26 colon cancer cells.b. high affinity SIRP α variant FD6-Fc blocks binding of wild type mouse SIRP α tetramer to mouse CD47 displayed on the surface of yeast.c. high affinity SIRP α variant CV1 binds to binding of mouse cd47.d.100nm wild type allele 2 SIRP α -Fc displayed on the surface of yeast, high affinity SIRP α -Fc or anti-CD 47 antibodies (clones B6H12 and 5F9) to canine sirk cells as detected IgG by flow cytometry using anti-human secondary antibodies.

Definition of

In the following description, a number of terms conventionally used in the field of cell culture are utilized. In order to provide a clear and consistent understanding of the specification and claims, as well as the scope to be given such terms, the following definitions are provided.

for the purposes of development, binding may be performed under experimental conditions, e.g., using an isolated protein as a binding partner, using a portion of the protein as a binding partner, using a yeast display of the protein or protein portion as a binding partner, etc.

for physiologically relevant purposes, binding of SIRP α to CD47 is generally an event between two cells, each of which expresses one of the binding partners.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, those encoded by the genetic code, and those amino acids that are later modified, such as hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine.

The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and/or being treated. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" encompass, but are not limited to, individuals having cancer. The subject may be a human, but also includes other mammals, particularly those used as laboratory models of human disease, e.g., mice, rats, etc.

The terms "cancer," "neoplasm," and "tumor" are used interchangeably herein and refer to cells that exhibit self-regulated growth such that they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic and non-metastatic cells. Cancer is known for almost every tissue. The phrase "cancer burden" refers to the amount of cancer cells or cancer volume in a subject. Reducing cancer burden accordingly refers to reducing the number of cancer cells or the volume of cancer in a subject. As used herein, the term "cancer cell" refers to any cell that is a cancer cell or is derived from a cancer cell, e.g., a clone of a cancer cell. Many types of cancer are known to those skilled in the art, including solid tumors, such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, and the like, as well as circulating cancers such as leukemias. Examples of cancer include, but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.

The "pathology" of cancer includes all phenomena that compromise the health of the patient. This includes, but is not limited to, abnormal or uncontrolled cell growth, metastasis, interference with normal function of adjacent cells, release of abnormal levels of cytokines or other secreted products, inhibition or exacerbation of inflammatory or immune responses, neoplasia, pre-malignancy, invasion of surrounding or distant tissues or organs such as lymph nodes, and the like.

As used herein, the terms "cancer recurrence" and "tumor recurrence" and grammatical variations thereof refer to the further growth of a tumor or cancer cells following diagnosis of cancer. In particular, recurrence may occur when further cancer cell growth occurs in the cancerous tissue. Similarly, "tumor spread" occurs when tumor cells disseminate into local or distal tissues and organs; thus, tumor spread includes tumor metastasis. "tumor invasion" occurs when tumor growth spreads locally outward to impair the function of the involved tissue by suppressing, destroying or preventing normal organ function.

As used herein, the term "metastasis" refers to the growth of a cancerous tumor in an organ or body part that is not directly connected to the organ of the original cancerous tumor. It is understood that metastasis includes micrometastases, which are the presence of undetectable amounts of cancer cells in organs or body parts that are not directly connected to the organ of the original cancerous tumor. Metastasis can also be defined as several steps of a process, such as detachment of cancer cells from the original tumor site and migration and/or invasion of cancer cells to other parts of the body.

The term "sample" in reference to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after they have been obtained, for example by treatment with reagents; washing; or enriching certain cell populations, such as cancer cells. The definition also includes samples that have been enriched for a particular type of molecule, e.g., nucleic acid, polypeptide, etc. The term "biological sample" encompasses clinical samples and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cultured cells, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. "biological sample" includes samples obtained from cancer cells of a patient, e.g., samples comprising polynucleotides and/or polypeptides obtained from cancer cells of a patient (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides); and a sample comprising cancer cells from the patient. A biological sample that includes cancer cells from a patient can also include non-cancer cells.

The term "diagnosis" is used herein to refer to the identification of a molecular or pathological state, disease or condition, for example, the identification of a molecular subtype of breast, prostate or other types of cancer.

The term "prognosis" is used herein to refer to predicting the likelihood of death or progression from cancer, including recurrence, metastatic spread, and drug resistance of neoplastic disease, such as ovarian cancer. The term "prediction" is used herein to refer to a predicted or estimated behavior based on observation, experience, or scientific reasoning. In one example, a physician can predict the likelihood that a patient will survive a surgical removal of a primary tumor and/or chemotherapy for a period of time without cancer recurrence.

As used herein, the terms "treatment", "treating" and the like refer to the administration of an agent or the performance of a procedure for the purpose of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of achieving a partial or complete cure of the disease and/or disease symptoms. As used herein, "treatment" may include treatment of tumors in mammals, particularly humans, and includes: (a) preventing the disease or disease symptoms from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease (e.g., including diseases that may be associated with or caused by the primary disease); (b) inhibiting the disease, i.e., stopping its development; and (c) alleviating the disease, i.e., causing the disease to regress.

Treatment may refer to any marker of success in the treatment or alleviation or prevention of cancer, including any objective or subjective parameter, such as elimination; (iii) mitigation; reduce symptoms or make the patient more resistant to the disease state; slowing the rate of degeneration or decline; or make the degradation endpoint more debilitating. Treatment or alleviation of symptoms can be based on objective or subjective parameters; including physician examination results. Thus, the term "treating" includes administering a compound or agent of the invention to prevent or delay, alleviate, or halt or inhibit the development of the symptoms or conditions associated with cancer or other diseases. The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptom, or disease side effect in a subject.

"combination with …", "combination therapy", and "combination product" refer in certain embodiments to the simultaneous administration of a first therapeutic agent and a compound used herein to a patient. When administered in combination, each component may be administered simultaneously or sequentially in any order at different time points. Thus, each component may be administered separately but close enough in time to provide the desired therapeutic effect.

in some embodiments, treatment is achieved by administering a high affinity SIRP α reagent of the invention in combination with a cytotoxic agentmitomycin, asparagine, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, Dacarbazine (DTIC), actinomycin D, docetaxel, doxorubicin, dronabinol, duocarmycin (duocarmycin), etoposide (etoposide), filgrastim (filgrastim), fludarabine (fludarabine), fluorouracil, gemcitabine (gemcitabine), granisetron (granisetron), hydroxyurea, noroxyphenicol, isocyclovir, interferon α, irinotecan, lansoprazole, levamisole, levofloxacin, mesterone, mestranol, metoclopramide (metoclopramide), metoclopramide, methotrexate, mitoxantrone (methamidone), methotrexate, mitoxantrone, methotrexate, and mitoxantrone^TM) Pilocarpine (pilocarpine), prochlorperazine (prochloroperazine), rituximab (rituximab), saprine, tamoxifen (tamoxifen), taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate.

in other embodiments, administration of the high affinity SIRP α agents of the invention is combined with an effective dose of an agent that increases the hematocrit of the patient, such as an Erythropoietin Stimulating Agent (ESA)(darbepotin alfa)), (darbepotin alfa) (darb (r,(alfa-epoetin),(peginesatide)、And the like.

Other combination therapies include administration with cell-specific antibodies, such as antibodies selective for tumor cell markers, radiation, surgery, and/or hormone deprivation (Kwon et al, proc.natl.acad.sci.s.a., 96: 15074-9, 1999). Angiogenesis inhibitors may also be combined with the methods of the invention.

Many antibodies are currently used clinically for the treatment of cancer, and others are in different stages of clinical development. For example, there are many antigens and corresponding monoclonal antibodies used to treat B cell malignancies. One target antigen is CD 20. Rituximab is a chemically unconjugated monoclonal antibody directed against the CD20 antigen. CD20 has important functional roles in B cell activation, proliferation and differentiation. The CD52 antigen was targeted by the monoclonal antibody alemtuzumab indicated for the treatment of chronic lymphocytic leukemia. CD22 is targeted by a number of antibodies and recently demonstrated efficacy in combination with toxins in chemotherapy-resistant hairy cell leukemia. Two new monoclonal antibodies tositumomab (tositumomab) and ibritumomab (ibritumomab) targeting CD20 have been submitted to the U.S. Food and Drug Administration (FDA). These antibodies are conjugated to radionuclides. Alemtuzumab (Campath) for the treatment of chronic lymphocytic leukemia; gemtuzumab ozogamicin (Mylotarg) for the treatment of acute myeloid leukemia; ibritumomab tiuxetan (Zevalin) for use in the treatment of non-hodgkin lymphoma; panitumumab (Vectibix) is used for the treatment of colon cancer.

Monoclonal antibodies that have been used in the methods of the invention for solid tumors include, but are not limited to, eculizumab (edrecolomab) and trastuzumab (herceptin). Epirubizumab targets the 17-1A antigen found in colon and rectal cancers and has been approved in europe for these indications. Trastuzumab targets the HER-2/neu antigen. This antigen is found in 25% to 35% of breast cancers. Cetuximab (Erbitux) is also of interest for use in the methods of the invention. The antibodies bind to the EGF receptor (EGFR) and have been used to treat solid tumors, including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).

for such purposes, the SIRP α agents of the invention are administered in combination with, for example, rituximab to deplete B cells in inflammatory diseases and autoimmune disorders, alemtuzumab against multiple sclerosis, OKT3 for immunosuppression, other therapeutic antibodies for bone marrow transplant conditioning, and the like.

"Simultaneous administration" of a cancer treatment drug, ESA or tumor targeting antibody and a pharmaceutical composition of the invention means administration of the high affinity SIRP α agent at a time when the drug, ESA or antibody and the composition of the invention will both have a therapeutic effect.

As used herein, the phrase "disease-free survival" refers to the absence of such tumor recurrence and/or spread and fate of the patient after diagnosis, relative to the effect of cancer on the patient's lifespan. The phrase "overall survival" refers to the fate of a patient after diagnosis, regardless of the likelihood that the patient's cause of death is not directly attributable to the effects of cancer. The phrases "likelihood of disease-free survival", "risk of recurrence", and variants thereof refer to the probability that a tumor will recur or spread in a patient after diagnosis of cancer, wherein the probability is determined according to the methods of the invention.

As used herein, the term "related" or "… related" and similar terms refer to a statistical association between instances of two events, wherein an event includes a number, a data set, and the like. For example, when an event includes a number, a positive correlation (also referred to herein as a "direct correlation") means that as one increases, the other also increases. Negative correlation (also referred to herein as "anti-correlation") means that when one increases, the other decreases.

"dosage unit" refers to physically discrete units suitable as unitary dosages for the particular individual to be treated. Each unit may contain a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for a unit dosage form may depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds.

"pharmaceutically acceptable excipients: means excipients that can be used in the preparation of pharmaceutical compositions, which are generally safe, non-toxic and satisfactory, and include excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or in the case of aerosol compositions, gaseous.

"pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed wherein the acidic protons present in the compound are capable of reacting with an inorganic or organic base. Suitable inorganic salts include those formed with alkali metals such as sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include those formed with organic bases such as amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane and arene sulfonic acids, such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compound, e.g. C_1-6An alkyl ester. When two acidic groups are present, the pharmaceutically acceptable salt or ester can be a mono-acid mono-salt or ester, or a di-salt or ester; and similarly, when more than two acidic groups are present, some or all of such groups may be salified or esterified. The compounds named in the present invention may be present in unsalted or unesterified form or in salified and/or esterified form, and the naming of such compounds is intended to include the original (unsalted and unesterified) compounds and pharmaceutically acceptable salts thereofAnd esters. Moreover, certain compounds named in the present invention may exist in more than one stereoisomeric form, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers.

The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent materials that can be administered to humans without producing undesirable physiological effects to the extent that administration of the composition would be prohibited.

By "therapeutically effective amount" is meant an amount sufficient to effect treatment of a disease when administered to a subject to treat the disease.

Detailed description of the embodiments

in one embodiment, the invention provides a soluble SIRP α variant polypeptide, wherein the polypeptide lacks a SIRP α transmembrane domain and includes at least one amino acid change relative to a wild-type SIRP α sequence, and wherein the amino acid change increases the affinity of the SIRP α polypeptide to bind CD47, e.g., by reducing the off-rate by at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or more.

in some embodiments, the amino acid change comprises a substitution, deletion, addition, insertion, etc. of one or more amino acids.

the high affinity SIRP α reagent of the present invention includes at least one amino acid modification within the d1 domain of SIRP α, as shown in SEQ ID NO:1, and corresponding to residues 31 to 149 of the native human full length human protein the high affinity SIRP α reagent may consist of all or a portion of the d1 domain, and may further include one or more amino acids from the SIRP α other than the d1 domain, or may include an amino acid sequence other than SIRP α including, but not limited to, an immunoglobulin Fc region sequence.

the high affinity SIRP α polypeptide may be at least about 100 amino acids in length, at least about 110, at least about 120, at least about 150, at least about 200 amino acids in length, up to the full length of the wild-type protein in the transmembrane domain, i.e., about 343 amino acids in length, and optionally fused to a heterologous polypeptide, such as an immunoglobulin Fc.

In other embodiments, short peptides within the d1 domain and comprising at least one amino acid change set forth herein are used, wherein such peptides typically comprise a contiguous string of amino acids from the sequences set forth herein, no more than 10 amino acids in length, no more than 15 amino acids in length, no more than 20, no more than 25, no more than 30 amino acids, no more than 35 amino acids, no more than 40 amino acids, no more than 45 amino acids, no more than 50 amino acids, no more than 55 amino acids, no more than 60 amino acids, no more than 65 amino acids, no more than 70 amino acids, no more than 75 amino acids, no more than 80 amino acids, no more than 85 amino acids, no more than 90 amino acids, no more than 95 amino acids, no more than 100 amino acids.

in some embodiments, amino acid changes of the high affinity SIRP α polypeptide are made at one or more amino acids within the hydrophobic core residue group of SIRP α, including but not limited to residues (numbered by the wild-type sequence of the d1 domain as shown in SEQ id no: 1) L4, V6, V27, I36, F39, L48, I49, Y50, F57, V60, M72, F74, I76, V92, F94 and F103.

In other embodiments, the amino acid change is made at one or more amino acids within a set of contact residues that interact with CD47, including but not limited to a29, L30, I31, P32, V33, G34, P35, Q52, K53, E54, S66, T67, K68, R69, F74, K93, K96, G97, S98 and D100(SEQ ID NO: 1).

In other embodiments, the amino acid changes are made at two or more, three or more, four or more, five or more, and no more than 14 amino acids within the combined set of contact residues and set of hydrophobic core residues defined above.

In some embodiments, the amino acid change is made at one or more of the amino acids within the group including, but not limited to: residues L4, V6, a21, V27, I31, E47, K53, E54, H56, S66, V63, K68, V92, F94, and F103, or combinations thereof, for example at two or more, three or more, four or more, five or more, six or more, seven or more, and no more than 15 residues.

in some embodiments, the high affinity SIRP α reagent comprises at least one amino acid change selected from the group consisting of (1) L4V, (L4I), (2) V6I, (V6L), (3) A21V, (4) V27I, (V27L), (5) I31T, (I31S), (I31F), (6) E47V, (E47L), (7) K53R, (8) E54Q, (9) H56P; H56R, (10) S66T, (S66G), (11) K68R, (12) V92I, (13) F94L, (F94V), (14) V63I, and (15) F103V, hi some embodiments, the high affinity SIRP α polypeptide comprises a modification selected from the group consisting of (1) to (15), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 11, 13, 15, and conservative combinations thereof.

The set of amino acid changes may include combinations of the above, for example:

V27I or V27L; K53R; S66T or S66G; K68R; and F103V.

L4V or L4I; V27I or V27L; E47V or E47L; K53R; E54Q; S66T or S66G; K68R; V92I; and F103V.

L4V or L4I; V6I or V6L; A21V; V27I or V27L; I31T, I31S or I31F; E47V or E47L; K53R; H56P or H56R; S66T or S66G; K68R; and F94L or F94V.

V6I or V6L; V27I or V27L; I31T, I31S or I31F; E47V or E47L; K53R; E54Q; H56P or H56R; S66T or S66G; V92I; and F94L or F94V.

L4V or L4I; A21V; V27I or V27L; I31T, I31S or I31F; E47V or E47L; K53R; E54Q; H56P or H56R; S66T or S66G; F94L or F94V; and F103V.

L4V or L4I; V6I or V6L; V27I or V27L; I31T, I31S or I31F; E47V or E47L; K53R; H56P or H56R; S66T or S66G; K68R; V92I; and F94L or F94V.

L4V or L4I; V6I or V6L; I31T, I31S or I31F; E47V or E47L; K53R; H56P or H56R; S66T or S66G; V92I; and F103V.

V6I; V27I; I31F; E47L; K53R; E54Q; H56P; and S66T.

L4V; V6I; V27I; I31F; E47V; K53R; E54Q; H56P; V63I; S66T; K68R; and V92I.

V6I; V27I; I31T; E47V; K53R; E54Q; H56P; S66G; K68R; V92I; and F103V.

V6I; V27I; I31F; E47V; K53R; E54Q; H56P; S66T; and V92I.

in some embodiments, the high affinity SIRP α polypeptide comprises the following sets of amino acid changes:

for example, { V27I; K53R; S66T; S66G; K68R; F103V };

for example, { L4V; V27L; E47V; K53R; E54Q; S66G; K68R; V92I };

for example, { L4V; V6I; A21V; V27I; I31T; E47L; K53R; H56P; S66T; K68R; F94L };

for example, { V6I; V27I; I31S; I31F; E47V; K53R; E54Q; H56P; S66G; V92I; F94L };

for example, { L4I; A21V; V27I; I31F; E47V; K53R; E54Q; H56R; S66G; F94V; F103V };

for example, { L4V; V6I; V27I; I31F; E47V; K53R; H56R; S66G; K68R; V92I; F94L }; or

For example, { L4V; V6L; I31F; E47V; K53R; H56P; S66G; V92I; F103V

For example, as shown in SEQ ID NO: 37, { V6I; V27I; I31F; E47L; K53R; E54Q; H56P; S66T }.

For example, as shown in SEQ ID NO: 38 { L4V; V6I; V27I; I31F; E47V; K53R; E54Q; H56P; V63I; S66T; K68R; V92I }.

For example, as shown in SEQ ID NO: 39, { V6I; V27I; I31T; E47V; K53R; E54Q; H56P; S66G; K68R; V92I; F103V }.

For example, as shown in SEQ ID NO:10, { V6I; V27I; I31F; E47V; K53R; E54Q; H56P; S66T; V92I }.

for the purposes of the present invention, the inventive agents include a portion of SIRP α normally located between the signal sequence and the transmembrane domain sufficient to bind CD47 with an identifiable affinity, e.g., high affinity, or a fragment thereof that retains binding activity the high affinity SIRP α agent will typically include at least the d1 domain of SIRP α (SEQ ID NO: 1) having the above-described modified amino acid residues the high affinity SIRP α polypeptide may include a wild-type SIRP α d1 polypeptide (SEQ ID NO: 1) or allelic variants thereof, i.e., at least amino acids 1-3, 7-20, 32-46, 69-91, 95-102 of the wild-type allele 2. the high affinity SIRP α polypeptide may also include portions of the native human SIRP α protein other than the d1 domain, including but not limited to residues 150 to 374 of the native protein (as set forth in SEQ ID NO: 2), or at least 10 contiguous amino acids, at least 20 contiguous amino acids, at least 50 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, at least 250 or more contiguous amino acids including but not limited to the sequence set forth in SEQ ID NO: 2.

such modifications may also include glycosylation modifications, such as those accomplished by modifying the glycosylation pattern of the polypeptide during polypeptide synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes.

in one embodiment, the high affinity sirpa agent has at least about 1x 10^-8Kinetics K of M against CD47_Din another embodiment, the high affinity sirpa agent has at least about 1x 10^-9Kinetics K of M against CD47_Din yet another embodiment, the high affinity sirpa agent has at least about 1x 10^-10Kinetics K of M against CD47_Din various embodiments described herein, a high affinity sirpa agent exhibits a kinetic K against CD47_Dkinetic K of a native human SIRPa polypeptide exemplified in SEQ ID NOs 1 and 2_Din some embodiments, a high affinity SIRP α reagent needleKinetics of CD 47K_Dis the kinetics K of a native SIRP α polypeptide_DAt least about 10 times, 50 times, 100 times, 500 times, 1000 times.

binding to CD47 can be determined, for example, by the ability of a SIRP α reagent to bind to CD47, CD47 coated on an assay plate, CD47 displayed on the surface of a microbial cell, CD47 in solution, etc. the binding activity of a SIRP α variant of the invention to CD47 can be determined by immobilizing a ligand, such as CD47 or a SIRP α variant, to a bead, substrate, cell, etc.

in some embodiments, the second polypeptide is part or all of an immunoglobulin Fc region.

in some embodiments, a high affinity SIRP α binding domain is provided, i.e., a SIRP α d1 domain modified as shown herein to provide high affinity binding to CD47, as a multimeric protein, i.e., 2, 3, 4, or more SIRP α binding domains covalently or non-covalently linked, e.g., as a fusion protein, disulfide bond, biotin through binding to avidin, streptavidin, etc., such multimeric high affinity SIRP α binding proteins can be used as a single agent to increase phagocytosis of CD 47-expressing cells, or in combination with other binding agents, e.g., cell-specific monoclonal antibodies.

in some such embodiments, the high affinity SIRP α binding domain is fused or otherwise bound to an immunoglobulin sequence to form a chimeric protein.

in such fusions, the encoded chimeric polypeptide may retain at least a functionally active hinge, the CH2 and CH3 domains of the immunoglobulin heavy chain constant region, the fusion may also be made at the C-terminus of the Fc portion of the constant domain, or just the N-terminus of the CH1 or light chain corresponding region of the heavy chain.

although an immunoglobulin light chain need not be present, an immunoglobulin light chain may be included, covalently bound to a SIRP α polypeptide-immunoglobulin heavy chain fusion polypeptide, or directly fused to a SIRP α polypeptide.

In other fusion protein constructs, the second polypeptide is a marker sequence, e.g., a peptide that facilitates purification of the fusion polypeptide. For example, the marker amino acid sequence may be a hexa-histidine peptide, such as the tags provided in, inter alia, the pQE vector (QIAGEN, inc., 9259Eton Avenue, Chatsworth, calif., 91311), many of which are commercially available. For example, as in Gentz et al, proc.natl.acad.sci.usa 86: 821-824, 1989, hexa-histidine provides convenient purification of the fusion protein. Another peptide tag, the "HA" tag, used for purification corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al, Cell 37: 767, 1984. The addition of peptide moieties to facilitate polypeptide processing is a technique that is well known and conventional in the art.

in other embodiments, a high affinity SIRP α binding domain is provided as a monomeric protein, which may be an isolated d1 domain, or a d1 domain fused to SIRP α or a chimeric sequence, for example, a monomeric SIRP α binding domain is used as an adjuvant to increase phagocytosis of CD 47-expressing cells in combination with cell-specific binding agents, such as antibodies, particularly tumor cell-specific antibodies as defined herein.

for example, variants of the invention also include analogs containing residues other than naturally occurring L amino acids, e.g., D amino acids, or non-naturally occurring synthetic amino acids.

As used herein, "cytotoxic moiety" refers to a moiety that inhibits cell growth or promotes cell death when in proximity to or taken up by a cell.

As used herein, an "imaging moiety" or detectable label refers to a moiety that can be used to increase contrast between a tumor and surrounding healthy tissue in visualization techniques, such as radiography, positron emission tomography, magnetic resonance imaging, direct or indirect visualization. Thus, suitable imaging moieties include radioimaging moieties such as heavy metal and radiation emitting moieties, positron emitting moieties, magnetic resonance contrast moieties, and optically visible moieties (e.g., fluorescent or visible spectrum dyes, visible particles, etc.. one of ordinary skill will appreciate that there is some overlap between the therapeutic moiety and the imaging moiety.

in general, the therapeutic or imaging agent may be conjugated to the high affinity SIRPa agent moiety by any suitable technique, with due consideration for pharmacokinetic stability and reduced overall patient toxicity requirements.

it will be apparent to those skilled in the art that a number of bifunctional or multifunctional reagents of both homo-and heterofunctionality (e.g., those described in the catalog of Pierce Chemical co., Rockford, iii.) can be employed as linker groups.

The carrier can carry the agent in a variety of ways, including covalent binding, either directly or through a linker group, and non-covalent association. Suitable covalently bound carriers include proteins such as albumin, peptides, and polysaccharides such as glycosaminoglycans, each of which has multiple sites for attachment of moieties. The carrier may also carry the agent by non-covalent association, for example by non-covalent association or by encapsulation.

Carriers and linkers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. Radionuclide chelates can be formed from chelating compounds, including those containing nitrogen and sulfur atoms as donor atoms for binding metals or metal oxides, radionuclides.

the radiographic moieties used as imaging moieties in the present invention include compounds and chelates having relatively large atoms such as gold, iridium, technetium, barium, thallium, iodine and isotopes thereof¹⁸F, which can be easily conjugated to high affinity sirpa agents by fluorination reactions.

The magnetic resonance contrast portion includes chelates of chromium (III), manganese (II), iron (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), and ytterbium (III) ions. Gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III) and iron (III) ions are particularly preferred because of their very strong magnetic moment.

The optically visible portion used as the imaging portion includes fluorescent dyes or dyes of the visible spectrum, visible particles and other visible label portions. Fluorescent dyes such as fluorescein, coumarin, rhodamine, bodipy Texas Red and cyanine dyes are useful when sufficient excitation energy can be provided to the site to be visually inspected. Endoscopic visualization may be more compatible with the use of such markers. Acceptable dyes include FDA approved non-toxic food dyes and colorants, but pharmaceutically acceptable dyes that have been approved for internal administration are preferred.

The effective amount of the imaging conjugate composition to be administered to a particular patient depends on a number of factors, several of which will vary from patient to patient. The attending clinician can determine an effective amount to facilitate visualization of the tumor. The dosage will depend on the tumor treatment, the route of administration, the nature of the therapeutic agent, the sensitivity of the tumor to the therapeutic agent, and the like. Using ordinary skill, the attending clinician is able to optimize the dosage of a particular therapeutic or imaging composition during routine clinical trials.

Typical dosages may be from 0.001 to 100 milligrams of conjugate per kilogram of subject body weight. For imaging conjugates with relatively non-toxic imaging moieties, relatively large doses, ranging from 0.1 to 10mg per kilogram of patient body weight, can be used. The amount used will depend on the sensitivity of the imaging method and the relative toxicity of the imaging moiety.

when the protein is produced by a prokaryotic cell, it may be further processed by unfolding, e.g., heat denaturation, DTT reduction, etc., and may be further refolded using methods known in the art.

The polypeptides may be prepared by cell-free translation systems or synthetic in vivo synthesis using conventional methods known in the art. Various commercially available synthesis devices are available, such as the automated synthesizers of Applied Biosystems, inc., foster city, CA, Beckman, and the like. By using a synthesizer, a naturally occurring amino acid can be substituted with an unnatural amino acid. The particular sequence and manner of preparation will be dictated by convenience, economics, desired purity, and the like.

The polypeptides may also be isolated and purified according to conventional methods of recombinant synthesis. Lysates of the expression hosts can be prepared and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. For the most part, the compositions used will contain at least 20% by weight of the desired product, more typically at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes typically at least about 99.5% by weight, in relation to contaminants associated with the process of product preparation and purification thereof. Typically, the percentages are based on total protein.

Methods well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/gene recombination. Alternatively, the RNA capable of encoding the polypeptide of interest may be chemically synthesized. Those skilled in the art can readily utilize well known codon usage tables and synthetic methods to provide appropriate coding sequences for any of the polypeptides of the invention. Nucleic acids can be isolated and obtained in substantial purity. Typically, a nucleic acid will be obtained as a DNA or RNA that is substantially free of other non-naturally occurring nucleic acid sequences, typically at least about 50%, usually at least about 90% pure, and is typically "recombinant," e.g., having one or more nucleotides at both ends that are not normally associated on a naturally occurring chromosome. The nucleic acids of the invention may be provided as linear molecules or within circular molecules, and may be provided within autonomously replicating molecules (vectors) or molecules without replicating sequences. Expression of a nucleic acid may be regulated by other regulatory sequences, either by themselves or as known in the art. The nucleic acids of the invention can be introduced into an appropriate host cell using a number of techniques available in the art.

in some embodiments, the pharmaceutical compositions of the invention comprise one or more therapeutic entities of the invention, or a pharmaceutically acceptable salt, ester, or solvate thereof.

The therapeutic entities of the present invention are often administered as pharmaceutical compositions comprising an active therapeutic agent and other pharmaceutically acceptable excipients. The preferred form depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also include a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic non-therapeutic non-immunogenic stabilizers and the like.

In yet some other embodiments, the pharmaceutical compositions of the invention may also comprise large, slowly metabolizing macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (e.g., latex-functionalized Sepharose)^TMAgarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).

Application method

methods are provided for treating, reducing or preventing cancer, including but not limited to lymphoma, leukemia, carcinoma, melanoma, glioblastoma, sarcoma, myeloma, and the like, as primary or metastatic cancers, by inhibiting the interaction between SIRP α and CD47, thereby increasing phagocytosis of tumor cells in vivo.

The effective dosage of the therapeutic entities of the invention, e.g., for treating cancer, will vary depending on a number of different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals can also be treated, e.g., companion animals such as dogs, cats, horses, etc., laboratory animals such as rabbits, mice, rats, etc., and the like. The therapeutic dose can be titrated to optimize safety and efficacy.

In some embodiments, the therapeutic dose range may be about 0.0001 to 100mg/kg and more usually 0.01 to 5mg/kg of host body weight. For example, the dose may be 1mg/kg body weight or 10mg/kg body weight or in the range of 1-10 mg/kg. Exemplary treatment regimens require administration once every two weeks or once a month or once every 3 to 6 months. The therapeutic entities of the present invention are typically administered on multiple occasions. The interval between single doses may be weekly, monthly or yearly. The intervals may also be irregular, as determined by measuring blood levels of the therapeutic entity in the patient. Alternatively, the therapeutic entities of the invention may be administered as sustained release formulations, in which case less frequent administration is required. The dose and frequency will vary depending on the half-life of the polypeptide in the patient.

In prophylactic applications, relatively low doses may be administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the remainder of the life. In other therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until disease progression is slowed or halted, and preferably until the patient exhibits partial or complete relief from the symptoms of the disease. Thereafter, a prophylactic regimen may be administered to the patient.

In yet other embodiments, the methods of the invention comprise treating, reducing or preventing tumor growth, tumor metastasis or tumor invasion of a cancer, including lymphoma, leukemia, carcinoma, melanoma, glioma, sarcoma, myeloma, and the like. For prophylactic use, a pharmaceutical composition or medicament is administered to a patient susceptible to or otherwise at risk of a disease, including biochemical, tissue and/or behavioral symptoms of the disease, complications thereof, and intermediate pathological phenotypes that arise during disease progression, in an amount sufficient to eliminate or reduce the risk of, reduce the severity of, or delay the onset of the disease.

the course of disease treatment can be monitored using the high affinity SIRP α polypeptides of the invention in vitro and in vivo, for example by measuring the increase or decrease in the number of cells expressing CD47, particularly cancer cells expressing CD47, which can determine whether a particular treatment regimen intended to alleviate the disease is effective.

the present invention relates to a method for detecting CD47 in a sample using a high affinity SIRP α polypeptide, and more particularly to a method for detecting CD47 in a sample using a high affinity SIRP α polypeptide.

the high affinity SIRPa polypeptides may be bound to a number of different carriers and used to detect the presence of CD47 expressing cells examples of well known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite.

There are many different labels and labeling methods known to those of ordinary skill in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. One of ordinary skill in the art will know or be able to determine other suitable labels for binding to the polypeptides of the invention using routine experimentation. Furthermore, the binding of these labels to the polypeptides of the invention can be carried out using standard techniques commonly used by those of ordinary skill in the art.

when present in biological fluids and tissues, cd47 may be detected by the high affinity SIRP α polypeptides of the invention any sample containing a detectable amount of CD47 may be used.

Another labeling technique that may lead to greater sensitivity consists of coupling the polypeptide to a low molecular weight hapten. These haptens can then be specifically detected by a second reaction. For example, haptens such as biotin, which is reactive with avidin, or dinitrophenol, pyridoxal or fluorescein, which is reactive with specific anti-hapten antibodies, are commonly used.

for example, administration within one hour prior to direct visual detection may be appropriate, or within 12 hours prior to an MRI scan may be appropriate.

The composition for treating cancer may be administered parenterally, topically, intravenously, intratumorally, orally, subcutaneously, intraarterially, intracranially, intraperitoneally, intranasally, or intramuscularly. The usual route of administration is intravenous or intratumoral, but other routes may be equally effective.

Generally, the compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection may also be prepared. As discussed above, the formulation may also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide, or copolymers to enhance the adjuvant effect. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of the invention may be administered in the form of long acting injections or implant formulations, which may be formulated in a manner that allows for sustained or pulsed release of the active ingredient. The pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practice (GMP) regulations of the U.S. food and drug administration.

toxicity of the high affinity SIRP α reagents described herein may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining LD₅₀(50% lethal dose of population) or LD₁₀₀(100% lethal dose of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic for human use. The dosages of the proteins described herein are preferably within a range of circulating concentrations that include an effective dose with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage may be selected by the individual physician in view of the patient's condition.

also within the scope of the invention are kits comprising a composition of the invention (e.g., a high affinity SIRP α reagent and formulations thereof) and instructions for use.

Having now fully described the invention, it will be apparent to those of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

Experiment of

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

The invention has been described in terms of specific embodiments discovered or suggested by the inventors to include the preferred modes of practicing the invention. Those skilled in the art will appreciate, in light of the present disclosure, that many modifications and changes may be made in the specific embodiments illustrated without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the base DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalence considerations, changes can be made in protein structure without affecting biological effects in terms of species and quantity. All such modifications are intended to be included within the scope of the appended claims.

protein expression and purification variants of the IgSF domain of human CD47 (residues 19-135) and the first IgSF domain of human SIRP α (residues 31-148) were cloned into pAcGP67 with a C-terminal 8x histidine tag and expressed in Hi5 cells using recombinant baculovirus.the free cysteine on the CD47 IgSF domain was mutated to glycine. the protein was purified by Ni-NTA chromatography and gel filtration through Superdex-75 column into HBS (10mM Hepes pH7.4, 150mM NaCl). to produce biotinylated protein, the C-terminal biotin receptor peptide (BAP) -LNDIFEAQKIEWHE tag was added and the protein was co-expressed with BirA ligase with excess biotin (100 μ M). to crystallize, the SIRP α variant FD6 was expressed in the periplasm of E.coli with the N-terminal Maltose Binding Protein (MBP) tag removed by treatment with 3C protease the IgDOSF domain was co-expressed in HindoSF 5 cells.

to express SIRP α variant proteins fused to human IgG4 and IgG2 Fc chains, SIRP α variants were cloned in-frame with the IL2 signal sequence into pFUSE-hIgG4-Fc2 and pFUSE-hIgG2-Fc2 vectors (Invitrogen).

FD 6: crystallization and structural determination of CD47 complex: coli-derived FD6 and deglycosylated insect-derived CD47 were combined at a ratio of 1: mix at 1 ratio and treat with carboxypeptidases A and B, followed by gel filtration through a Superdex-75 column into HBS. The complex was concentrated to 22mg/mL and crystallized by vapor diffusion in sitting drops (sitting drops) by adding 0.1. mu.L of protein to an equal volume of 2.0M ammonium sulfate, 0.1M Tris (pH 7.3). Diffraction studies were performed at Advanced Light Source. The crystal structure was resolved by molecular replacement with PHASER and refined using pheenix and COOT.

Surface plasmon resonance: SPR experiments were performed on a Biacore T100 instrument at 25 ℃. The experiment used a Biacore SA sensor chip (GE Healthcare) to capture biotinylated CD47 at a surface density of approximately 150 RU. An irrelevant biotinylated protein was immobilized as a reference surface of the SA sensor chip with RU matched to the experimental surface. Make it unprocessedbiotinylated SIRP α variant in running buffer [1xHBS-P (GE Healthcare)]The serial dilutions of (1) were run through the chip at a rate of 50. mu.L/min. Utilizing three 30 second 2M MgCl₂Regenerated CD47 was injected. Use was made of a catalyst having 1: 1 Biacore T100 evaluation software version 2.0 of Langmuir binding model the data was analyzed.

Cancer cell lines and culture conditions: jurkat cells (ATCC) and DLD-1 cells (ATCC) were maintained in RPMI (Invitrogen) + 10% fetal bovine serum (Omega Scientific) + 1% GlutaMax (Invitrogen) + 1% penicillin/streptomycin solution (Invitrogen). Jurkat cells were maintained in suspension, while DLD-1 cells were maintained as an adherent monolayer. Both cell lines were passaged at regular intervals of 3-4 days before reaching confluence. To prepare a GFP-luciferase + cell line, Jurkat and DLD-1 cells were transduced with lentiviruses expressing luciferase 2(Promega) -eGFP (derived from pCDH-CMV-MCS-EF1-puro HIV-based lentiviral vectors (Systems Bioscience)). GFP + cells among the cells were sorted using BDFACSAriaII flow cytometric analyzer and maintained in culture in the same manner as the parental cell line.

evaluation of SIRP α variants bound to human cancer cells GFP + Jurkat cells were washed in PBS and then incubated with titrated concentrations of biotinylated SIRP α variants for 30 minutes on ice, wild-type SIRP α tetramers were pre-formed by incubating biotinylated wild-type SIRP α with streptavidin for 15 minutes on ice at a 4: 1 molar ratio, the cells were washed in FACS buffer (PBS + 2% FBS) and then incubated with 50nM streptavidin conjugated to Alexa Fluor647 for 20 minutes on ice, the cells were washed twice and then analyzed for SIRP α variant binding by flow cytometry using Accuri C6 flow cytometer, SIRP α variants were analyzed for binding by flow cytometry, to evaluate the blocking of wild-type SIRP α to cD47, 50nM wild-type SIRP α tetramers were incubated with titrated concentrations of cD47 blocker on ice for 30 minutes, the maximum percent binding of SIRP α variants was measured for each fluorescence intensity of the phdsp variants using anti-cD 47 clone B6H12 (bioscience) as a positive control for blocking.

Macrophage derivation and culture: to derive human macrophages, leukopenia system chambers from anonymous donors were obtained from stanford blood centers. Peripheral blood mononuclear cells were obtained by density gradient centrifugation on Ficoll-Paque Premium (GE Healthcare). CD14+ monocytes were purified using CD14 microbeads (Miltenyi) and an AutoMACS Pro separator (Miltenyi). Monocytes were differentiated into macrophages by culturing for 7 days in IMDM + GlutaMax (Invitrogen) + 10% AB human serum (Invitrogen) + 1% penicillin/streptomycin, at which time they were used for phagocytosis assays.

Mouse macrophages were derived by isolating bone marrow from C57Bl/Ka Rosa26-mRFP1 transgenic mice and culturing in RPMI (Invitrogen) + 10% FBS + 1% GlutaMax + 1% penicillin/streptomycin supplemented with 10. mu.g/mL mouse M-CSF (Peprotech). After 7 days of differentiation, macrophages were harvested and used for phagocytosis assay.

in vitro phagocytosis assay using mouse and human macrophages in vitro phagocytosis assay for evaluation by flow cytometry approximately 50,000 macrophages per well were added to 96 well tissue culture plates 200,000 GFP + tumor cells were preincubated with antibody or SIRP α variant therapy in serum free medium for 30 minutes and then added to macrophages as described, anti-CD 47 clone 2D3(eBioscience) and cetuximab (Bristol-Myers Squibb) were used for conditioning, macrophages and target cells were incubated in a humidified 37 ° incubator containing 5% carbon dioxide for 2 hours after incubation, cells were washed, removed from the plate and prepared for flow cytometry after phagocytosis assay excluding dead cells from the assay by staining with DAPI (initen), human macrophages were identified by staining with Alexa Fluor647 anti-human CD14(BioLegend) conjugated macrophage assay 7.6.4 percent phagocytosis assay using a Biosciences with high throughput BD.

for visual phagocytosis in vitro, 50,000 macrophages were plated in 24-well plates tumor cells were labeled with 5 μ M CFSE (Invitrogen) according to the manufacturer's protocol 200,000 CFSE + tumor cells were treated with antibody or SIRP α variant in serum-free medium for 30 minutes, added to the macrophages, and then incubated at 37 ° for 2 hours after incubation.

the results are comparable to those observed with hu5F9 anti-CD 47 ab. these data show that the high affinity soluble SIRPa reagent is comparable to the anti-CD 47 antibody in effectively blocking "eat-me-not signal" and allowing phagocytosis and clearance of cells by binding to CD47 on HL60 cells.

Example 2

high affinity sirpa reduces the threshold for macrophage phagocytosis by cancer cells.

the ability of tumors to evade the immune system is an emerging cancer marker, and new therapeutic strategies to direct the immune response to cancer cells have shown promise in both experimental and clinical settings macrophage often infiltrates into tumors, and recent studies have identified CD47 as an "don't eat me" signal against phagocytosis that is highly expressed on many types of cancer to evade macrophage-mediated destruction.

We generated a SIRP α variant that binds CD47 with approximately a 50,000 fold increase in affinity relative to wild-type SIRP α when produced as a multimeric high affinity SIRP α -Fc fusion protein, this variant acted as a single agent to induce phagocytosis in vitro and reduce human tumor growth in vivo.

to generate the ideal CD47 antagonist, protein engineering was used to increase the affinity of soluble SIRP α for CD47 (fig. 1A) we generated a mutant library of N-terminal group V Ig domains of SIRP α conjugated to Aga2p for yeast surface display (fig. 1B) we performed two 'generations' of in vitro evolution using CD47 IgSF domain as selection reagent the first generation required five rounds of selection from a pooled mutant library containing randomized pairs of two classes of SIRP α residues-those contacting CD47 or those remaining in the hydrophobic core (fig. 5A) the first generation SIRP α variants obtained bind to CD47 with 20-100 times higher affinity than wild-type SIRP α as measured by surface plasmon resonance, the second generation directed evolution was performed by constructing a library that achieves full coverage of 13 residues mutated in the first generation selector, after five rounds of additional selection we obtained the CD47 binding variants with low dissociation constants of our (K34) that we had from their binding constants of pms of CD 34_D) And a decay half-life (t) of up to 44 minutes_1/2) in contrast, wild-type SIRP α has a K of 0.3-0.5. mu.M_DAnd t of 1.8 seconds_1/2when we grafted these nine conservative substitutions onto the dominant wild-type SIRP α allele (allele 2), the resulting variant (designated CV1, consensus variant 1) bound CD47 with an affinity of 11.1pM (fig. 1C).

CV1 sequence has the following amino acid changes relative to the wild type allele: V6I; V27I; I31F; E47V; K53R; E54Q; H56P; S66T; V92I. CV1 may include, for example, the d1 domain amino acid sequence as follows:

(SEQ ID NO：10)EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIYNQRQGPFPRV TTVSDTTKRN NMDFSIRIGN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS

to see if the high affinity SIRP α variants retained a CD47 binding geometry similar to the wild-type protein, we determined the crystal structure of the complex between the high affinity variant FD6 and CD47 IgSF domains (fig. 1D). the root mean square difference of the overlap of the FD 6: CD47 complex with the wild-type SIRP α: CD47 complex was onlythe high degree of structural similarity is indicated and confirms that our efforts to retain the geometry of the wild-type interaction (figure 1E) the overlapping binding pattern of FD6 and wild-type SIRP α indicate that they will compete for the same CD47 epitope, providing the greatest possible antagonistic mechanism as a significant difference, the C 'D loop of FD6 contains three of the four contact mutations present in the consensus sequence (figure 1E) we conclude that these mutations stabilize the C' D loop, which localizes the positive charge of Arg53 in the glutamic acid cluster on CD47 (figure 1E), the remainder of the binding interface between FD6 and CD47 is highly similar to the wild-type sirpa: CD47 interface, with the most significant exception of Ile31 mutated to phe.

to test the functional properties of high affinity SIRP α variants, we first examined their ability to bind and antagonize CD47 on the surface of cancer cells we found that SIRP α variants with increased CD47 affinity show greater potency in binding (fig. 8a, c) and blocking cell surface CD47 (fig. 2a and 8B.) as single domain monomers, FD6 and CV1 variants show potent antagonism relative to wild-type SIRP α. importantly, both high affinity variants are more potent CD47 antagonists than Fab fragments generated from anti-CD 47 antibody clone B6H12, and anti-CD 47 antibody clone B6H12 is a well characterized CD47 antagonist demonstrating therapeutic efficacy in vitro and in vivo (fig. 8 a).

As a fusion protein with the Fc fragment of human IgG4(hIgG4), as seen microscopically, the high affinity SIRPa variant leads to a significant increase in phagocytosis by cancer cells (FIG. 2 b). to obtain a quantitative measure of phagocytosis, primary human macrophages and GFP were evaluated⁺tumor cells were co-cultured with CD47 blockers and then analyzed by flow cytometry (fig. 2c) using various cancer cell lines representing solid and hematological malignancies, we found that treatment with saturating concentrations of the high affinity SIRP α -hIgG4 variant produced significantly increased phagocytosis relative to the wild-type SIRP α -hIgG4 control (fig. 2d) although the high affinity SIRP α -hIgG4 variant and isotype-matched anti-CD 47 antibody produced comparable levels of phagocytosis at saturating concentrations (fig. 2d), the high affinity SIRP α variant demonstrated significant advantages when titrated to produce a dose-response curve (fig. 2e), the high affinity of FD6-hIgG4 and 1-hIgG4 corresponded to reduced EC CV (fig. 2e)₅₀Indicating more efficient phagocytosis induction.

interestingly, no significant increase in phagocytosis was observed after treatment with saturating concentrations of high affinity sirpa monomers (fig. 2d), indicating that blocking CD47 alone was not sufficient to induce maximal phagocytosis.

treatment with anti-CD 47 clone 2D3 or anti-EpCam antibodies that bind CD47 but do not block SIRP α interaction, however, macrophages exhibit a significant increase in phagocytosis after the addition of high affinity SIRP α monomer FD6 to both antibody treatments (fig. 2 f).

to illustrate the clinical significance of this principle, we examined the ability of high affinity SIRP α monomers to enhance the efficacy of established monoclonal antibodies currently used as cancer therapies.first, phagocytosis assays were performed using DLD-1 colon cancer cells treated with the anti-EGFR antibody cetuximab-the combination of cetuximab in response to titrating concentrations of cetuximab alone, in combination with wild-type SIRP α monomers, or in combination with high affinity SIRP α monomers was evaluated to produce a significant increase in cetuximab maximum efficacy and potency relative to cetuximab alone or in combination with wild-type SIRP α monomers (fig. 2 g). similar effects were observed when evaluated with rasji lymphoma cells treated with titrating concentrations of rituximab (an anti-CD 20 antibody) (fig. 2 h). again, high affinity SIRP α monomers increased the maximum efficacy and potency of rituximab.

Next, we evaluated the in vivo efficacy of the high affinity SIRP α variants using a mouse tumor model As an active model for advanced human colon cancer, GFP-luciferase⁺DLD-1 cells were implanted into the abdominal cavity of NSG mice (fig. 3a), after implantation was confirmed by bioluminescence imaging, daily treatment was initiated with vehicle control or high affinity SIRP α variant CV1-hIgG4 as monotherapy. bioluminescence monitoring of total flux revealed a modest decrease in tumor growth rate during the first few weeks of treatment with CV1-hIgG4 (fig. 9), which resulted in significant survival benefit over time (fig. 3 b). since red blood cell loss was the major side effect observed following treatment with anti-mouse CD47 antibody, we examined a similar decrease in blood of CV1-hIgG4 treated miceAll cells in the fluid (fig. 3c) and resulted in a modest decrease in hematocrit (fig. 3 d). However, as previously observed, prolonged treatment did not cause further toxicity.

Since CV1-hIgG4 exhibited anti-tumor efficacy as a single agent, we next evaluated its efficacy in a human bladder cancer model, which is a type of cancer for which no targeted biologic currently exists. GFP-luciferase⁺639-V bladder cancer cells were injected into the dorsal subcutaneous tissue of NSG mice. Implantation was confirmed by bioluminescence imaging and mice were randomized into groups treated daily with vehicle control or CV1-hIgG 4. Treatment with CV1-hIgG4 significantly reduced tumor growth rate as assessed by bioluminescence imaging (fig. 3e, f). Tumor volume was assessed just before death in the first control-treated mice, when large tumors were measurable in all control mice, whereas no significant tumor was palpable in CV1-hIgG 4-treated mice (fig. 3 g). Thus, a clear survival benefit was observed even after discontinuation of treatment at the time of death of all control mice (fig. 3 h).

As previously observed, treatment with CV1-hIgG4 resulted in a decrease in the red blood cell index (fig. 10 a). CV1-hIgG4 treated mice also developed palpable stromal tissue around the tumor implantation site. Histopathological examination of the tissue revealed small tumor nodules embedded in a broad inflammatory infiltrate containing macrophages (fig. 10b, c).

following in vivo engraftment of GFP-luciferase + Raji cells in a local model of human lymphoma, mice were randomly grouped and daily treatments with vehicle, CV1 monomer alone, rituximab alone, or a combination of rituximab plus CV1 monomer for three weeks after engraftment was confirmed by bioluminescence imaging treatment with CV1 monomer alone or rituximab alone reduced tumor growth only, while treatment with combination therapy significantly eliminated tumors in most mice (fig. 4a, b, c), no significant red cell reduction was found during treatment (fig. 11a, b, c), the effect of each therapy was translated into a respective survival curve trend (fig. 4d), it is evident that the synergistic effect of combining high affinity sirpa monomers with tumor specific monoclonal antibodies resulted in long lasting cure in most animals (fig. 4 d).

while previous studies have demonstrated the value of targeting the CD47-SIRP α interaction as an immune intervention for cancer, here we further manipulated the system to produce highly potent and potent CD47 antagonists that exhibit optimal properties as therapeutics.

similarly, when CD47 signals inhibition by free transduction of SIRP α on macrophages are inhibited, however, when CD47 is blocked by high affinity SIRP α monomers in the presence of surface bound antibody Fc, macrophages are strongly stimulated.

in contrast to CD47 antagonism, the effects observed by others may be mediated primarily by phagocytophilic action of Fc, since phagocytosis is only evident when macrophages are pre-activated with endotoxin and interferon- γ.

the high affinity SIRP α reagents constitute a new class of anti-tumor biologics that can be further engineered.furthermore, because many tumors overexpress CD47 and expression levels are associated with poor patient outcomes, high affinity SIRP α variants can be adapted as non-invasive imaging agents for cancer. CD47 is commonly utilized by tumor cells to evade the immune system, and thus, high affinity SIRP α variants may be a valuable therapeutic agent for many human cancers.

Method of producing a composite material

protein expression and purification CD47 IgSF domain (residues 1-117) with a C15G mutation and a C-terminal 8 histidine tag was secreted from Trichoplusia Ni (Hi-5) cells using baculovirus and purified by Ni-NTA and size exclusion chromatography with Superdex-75 column to produce glycan-minimized CD47 for crystallography, CD47 was co-expressed with endoglycosidase-H (endoH) in the presence of kifunensine using a modified pMal-p2X expression vector (New England Biobs) containing a rhinovirus 3C protease cleavage site following the MBP tag and the C-terminal 8 histidine tag to express the monomeric SIRP α variant (residual England B) as an MBP-fusion in the periplasm of BL-21(DE3) E.coliGroups 1-118). OD of 0.8 with 1mM IPTG₆₀₀cells were induced and incubated with shaking at 22 ℃ for 24H. periplasmic proteins were obtained by osmotic shock and MBP-fusion proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography the eluted proteins were digested with 3C protease at 4 ℃ for 12H to remove MBP and further purified by an additional Ni-NTA chromatography step followed by size exclusion chromatography with a Superdex-S75 column for in vitro phagocytosis assay and in vivo experiments triton x-114 was used as described before to remove endotoxin and endotoxin removal was confirmed using the toxinsensochromogenic LAL assay kit (genpt). sirpa-Fc bodies were generated by cloning sirpa variants into modified pFUSE-hv 4-invitfc vectors (gengen) with IL-2 signal sequence and engineered Ser228Pro mutations sirpa-Fc bodies were generated by transient transfection of proteins in Freestyle 293-F cells (traiten) and transient expression of proteins in highgehrip (gezacho) in heizacho 3C-expressing recombinant proteins (gehrig) and expressing them in a gehrifc (gehrigh-2).

to obtain biotinylated CD47 and SIRP α, proteins with a carboxy-terminal biotin receptor peptide tag (GLNDIFEAQKIEWHE) were expressed and purified as described above.

Preparation of Fab fragment of B6H 12. The B6H12 antibody was desalted into 20mM sodium citrate (pH6.0), 25mM cysteine, 5mM EDTA and diluted to a concentration of 4 mg/mL. The antibody was then mixed with 250. mu.L of immobilized ficin resin (Thermo Scientific) per mL of antibody and incubated for 5 hours at 37 ℃ with rotation. The digested fragments were purified by passing the reaction mixture through a monoQ column (passing through the B6H12 Fab resident in the liquid) followed by gel filtration through a Superdex-200 column.

as described above, the N-terminal V-group domain of SIRP α (residues 1-118) as a C-terminal fusion with Aga2 was displayed on the surface of saccharomyces cerevisiae strain EBY100 using the pCT302 vector, a pooled first generation library was generated by two separate combinatorial PCR reactions, which randomize the CD47 contact residue and the hydrophobic 'core' residue of SIRP α, respectively, using a primer set with degenerate codons, a contact residue PCR primer set, randomized Ser29 ═ RST, Leu30 ═ NTT, Ile31 ═ NTT, Pro32 ═ CNT, Val33 ═ NTT, Gly34 ═ Lys, Pro35 ═ CNT, Gln52 ═ SAW, Lys53 ═ ARG, Glu54 ═ SAW, Ser66 ═ RST 6 ═ RST, thrst, Lys68, Phe, ARG 4642 ═ sgt, and Phe 97 ═ rgt:

(SEQ ID NO：11)5′GAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTATCAGTTGCAGCT3′；(SEQ ID NO：12)5′GGTCACAGTGCAGTGCAGAATGGCCGACTCTCCAGCTGCAACTGATACGGA3′；(SEQID NO：13)5′CTGCACTGCACTGTGACCRSTNTTNTTCNTNTTRSTCNTATCCAGTGGTTCAGAGGA3′；(SEQID NO：14)5′ATTGTAGATTAATTCCCGGGCTGGTCCAGCTCCTCTGAACCACTGGAT3′；(SEQ ID NO：15)5′CGGGAATTAATCTACAATSAWARGSAWGGCCACTTCCCCCGGGTAACAACTGTTTCAGAG3′；(SEQ ID NO：16)5′GTTACTGATGCTGATGGAAANGTCCATGTTTTCCYTCYTASYASYCTCTGAAACAGTTGTTAC3′；(SEQID NO：17)5′TCCATCAGCATCAGTAACATCACCCCAGCAGATGCCGGCACCTACTACTGTGTG3′；(SEQ IDNO：18)5′TCCAGACTTAAACTCCGTWTYAGGASYASYCNTCCGGAACNTCACACAGTAGTAGGTGCC3′；(SEQID NO：19)5′ACGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGTGCCAAACCCTCT3′

'core' residue PCR primers, randomized Leu4, Val6, Val27, Ile36, Phe38, Leu47, Ile49, Tyr50, Phe57, Val60, Met72, Phe74, Ile76, V92, Phe94, and Phe103 to NTT:

(SEQ ID NO：20)5′GGATCCGAGGAGGAGNTTCAGNTTATTCAGCCTGACAAGTCCGTATCAGTTGCAGCTGGAGAG3′；(SEQ ID NO：21)5′GGGCCCCACAGGGATCAGGGAGGTAANAGTGCAGTGCAGAATGGCCGACTCTCCAGCTGCAAC3′；(SEQ ID NO：22)5′CTGATCCCTGTGGGGCCCNTTCAGTGGNTTAGAGGAGCTGGACCAGCCCGGGAA3′；(SEQ ID NO：23)5′GTGGCCTTCTTTTTGATTAANAANAANTTCCCGGGCTGGTCCAGC3′；(SEQ ID NO：24)5′AATCAAAAAGAAGGCCACNTTCCCCGGNTTACAACTGTTTCAGAGTCCACAAAGAGAGAAAAC3′；(SEQ ID NO：25)5′GCCGGCATCTGCTGGGGTGATGTTACTGATGCTAANGGAAANGTCAANGTTTTCTCTCTTTGTGGA3′；(SEQ ID NO：26)5′ACCCCAGCAGATGCCGGCACCTACTACTGTNTTAAGNTTCGGAAAGGGAGCCCTGACACGGAG3′，(SEQ ID NO：27)5′AGAGGGTTTGGCACGCACAGACAGCTCAGTGCCTGCTCCAGACTTAANCTCCGTGTCAGGGCTCCC3′。

the PCR product was further amplified with primers containing homology to the pCT302 vector, combined with linearized pCT302 vector DNA, and co-electroporated into EBY100 yeast. The resulting library contained 4.0X 10⁸And (4) a transformant.

The second generation library was generated and transformed identically to the first generation library, but in combination with the following primers: randomized Leu4 ═ NTT, Val6 ═ NTT, Val27 ═ NTT, Ile31 ═ WYT, Glu47 ═ SWA, Lys53 ═ ARG, Glu54 ═ SAK, His56 ═ CNT, Ser66 ═ RST, Lys68 ═ ARG, Val92 ═ NTT, Phe94 ═ NTT, Phe103 ═ NTT:

(SEQ ID NO: 28)5 'GGATCCGAGGAGGAGNTTCAGNTTATTCAGCCTGACAAGTCCGTATC 3'; (SEQ ID NO: 29)5 'GTGCAGTGCAGAATGGCCGACTCTCCAGCTGCAACTGATACGGACTTGTCAGGCTGAA 3'; (SEQ ID NO: 30)5 'CATTCTGCACTGCACTNTTACCTCCCTGWYTCCTGTGGGGCCCATCCAG 3'; (SEQ ID NO: 31)5 'CGGGCTGGTCCAGCTCCTCTGAACCACTGGATGGGCCCCACAGG 3'; (SEQ ID NO: 32) 5' GAGCTGGACCAGCCCGGSWATTAATCTACAATCAAARGSAKGGCCNTTTCCCCCGGGTAACAACTGTTTCAGAG 3; (SEQ ID NO: 33)5 'GAAAAGTCCATGTTTTCTCTCYTTGTASYCTCTGAAACAGTTGTTAC 3'; (SEQ ID NO: 34)5 'AGAGAAAACATGGACTTTTCCATCAGCATCAGTAACATCACCCCAGCAGATGCC GGCAC 3'; (SEQ ID NO: 355 'CTCCGTGTCAGGGCTCCCTTTCCGAANCTTAANACAGTAGTAGGTGCCGGCATC TGCTG 3', (SEQ ID NO: 36)5 'GAGCCCTGACACGGAGNTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGTGCCAAACCCTCT 3'⁸And (4) a transformant.

Selection of the first generation library transformed yeast were expanded in SDCAA liquid medium at 30 ℃ and induced in SGCAA liquid medium at 20 ℃. All selection steps were performed at 4 ℃. For the first round of selection, 4.10 covering ten times the number of transformants representing the library⁹The individual induced yeast were resuspended in 5mL PBE (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.5mM EDTA). The yeast was mixed with 500 μ L paramagnetic streptavidin microbeads (Miltenyi) pre-coated with biotinylated CD47 and the mixture was incubated for 1 hour with rotation. The yeast was pelleted by centrifugation at 5,000g for 5 minutes and washed twice with 10mL PBE. Magnetically labeled yeast were resuspended in 5mL PBE and isolated using LS MACS columns according to the manufacturer's instructions (Miltenyi). The eluted yeast was pelleted, resuspended in SDCAA media, and expanded for the next round of selection. Additional four-wheel selections were made similar to the first wheel, with the following adjustments: will be 1 × 10⁸Each yeast was resuspended in 500. mu.L PBE containing FITC-labeled anti-c-Myc antibody (Miltenyi) or biotinylated CD47 protein at successively lower concentrations from 1. mu.M to 100 nM. After 1 hour incubation, the yeast were washed with PBE and used for selection with CD47, labeled with streptavidin-PE (Invitrogen) or streptavidin-Alexa Fluor647 (prepared internally) for 15 minutes. The yeast were washed two additional times with PBE and magnetically labeled with 50. mu.L of appropriate anti-fluorophore microbeads (anti-FITC, anti-PE or anti-Alexa Fluor 647; Miltenyi) for 15 minutes. The yeast was washed once, resuspended in 3mL PBE, and separated with LS column as in the first round.

For the first two rounds of selection of the second generation library, yeast were selected with the monomeric biotinylated CD47 protein as in rounds 2 to 5 of the first generation selection. The first round was selected with 20nM biotinylated CD47 and the second round was selected with 1nM biotinylated CD47, using a larger staining volume (10mL PBE) to avoid ligand depletion. For all subsequent rounds of selection, kinetic selection was performed. Briefly, yeast were stained with 20nM biotinylated CD47 for 1 hour, washed with PBE, and resuspended in 500. mu.L PBE containing 1. mu.M non-biotinylated CD 47. Cells were incubated at 25 ℃ for 90 min (third round) or 300 min (fourth and fifth rounds), after which they were washed with ice-cold PBE and stained with fluorescently labeled streptavidin. For rounds 1 to 4, yeasts were isolated using MACS as described for the first generation library. For the fifth selection, yeast were co-labeled with FITC-labeled anti-c-Myc and streptavidin-Alexa Fluor647 and selected using a FACSAria cell sorter (BDbiosciences).

Surface Plasmon Resonance (SPR). at 25 deg.CThe experiment was performed with Biacore T100. Protein concentration was quantified by Nanodrop2000 spectrometer (Thermo Scientific) at absorbance of 280 nm. Capture of biotinylated CD47 (R) using Biacore SA sensor chip (GE Healthcare)_maxabout 150 RU.) irrelevant biotinylated proteins were immobilized, with RU values matched to the reference surface of non-specific binding controls measured using serial dilutions of sirpa variants in HBS-P + buffer (GEHealthcare) — three 60 second 2M MgCl₂Regenerated CD47 surface was injected. The following compositions were used: 1Langmuir binding model all data were analyzed using Biacore T100 evaluation software version 2.0.

FD 6: CD47 with minimized glycans and e.coli-derived FD6 were measured at a ratio of 1: mix at 1 ratio and digest with carboxypeptidases a and B to remove the C-terminal 8x histidine tag. Digested FD6 was purified by gel filtration into HEPES buffered saline (HBS; 10mM HEPES (pH7.4), 150mM NaCl) with Superdex-75 column: CD47 complex, and concentrated to 22 mg/mL. Crystals were obtained by adding 0.1. mu.L of protein to equal volumes of 2.0M ammonium sulfate and 0.1M Tris (pH7.3) and cryoprotected in paraffin oil. Diffraction studies were performed in an Advanced Light Source (Berkeley, Calif., USA) with beam 8-2. Obtaining anisotropythe dataset was processed with HKL-3000, the FD 6: CD47 complex was resolved by individual model molecule replacement with CD47 and SIRP α from Protein database (Protein Data Bank) registration code 2JJS, refinement with PHENIX and model tuning with COOT solvent correction with bulk solvent flattening initial refinement using rigid body, co-ordination, and real space refinements, and individual atom replacement parameter refinements after which TLS refinement was added in refinement iterations.

Cell lines and GFP-luciferase + transduction. DLD-1 cells (ATCC), HT-29 cells (ATCC), Raji cells (ATCC), Jurkat cells (ATCC) were cultured in RPMI + GlutaMax (Invitrogen) supplemented with 10% fetal bovine serum (Omega Scientific), 100U/mL penicillin, and 100. mu.g/mL streptomycin (Invitrogen)And 639-V cells (DSMZ). GFP-luciferase production by transduction with pCDH-CMV-MCS-EF1puro HIV-based lentiviral vectors (Systems Biosciences) engineered to express eGFP-luciferase 2(pgl4) fusion proteins⁺A cell line. Stable cell lines were generated by sorting GFP expression on a FACSAria II cell sorter (BD Biosciences).

cell-based CD47 binding assays various concentrations of biotinylated SIRP α monomer, SIRP α -hIgG4 fusion protein, or anti-CD 47 antibody were incubated with indicated cancer cells the binding of biotinylated monomers was detected using 100nM Alexa Fluor647 conjugated streptavidin as a second staining reagent and analyzed on an Accuri C6 flow cytometer (BDBiosciences) the binding of SIRP α -hIgG4 fusion protein or anti-CD 47 antibody was detected using goat anti-human IgG antibody (Invitrogen) and analyzed on lsrforta (BD Biosciences) with a high-throughput sampler.

cell-based CD47 blocking assay biotinylated WTa1d1 SIRP α was incubated with Alex Fluor647 conjugated streptavidin to form a WTa1d1 SIRP α tetramer 100nM WTa1d1 SIRP α tetramer was combined with titrated concentrations of CD47 antagonist and added simultaneously to 50,000 GFP-luciferase₊Raji cells. Cells were incubated at 4 ℃ for 30 minutes and then washed to remove unbound tetramer. Samples were stained with DAPI (Sigma) to exclude dead cells and fluorescence was measured using LSRFortessa (BD Biosciences) with a high-throughput sampler. Data represent geometric mean fluorescence intensity analyzed using flowjov9.4.10(Tree Star), normalized to maximum tetramer binding, and fitted to a sigmoidal dose-response curve using Prism 5 (Graphpad).

Macrophage derivation and phagocytosis assays. Leukopenia system (LRS) chambers from anonymous donors were obtained from stanford blood centers and enriched for peripheral blood mononuclear cells by density gradient centrifugation on Ficoll-Paque Premium (GE Healthcare). anti-CD 14 microbeads (Miltenyi) were used in AuMonocytes were purified on toMACS (Miltenyi) and differentiated into macrophages by culturing for 7-10 days in IMDM + GlutaMax (Invitrogen) supplemented with 10% AB human serum (Invitrogen) and 100U/mL penicillin and 100 μ g/mL streptomycin (Invitrogen). Phagocytosis assays were performed by co-culturing 50,000 macrophages with 100,000 GFP + tumor cells for 2 hours, and then analyzed using a LSRFortessa cell analyzer (BD Biosciences) with a high-throughput sampler. Antibodies for use in therapy include: mouse IgG1 isotype control (eBioscience), anti-CD 47 clone 2D3(eBioscience), anti-EpCam (BioLegend), cetuximab (Bristoll-Myers Squibb), and rituximab (Genentech). Macrophages were identified by flow cytometry using anti-CD 14, anti-CD 45, or anti-CD 206 antibodies (BioLegend). Dead cells were excluded from the assay by staining with DAPI (Sigma). Phagocytosis was assessed as GFP using FlowJo v9.4.10(Tree Star)⁺The percentage of macrophages, and normalized to the maximum response of each independent donor to each cell line. Statistical significance was determined by 2-way ANOVA with Bonferroni post-hoc test, and when indicated, data were fitted to sigmoidal dose-response curves using Prism 5 (Graphpad).

Phagocytosed living cells are imaged. As previously described, RFP is generated⁺Mouse macrophages and evaluated in live cell imaging assays. Briefly, from C57BL/K_aRosa26 mRFP1 transgenic mice bone marrow cells were isolated and differentiated in 10ng/mL murine M-CSF (Peprotech). 500,000 Raji cells were labeled with 0.5M CFSE (Invitrogen) and matched with 50,000 RFPs₊Macrophages were co-cultured and imaged using biostate IMQ (Nikon) equilibrated to 37 ℃ and 5% carbon dioxide.

A mouse. Cg-Prkdc^scidIL2rgtm1wjl/SzJ (NSG) mice were used for all in vivo experiments. Mice were implanted with tumors at approximately 6-10 weeks of age and experiments were performed with age and gender matched groups of 8-15 mice. Mice were maintained in a barrier facility, cared for by Stanford Veterinary Services Center (Stanford Veterinary Services Center) and treated according to protocols approved by the university of Stanford administration for care of laboratory animals.

Tumor models. To simulate human colon cancer, 1-10⁵Individual GFP-luciferase + DLD-1 cells were injected into the abdominal cavity of NSG mice. Tumor nodules were observed on an M205FA fluorescence dissection microscope (Leica) equipped with a DFC 500 camera (Leica). By mixing 1.25-10⁵GFP-luciferase⁺639-V cells were implanted into dorsal subcutaneous tissue of NSG mice in 25% Matrigel (BD Biosciences) to mimic bladder cancer. For the local model of human lymphoma, 1 · 10 will be used⁶GFP-luciferase⁺in all models, treatment was initiated and continued as indicated after confirmed implantation, 200 μ g of SIRP α variant or antibody was administered by intraperitoneal injection on a daily schedule for all treatments, tumor growth was monitored by bioluminescence imaging, and tumor size was measured to follow the ellipsoid formula (pi/6-length-width)²) The volume is calculated. Statistical significance was determined by Mann-Whitney test or Kruskal-Wallis with Dunn post hoc test (as applicable). Survival was analyzed by the Mantel-Cox test.

blood analysis blood was drawn from the orbital plexus and collected in dipotassium-EDTA microtubes (BD Biosciences) blood parameters were evaluated using a HemaTrue analyzer (Heska) statistical significance was determined by 2-way ANOVA with Bonferroni post test.

Bioluminescence imaging. Anesthetized mice were injected with 16.67mg/mL reconstituted 200 μ L D-fluorescein (firefly) potassium salt (Biosynth) in sterile PBS. Bioluminescence imaging was performed using IVIS spectroscopy (Caliper Life Sciences) over 20 minutes to record maximum radiation. The total flux peak from the anatomical region of interest was assessed using Living Image 4.0(Caliper Life Sciences) and used for analysis.

Among the proteins used in the examples described herein, the following are included:

FD6-hIgG4(FD6 underlined, human IgG 4S 228P bold) which includes the CH2, CH3 and hinge regions of human IgG4 and the d1 domain of high affinity SIRP α FD 6:

(SEQ ID NO：40)

EEEVQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTISETTRR ENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPSAAAPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

CV1-hIgG4(CV-1 underlined, human IgG 4S 228P bold) which includes the CH2, CH3 and hinge regions of human IgG4 and the d1 domain of high affinity SIRP α CV1 note that CV1 amino acid substitutions were "constructed" in human wild-type allele 2:

(SEQ ID NO：41)

EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKR NNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSAAAPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

FD6-hIgG2(FD6 underlined, human IgG2 bold) which includes the CH2, CH3 and hinge regions of human IgG2 and the d1 domain of high affinity SIRP α FD 6:

(SEQ ID NO：42)

EEEVQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTISETTRR ENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPSAAAVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

CV1-hIgG2(CV-1 underlined, human IgG2 bold) which includes the CH2, CH3 and hinge regions of human IgG2 and the d1 domain of high affinity SIRP α CV-1:

(SEQ ID N0：43)

EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKR NNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSAAAVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

GCN4 leucine zipper fusion:

FD 6-zipper (FD6 underlined, GCN4 leucine zipper bold), which includes the d1 domain of FD6 fused to GCN4, and which utilizes leucine zipper function to dimerize:

(SEQ ID NO：44)

EEEVQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTISETTRRENMDFSIS ISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPSAAA

concatamer (concatamer) constructs:

FD6 concatemer (FD6 underlined, GGGGSGGGGS linker, FD6 underlined)

(SEQ ID NO：45)

EEEVQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTISETTRRENMDFSIS ISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPSGGGGSGGGGSEEEVQIIQPDKSVSVAAGESAILHC TITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTISETTRR ENMDFSISISNITPADAGTYYCIKFRKGSPDT EFKSGAGTELSVRAKPS

Table of selected sequences

SEQ ID NO：1	Native sequence, d1 Domain	118 amino acid protein
			SEQ ID NO：2	Native sequence, full-length protein	504 amino acid protein
SEQ ID NO：3	1D4 mutant D1 Domain	118 amino acid protein
			SEQ ID NO：4	1A5 mutant d1 Domain	118 amino acid protein
SEQ ID NO：5	2D3 mutant D1 Domain	118 amino acid protein
			SEQ ID NO：6	2A10 mutant d1 Domain	118 amino acid protein
SEQ ID NO：7	2B5 mutant d1 Domain	118 amino acid protein
			SEQ ID NO：8	2A2 mutant d1 Domain	118 amino acid protein
SEQ ID NO：9	2F5 mutant d1 Domain	118 amino acid protein
			SEQ ID NO：10	CV1 mutant d1 Domain	119 amino acid protein
SEQ ID NO：37	FB3 mutant d1 Domain	118 amino acid protein
			SEQ ID NO：38	FD6 mutant d1 Domain	118 amino acid protein
SEQ ID NO：39	FA4 mutant d1 Domain	118 amino acid protein
			SEQ ID NO：40	FD6-hIgG4	345 amino acid protein
SEQ ID NO：41	CV1-hIgG4	346 amino acid protein
			SEQ ID NO：42	FD6-hIgG2	344 amino acid protein
SEQ ID NO：43	CV1-hIgG2	345 amino acid protein
			SEQ ID NO：44	GCN4 leucine zipper	Protein of 158 amino acids
SEQ ID NO：45	FD6 concatemer	Protein of 246 amino acids

Claims (15)

1. a high affinity SIRP α polypeptide, wherein the polypeptide lacks a SIRP α transmembrane domain, comprising a d1 domain of human SIRP α, the d1 domain comprising a set of amino acid substitution mutations selected from the group consisting of:

·V27I；K53R；S66T；K68R；F103V；

·L4V；V27L；E47V；K53R；E54Q；S66G；K68R；V92I；

·L4V；V6I；A21V；V27I；I31T；E47L；K53R；H56P；S66T；K68R；F94L；

·V6I；V27I；I31S；E47V；K53R；E54Q；H56P；S66G；V92I；F94L；

·L4I；A21V；V27I；I31F；E47V；K53R；E54Q；H56R；S66G；F94V；F103V；

L4V; V6I; V27I; I31F; E47V; K53R; H56R; S66G; K68R; V92I; F94L; or

·L4V；V6L；I31F；E47V；K53R；H56P；S66G；V92I；F103V；

·V6I；V27I；I31F；E47L；K53R；E54Q；H56P；S66T；

·L4V；V6I；V27I；I31F；E47V；K53R；E54Q；H56P；V63I；S66T；K68R；V92I；

V6I; V27I; I31T; E47V; K53R; E54Q; H56P; S66G; K68R; V92I; F103V; or

V6I, V27I, I31F, E47V, K53R, E54Q, H56P, S66T, V92I, wherein the set of amino acid substitutions increases the affinity of the high affinity SIRP α polypeptide to bind CD47 relative to a wild type SIRP α polypeptide, and wherein the high affinity SIRP α polypeptide has a K for CD47 of equal to or less than 11.6nM_D。

2. A high affinity SIRPa polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs 3-10 and 37-39.

3. the polypeptide of claim 1 or 2, consisting of the SIRP α d1 domain.

4. the polypeptide of claim 1 or 2, further comprising an amino acid sequence from sirpa that is outside the d1 domain.

5. The polypeptide of claim 2, wherein said polypeptide comprises the amino acid sequence of SEQ ID No. 10.

6. The polypeptide of any one of claims 1, 2 and 5, which is fused to an immunoglobulin Fc sequence.

7. the polypeptide of any one of claims 1, 2, and 5, wherein the high affinity SIRP α polypeptide is multimeric.

8. the polypeptide of any one of claims 1, 2, and 5, wherein the high affinity SIRP α polypeptide is monomeric.

9. A therapeutic formulation comprising the polypeptide of any one of claims 1, 2 and 5.

10. The polypeptide of any one of claims 1, 2 and 5, wherein the polypeptide further comprises a detectable label.

11. Use of the formulation of claim 9 for the preparation of a medicament for the treatment of a CD47 expressing cancer.

12. The use of claim 11, further comprising contacting the CD 47-expressing cancer with a tumor-specific antibody.

13. The use of claim 11, wherein the use is in vitro.

14. The use of claim 11, wherein the use is in vivo.

15. Use of a polypeptide according to claim 10 for the preparation of an imaging agent for imaging a tumor.