WO2013109752A1 - High affinity sirp-alpha reagents - Google Patents
High affinity sirp-alpha reagents Download PDFInfo
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- WO2013109752A1 WO2013109752A1 PCT/US2013/021937 US2013021937W WO2013109752A1 WO 2013109752 A1 WO2013109752 A1 WO 2013109752A1 US 2013021937 W US2013021937 W US 2013021937W WO 2013109752 A1 WO2013109752 A1 WO 2013109752A1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
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- C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
Definitions
- the cell surface protein CD47 on healthy cells and its engagement of a phagocyte receptor, SIRPa constitutes a key "don't eat- me" signal that can turn off engulfment mediated by multiple modalities, including apoptotic cell clearance and FcR mediated phagocytosis.
- Blocking the CD47 mediated engagement of SIRPa on a phagocyte, or the loss of CD47 expression in knockout mice, can cause removal of live cells and non-aged erythrocytes.
- SIRPa recognition also allows engulfment of targets that are not normally phagocytosed.
- CD47 is a broadly expressed transmembrane glycoprotein with a single Ig-like domain and five membrane spanning regions, which functions as a cellular ligand for SIRPa with binding mediated through the NH2-terminal V-like domain of SIRPa.
- SIRPa is expressed primarily on myeloid cells, including macrophages, granulocytes, myeloid dendritic cells (DCs), mast cells, and their precursors, including hematopoietic stem cells.
- Structural determinants on SIRPa 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 SIRPa cis dimerization in CD47 binding is discussed by Lee et al. (2010) J. B.C. 285:37953-63.
- CD47 In keeping with the role of CD47 to inhibit phagocytosis of normal cells, there is evidence that it is transiently upregulated on hematopoietic stem cells (HSCs) and progenitors just prior to and during their migratory phase, and that the level of CD47 on these cells determines the probability that they are engulfed in vivo. CD47 is also constitutively upregulated on a number of cancers. Overexpression of CD47 by tumor cells may increase pathogenicity by allowing the cell to evade phagocytosis. SUMMARY OF THE I NVENTION
- High affinity SIRPa polypeptides and analogs thereof are provided, which are referred to herein as high affinity SIRPa reagents.
- the reagents are sequence variants of the native human SIRPa protein, and have utility for in vivo and in vitro methods that block the interaction between native SIRPa protein and its receptor, CD47.
- the amino acid changes that provide for increased affinity are localized in the d1 domain, and thus the high affinity SIRPa reagents of the invention comprise a d1 domain of human SIRPa, with at least one amino acid change relative to the wild-type sequence within the d1 domain.
- Such a high affinity SIRPa reagent optionally comprises additional amino acid sequences, for example antibody Fc sequences; portions of the wild-type human SIRPa protein other than the d1 domain, including without limitation residues 150 to 374 of the native protein or fragments thereof, usually fragments contiguous with the d1 domain; and the like.
- High affinity SIRPa reagents may be monomeric or multimeric, i.e. dimer, trimer, tetramer, etc.
- the invention provides soluble high affinity SIRPa reagents, which lack the SIRPa transmembrane domain, and comprise at least one amino acid change in the d1 domain relative to wild-type human SIRPa, which amino acid change increases the affinity of the SIRPa polypeptide binding to CD47.
- the high affinity SIRPa polypeptide may be post- translationally modified, for example by glycosylation, pegylation, etc.
- the high affinity SIRPa reagent is a fusion protein comprising an Fc domain.
- the invention also includes pharmaceutical formulations of high affinity SIRPa reagents in combination with a pharmaceutically acceptable excipient.
- Such formulations may be provided as a unit dose, e.g. a dose effective to increase targeted phagocytosis in an individual.
- Pharmaceutical formulations also include lyophilized or other preparations of the high affinity SIRPa reagents, which may be reconstituted for use.
- methods are provided to manipulate targeted phagocytosis of cells, e.g. by macrophages or other mammalian phagocytic cells.
- a cell expressing CD47 is contacted with a high affinity SIRPa reagent of the invention in a dose effective to block the interaction between endogenous SIRPa and CD47. Blocking this interaction allows engulfment of targets that are not normally phagocytosed.
- the contacting may be performed in vivo, e.g. for therapeutic purposes, and in vitro, e.g. for screening assays and the like.
- the high affinity SIRPa reagent for these purposes may be multimeric; or monomeric. Monomeric reagents find particular use for administration in combination with an antibody that selectively binds to the cell targeted for phagocytosis.
- tumor cells e.g. solid tumors such as carcinomas, sarcomas, melanomas, etc.; leukemias; lymphomas, etc. are targeted for phagocytosis by contacting the tumor cell with a dose of a high affinity SIRPa polypeptide that is effective to block, or mask CD47 on the cell surface, allowing engulfment of targets that are not normally phagocytosed.
- a high affinity SIRPa polypeptide that is effective to block, or mask CD47 on the cell surface, allowing engulfment of targets that are not normally phagocytosed.
- Administration of an effective dose of high affinity SIRPa polypeptide to a patient prevents interaction between CD47 and SIRPa, which increases the clearance of tumor cells via phagocytosis.
- a high affinity SIRPa variant in the presence of an immunoglobulin Fc bound to the cell targeted for phagocytosis, which provides a pro-phagocytic signal.
- the high affinity SIRPa polypeptide can be combined with monoclonal antibodies directed against one or more additional tumor cell markers, which compositions can be synergistic in enhancing phagocytosis and elimination of tumor cells as compared to the use of single agents.
- Monomeric high affinity SIRPa reagents are advantageous for this purpose as they have low red blood cell toxicity.
- a SIRPa fusion construct comprising an immunoglobulin Fc region, e.g. one that provides a pro- phagocytic signal, may be administered.
- the high affinity SIRPa reagent comprises a detectable label.
- Such a labeled reagent can be used for imaging purposes in vitro or in vivo, e.g. in the imaging of a tumor.
- FIG. 1 Figures 1 A-1 E. Directed evolution of high-affinity SIRPa variants.
- (Right) Soluble, high-affinity SIRPa protein competitively antagonizes CD47 and prevents engagement with SIRPa on macrophages, thereby disinhibiting phagocytosis.
- C Representative plots showing phagocytosis assays analyzed by flow cytometry. Human macrophages were co-cultured with GFP+ DLD-1 cells and the indicated treatments. Gates were used to evaluate GFP+ macrophages (red) as a percentage of the total macrophage population (blue).
- D Representative plots showing phagocytosis assays analyzed by flow cytometry. Human macrophages were co-cultured with GFP+ DLD-1 cells and the indicated treatments. Gates were used to evaluate GFP+ macrophages (red) as a percentage of the total macrophage
- WT SIRPa or high-affinity SIRPa variant FD6 monomers were used at 1 ⁇ and combined as indicated.
- FIGS 3A-3H High-affinity SIRPa-Fc fusion proteins are effective as single agents for cancer.
- A Representative image of GFP-luciferase+ DLD-1 cells engrafted into the peritoneal cavity, forming disseminated tumor nodules amongst surrounding bowel loops.
- B Survival of mice engrafted with GFP-luciferase+ DLD-1 cells upon treatment with vehicle control (PBS, red) or high-affinity SI RPa-hlgG4 fusion protein (CV1 -hlgG4, blue).
- C Representative analysis of human Fc bound to the surface of whole blood cells from animals treated in b.
- D Blood analysis of treated animals in b showing mean and standard deviation from four animals per cohort over time.
- Dotted line shows lower limit of normal values.
- E Growth of GFP-luciferase+ 639-V bladder cancer cells in the dorsal subcutaneous tissue of NSG mice upon treatment with the indicated therapies as evaluated by bioluminescence imaging. Bars depict median values, points show values from individual mice.
- F Representative bioluminescence images of 639-V engrafted mice from each treatment group on day 37 post-engraftment.
- G Tumor volumes of 639-V engrafted mice on day 38 post-engraftment. Bars depict median values, points show values from individual mice.
- FIGS 4A-4F High-affinity SIRPa monomers enhance the efficacy of monoclonal antibodies in vivo.
- B Representative bioluminescence images of mice on day 29 post-engraftment. Red circles indicate sites of primary tumors, red arrow indicates site of metastases to axillary lymph nodes.
- Figures 5A-5B Library design and sequences from first generation selections. A. Left:
- SIRPa Summary of sequences of SIRPa variants obtained after the first generation of selections. The position of the mutated residues and their corresponding sequence in wild-type allele 1 is denoted at the top of the table. Blue shading indicates 'contact' mutations occurring at the SIRPa:CD47 interface. Italic font indicates mutations at positions that were not randomized in the pooled library (Glu47 and His56).
- FIG. 7 Representative electron density map of FD6:CD47 complex. 2mFo-DFc electron density map contoured at 2.0 ⁇ . Modeled amino acid residues are depicted as sticks, with FD6 residues in yellow and CD47 residues in green. Pyroglutamic acid residue 1 of CD47 is indicated as PCA1 above the corresponding residue and density.
- FIGS 8A-8C High-affinity SIRPa variants potently bind and block CD47 on cancer cells.
- B CD47 blocking assay on Jurkat cells. CD47 antagonists were added in competition with Alexa Fluor 647-conjugated wild-type SIRPa tetramer.
- Blocking was tested with a first generation SIRPa mutant as a monomer (1 A5 mono, teal), a second generation SIRPa mutant as a monomer (FD6 mono, green), a second generation SIRPa mutant as an Fc fusion with human lgG4 (FD6-hlgG4, blue), and anti-CD47 clone B6H12 (orange). Error bars indicate standard deviation.
- FIG. 9 High-affinity SIRPa-Fc variants restrict growth of DLD-1 colon cancer cells in vivo. Tumor growth curves upon treatment with vehicle (PBS, red) or high affinity SIRPohlgG4 fusion protein (CV1 -hlgG4, blue), as measured by bioluminescence imaging of the peritoneal cavities of treated mice. Points indicate values from individual mice, lines depict median values. * * p ⁇ 0.01 by 2-way ANOVA with Bonferroni post-test.
- FIGS. 10A-10D Treatment with high-affinity SIRPa-Fc variants causes macrophage infiltration and affects red cell indices.
- B Hematoxylin and eosin staining of palpable subcutaneous tissue mass from a CV1 - hlgG4 treated mouse, demonstrating the presence of infiltrating macrophages.
- a tumor nodule is visible in the top left of the image with an inflammatory infiltrate surrounding it.
- Inset shows representative macrophages in the area outlined by the dashed box.
- C Immunohistochemical staining for F4/80, a mouse macrophage marker, in palpable subcutaneous tissue mass from a CV1 -hlgG4 treated mouse.
- a tumor nodule is visible in the right portion of the image, with infiltrating macrophages stained on the left.
- Inset shows representative macrophages in the area outlined by the dashed box, with evidence of macrophages in the process of phagocytosis (black arrows) and successful engulfment of tumor cells (red arrows).
- D
- FIG. 1 1A-1 1 C Treatment with high-affinity SIRPa monomers does not cause red blood cell toxicity.
- A-C. ns not significant. Black arrows indicate the start and stop of daily treatment.
- Cynomolgus monkeys were treated with a single intravenous injection of high-affinity SI RPa-Fc (FD6-hlgG4, red) or a dose-escalation series of high-affinity SIRPa monomer (FD6 mono, blue). Dotted lines depict days of treatment with doses indicated above in mg/kg. Moderate toxicity to red blood cells was observed as a drop in hematocrit upon treatment with FD6- hlgG4, and no loss of red blood cells was observed with FD6 monomer therapy. No toxicity to other organ systems was observed.
- FIGS 13A-13C Radiolabeled high-affinity SIRPa variants are effective as noninvasive imaging agents for cancer.
- A. NSG mice were subcutaneously engrafted with DLD-1 human colon cancer cells on the upper right flank. Tumor bearing mice were injected with Cu- 64-labeled FD6 monomer and imaged by PET scan. Red arrowheads indicate uptake by tumor, black arrowheads indicate uptake by kidneys. Upper panels show dorsal images, and lower panels show transverse sections through tumors. M2, M3 represent images from two independent animals.
- B Quantification of signal from animals over time. FD6 persists in the tumor for at least 24 hours.
- FIGS 14A-14F Fluorescence-activated cell sorting of macrophages demonstrates high-affinity SIRPa variants induce phagocytosis of cancer cells.
- a Primary human macrophages and GFP+ DLD-1 colon cancer cells were co-cultured in the presence of 100 nM CV1 -hlgG4. Phagocytosis was quantified as the percentage of CD45+ macrophages that became GFP+. Macrophage populations were sorted by flow cytometry for those that were GFP negative (blue), GFP low (red), or GFP high (green).
- B-F Scale bar represents 100 ⁇ .
- Figures 16A-D High-affinity SIRPa variants induce maximal efficacy in the presence of tumor-bound antibody Fc chains.
- B Analytical gel filtration showing high-affinity SIRPa-Fc fusion proteins are purified as a single species (elution volume 13.44) with limited aggregation.
- CD47 blockade with High-affinity SIRPa variant CV1 monomer or anti-CD47 Fab fragments produced marginal increases in phagocytosis, while treatment with high-affinity SIRPa-Fc or intact anti-CD47 antibodies produced elevated phagocytosis.
- D Oligomerization of CD47 does not induce phagocytosis.
- Phagocytosis assay performed with primary human macrophages and GFP+ DLD-1 human colon cancer cells. Treatment with high-affinity SIRPa dimers that lack a pro-phagocytic stimulus do not induce substantial levels of phagocytosis.
- Figures 17A-17D High-affinity SIRPa variants bind and block other mammalian CD47 orthologs.
- C. High-affinity SIRPa variant CV1 binds mouse CD47 displayed on the surface of yeast.
- inhibitors refer to molecules that prevent the binding of SIRPa and CD47.
- the binding may be performed under experimental conditions, e.g. using isolated proteins as binding partners, using portions of proteins as binding partners, using yeast display of proteins or portions of proteins as binding partners, and the like.
- the binding of SIRPa and CD47 is usually an event between two cells, where each cell expresses one of the binding partners.
- SIRPa on phagocytic cells, such as macrophages
- CD47 on cells that could be targets for phagocytosis, e.g. tumor cells, circulating hematopoietic cells, and the like.
- Inhibitors may be identified using in vitro and in vivo assays for receptor or ligand binding or signaling.
- polypeptide As well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
- 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.
- Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma- carboxyglutamate, and O-phosphoserine.
- Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an .alpha, carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
- Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
- subject means a mammal being assessed for treatment and/or being treated.
- the mammal is a human.
- the terms "subject,” “individual,” and “patient” encompass, without limitation, individuals having cancer. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g. mouse, rat, etc.
- cancer refers to cells which exhibit autonomous, unregulated growth, such that they exhibit an aberrant 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. Cancers of virtually every tissue are known.
- cancer burden refers to the quantum of cancer cells or cancer volume in a subject. Reducing cancer burden accordingly refers to reducing the number of cancer cells or the cancer volume in a subject.
- cancer cell refers to any cell that is a cancer cell or is derived from a cancer cell e.g. clone of a cancer cell.
- cancers include solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, etc., and circulating cancers such as leukemias.
- cancer examples 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, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.
- the "pathology" of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
- cancer recurrence and “tumor recurrence,” and grammatical variants thereof, refer to further growth of neoplastic or cancerous cells after diagnosis of cancer. Particularly, recurrence may occur when further cancerous cell growth occurs in the cancerous tissue.
- Tuor spread similarly, occurs when the cells of a tumor disseminate into local or distant tissues and organs; therefore tumor spread encompasses tumor metastasis.
- Tuor invasion occurs when the tumor growth spread out locally to compromise the function of involved tissues by compression, destruction, or prevention of normal organ function.
- metastasis refers to the growth of a cancerous tumor in an organ or body part, which is not directly connected to the organ of the original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of an undetectable amount of cancerous cells in an organ or body part which is not directly connected to the organ of the original cancerous tumor. Metastasis can also be defined as several steps of a process, such as the departure of cancer cells from an original tumor site, and migration and/or invasion of cancer cells to other parts of the body.
- sample with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
- the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as cancer cells.
- the definition also includes sample that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc.
- biological sample encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like.
- a “biological sample” includes a sample obtained from a patient's cancer cell, e.g., a sample comprising polynucleotides and/or polypeptides that is obtained from a patient's cancer cell (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides); and a sample comprising cancer cells from a patient.
- a biological sample comprising a cancer cell from a patient can also include non-cancerous cells.
- diagnosis is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of a molecular subtype of breast cancer, prostate cancer, or other type of cancer.
- prognosis is used herein to refer to the prediction of the likelihood of cancer- attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease, such as ovarian cancer.
- prediction is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning. In one example, a physician may predict the likelihood that a patient will survive, following surgical removal of a primary tumor and/or chemotherapy for a certain period of time without cancer recurrence.
- treatment refers to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect.
- the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease.
- Treatment may include treatment of a tumor in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
- Treating may refer to any indicia of success in the treatment or amelioration or prevention of an cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
- the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician.
- treating includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with cancer or other diseases.
- therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
- each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
- treatment is accomplished by administering a combination of a high affinity SIRPa reagent of the invention with a cytotoxic agent.
- cytotoxic agents include chemotherapeutic agents.
- chemotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan,
- administering is combined with an effective dose of an agent that increases patient hematocrit, for example erythropoietin stimulating agents (ESA).
- ESA erythropoietin stimulating agents
- agents include, for example, Aranesp® (darbepoetin alfa), Epogen®NF/Procrit®NF (epoetin alfa), Omontys® (peginesatide), Procrit®, etc.
- combination therapies include administration with cell-specific antibodies, for example antibodies selective for tumor cell markers, radiation, surgery, and/or hormone deprivation (Kwon et ai, Proc. Natl. Acad. Sci U.S.A., 96: 15074-9, 1999).
- Angiogenesis inhibitors can also be combined with the methods of the invention.
- a number of antibodies are currently in clinical use for the treatment of cancer, and others are in varying stages of clinical development.
- antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies.
- One target antigen is CD20.
- Rituximab is a chimeric unconjugated monoclonal antibody directed at the CD20 antigen.
- CD20 has an important functional role in B cell activation, proliferation, and differentiation.
- the CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for treatment of chronic lymphocytic leukemia.
- CD22 is targeted by a number of antibodies, and has recently demonstrated efficacy combined with toxin in chemotherapy-resistant hairy cell leukemia.
- Monoclonal antibodies useful in the methods of the invention that have been used in solid tumors include, without limitation, edrecolomab and trastuzumab (herceptin).
- Edrecolomab targets the 17-1 A antigen seen in colon and rectal cancer, and has been approved for use in Europe for these indications.
- Trastuzumab targets the HER-2/neu antigen. This antigen is seen on 25% to 35% of breast cancers.
- Cetuximab (Erbitux) is also of interest for use in the methods of the invention.
- the antibody binds to the EGF receptor (EGFR), and has been used in the treatment of solid tumors including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).
- EGFR EGF receptor
- the SIRPa reagents of the invention are useful in other therapies in which monoclonal antibodies are administered for the purpose of depleting cells, e.g. in the treatment of inflammatory diseases by depletion immune cells.
- the SIRPa reagent of the invention is administered in combination with a therapeutic antibody, e.g. with rituximab for depletion of B cells in inflammatory diseases and autoimmune conditions; alemtuzumab for muliple sclerosis; OKT3 for immunosuppression; others for bone marrow transplant conditioning; and the like.
- Concomitant administration of a cancer therapeutic drug, ESA or tumor-directed antibody with a pharmaceutical composition of the present invention means administration with the high affinity SIRPa reagent at such time that both the drug, ESA or antibody and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug, ESA or antibody with respect to the administration of a compound of the invention.
- a person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.
- the phrase “disease-free survival,” refers to the lack of such tumor recurrence and/or spread and the fate of a patient after diagnosis, with respect to the effects of the cancer on the life-span of the patient.
- the phrase “overall survival” refers to the fate of the patient after diagnosis, despite the possibility that the cause of death in a patient is not directly due to the effects of the cancer.
- the phrases, "likelihood of disease-free survival", “risk of recurrence” and variants thereof, refer to the probability of tumor recurrence or spread in a patient subsequent to diagnosis of cancer, wherein the probability is determined according to the process of the invention.
- correlates refers to a statistical association between instances of two events, where events include numbers, data sets, and the like.
- a positive correlation also referred to herein as a "direct correlation” means that as one increases, the other increases as well.
- a negative correlation also referred to herein as an "inverse correlation” means that as one increases, the other decreases.
- Dosage unit refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier.
- the specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).
- “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, 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 where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the 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 and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the 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 compounds, e.g., d- 6 alkyl esters.
- a pharmaceutically acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified.
- Compounds named in this invention can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically acceptable salts and esters.
- compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
- a “therapeutically effective amount” means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.
- High affinity SIRPa polypeptides and analogs thereof are provided, which may be referred to generically as high affinity SIRPa reagents.
- the reagents are variants of the native human SIRPa protein.
- the present invention provides a soluble SIRPa variant polypeptide, wherein the polypeptide lacks the SIRPa transmembrane domain and comprises at least one amino acid change relative to the wild-type SIRPa sequence, and wherein the amino acid change increases the affinity of the SIRPa polypeptide binding to CD47, for example by decreasing 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.
- amino acid changes include any naturally occurring or man-made amino acid modifications known or later discovered in the field.
- amino acid changes include, e.g., substitution, deletion, addition, insertion, etc. of one or more amino acids.
- amino acid changes include replacing an existing amino acid with another amino acid.
- amino acid changes include replacing one or more existing amino acids with non-natural amino acids, or inserting one or more non-natural amino acids.
- Amino acid changes may be made in at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more amino acid residues relative to a native sequence.
- the one or more amino acid changes alter properties of SIRPa, e.g., affecting the stability, binding activity and/or specificity, etc.
- the high affinity SIRPa reagent of the invention comprises at least one amino acid modification within the d1 domain of SIRPa, which domain is set forth in SEQ ID NO:1 , and corresponds to residues 31 to 149 of the native human full-length human protein.
- the high affinity SIRPa reagent can consist of all or a portion of the d1 domain; and may further comprise one or more amino acids from SIRPa outside of the d1 domain; or may comprise amino acid sequences other than SIRPa, which include without limitation immunoglobulin Fc region sequences.
- High affinity SIRPa polypeptides can be at least about 100 amino acids in length, at least about 1 10, 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 at the transmembrane domain, i.e. about 343 amino acids in length, and is optionally fused to a heterologous polypeptide, e.g. immunoglobulin Fc.
- a heterologous polypeptide e.g. immunoglobulin Fc.
- shorter peptides within the d1 domain and comprising at least one amino acid change set forth herein find use, where such peptides usually comprise a contiguous stretch of amino acids from a sequence set forth herein of not more than 10 amino acids in length, not more than 15 amino acids in length, not more than 20, not more than 25, not more than 30 amino acids, not more than 35 amino acids, not more than 40 amino acids, not more than 45 amino acids, not more than 50 amino acids, not more than 55 amino acids, not more than 60 amino acids, not more than 65 amino acids, not more than 70 amino acids, not more than 75 amino acids, not more than 80 amino acids, not more than 85 amino acids, not more than 90 amino acids, not more than 95 amino acids, not more than 100 amino acids.
- amino acid changes in the high affinity SIRPa polypeptide are made at one or more of the amino acids within the set of hydrophobic core residues of SIRPa, which include, without limitation, residues (numbering defined by the wild type sequence of the d1 domain, set forth as SEQ ID NO:1 ) L4, V6, V27, I36, F39, L48, I49, Y50, F57, V60, M72, F74, I76, V92, F94 and F103.
- the amino acid changes are made on the wild-type allele 2 sequence, e.g. as shown for CV1 .
- amino acid changes are made at one or more of the amino acids within the set of contact residues that interact with CD47, which include, without limitation, A29, L30, 131 , P32, V33, G34, P35, Q52, K53, E54, S66, T67, K68, R69, F74, K93, K96, G97, S98, and D100 (SEQ ID NO:1 ).
- amino acid changes are made at two or more, three or more, four or more, five or more, and not more than 14 amino acids within the combined set of contact residues and the set of hydrophobic core residues defined above.
- amino acid changes are made at one or more of the amino acids within the set that includes, without limitation, residues L4, V6, A21 , V27, 131 , E47, K53, E54, H56, S66, V63, K68, V92, F94, and F103, or a combination thereof, for example at two or more, three or more, four or more, five or more, six or more, seven or more, and not more than 15 residues.
- the high affinity SIRPa reagent comprises at least one amino acid change selected from (1 ) L4V; L4I ; (2) V6I; V6L; (3) A21 V; (4) V27I; V27L; (5) I31 T; I31 S; 131 F; (6) E47V; E47L; (7) K53R; (8) E54Q; (9) H56P; H56R; (10) S66T; S66G; (1 1 ) K68R; (12) V92I; (13) F94L; F94V; (14) V63I; and (15) F103V.
- the high affinity SIRPa polypeptide comprises modifications selected from (1 ) to (15) above, at two, three, four, five, six, seven, eight, nine, 10, 1 1 , 12, 13, 14 or all 15 of the residues; and combinations and conservative equivalents thereof.
- Sets of amino acid changes may include combinations of the above, for example:
- V6I or V6L V27I or V27L; 131 T, 131 S, or 131 F; E47V or E47L; K53R; E54Q; H56P or H56R; S66T or S66G; V92I; and F94L or F94V.
- S66T or S66G; V92I; and F103V.
- V6I V27I; 131 F; E47V; K53R; E54Q; H56P; S66T; and V92I.
- the high affinity SIRPa polypeptide comprises the set of amino acid changes:
- a reagent of the invention comprises the portion of SIRPa that is sufficient to bind CD47 at a recognizable affinity, e.g., high affinity, which normally lies between the signal sequence and the transmembrane domain, or a fragment thereof that retains the binding activity.
- the high affinity SIRPa reagent will usually comprise at least the d1 domain of SIRPa (SEQ ID NO:1 ) with modified amino acid residues as described above.
- the high affinity SIRPa polypeptide may comprise at least amino acids 1 -3, 7-20, 32-46, 69-91 , 95- 102 of a wild-type SIRPa d1 polypeptide (SEQ ID NO:1 ) or an allelic variant thereof, i.e.
- the high affinity SIRPa polypeptide may further comprise portions of the native human SIRPa protein other than the d1 domain, including without limitation residues 150 to 374 of the native protein (set forth as SEQ ID NO:2), or including, without limitation, at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO:2, 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 contiguous amino acids or more.
- High affinity SIRPa reagents of the present invention can be further modified, e.g., joined to a wide variety of other oligopeptides or proteins for a variety of purposes.
- post-translationally modified for example by prenylation, acetylation, amidation, carboxylation, glycosylation, pegylation, etc.
- modifications can also include modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes.
- SIRPa variants of the present invention include SIRPa variants having phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.
- the high affinity SIRPa reagent has a kinetic K D of at least about 1 x 10 ⁇ 8 M for CD47. In another embodiment, the high affinity SIRPa reagent has a kinetic K D of at least about 1 x 10 ⁇ 9 M for CD47. In yet another embodiment, the high affinity SIRPa reagent has a kinetic K D of at least about 1 x 10 ⁇ 10 M for CD47. In various embodiments described herein, the high affinity SIRPa reagent exhibits a kinetic K D to CD47 that is at least about 5-fold greater than the kinetic K D of the native human SIRPa polypeptide, exemplified in SEQ ID NO:1 and 2.
- the high affinity SIRPa reagent has a kinetic K D to CD47 that is at least about 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold greater than the kinetic K D of the native SIRPa polypeptide.
- the to bind to CD47 can be determined, for example, by the ability of the SIRPa reagent to bind to CD47 coated on an assay plate; displayed on a microbial cell surface; in solution; etc.
- the binding activity of SIRPa variants of the present invention to CD47 can be assayed by immobilizing the ligand, e.g., CD47 or the SIRPa variant, to a bead, substrate, cell, etc.
- Agents can be added in an appropriate buffer and the binding partners incubated for a period of time at a given temperature. After washes to remove unbound material, the bound protein can be released with, for example, SDS, buffers with a high pH, and the like and analyzed.
- a SIRPa variant of the present invention is a fusion protein, e.g., fused in frame with a second polypeptide.
- the second polypeptide is capable of increasing the size of the fusion protein, e.g., so that the fusion protein will not be cleared from the circulation rapidly.
- the second polypeptide is part or whole of an immunoglobulin Fc region.
- the second polypeptide is any suitable polypeptide that is substantially similar to Fc, e.g., providing increased size, multimerization domains, and/or additional binding or interaction with Ig molecules.
- These fusion proteins can facilitate purification, multimerization, and show an increased half-life in vivo. Fusion proteins having disulfide-linked multimeric structures can also be more efficient in binding and neutralizing other molecules than a monomeric SIRPa.
- the high affinity SIRPa binding domain i.e. a SIRPa d1 domain modified as set forth herein to provide for high affinity binding to CD47
- a multimeric protein i.e. two, three, four or more SIRPa binding domains are covalently or non- covalently linked, e.g. as a fusion protein; disulfide bonded; through biotin binding to avidin, streptavidin, etc.
- Such multimeric high affinity SIRPa binding proteins are useful as single agents to increase phagocytosis of cells expressing CD47; or in combination with other binding agents, e.g. cell-specific monoclonal antibodies.
- the high affinity SIRPa binding domain is fused or otherwise joined to an immunoglobulin sequence to form a chimeric protein.
- the immunoglobulin sequence preferably, but not necessarily, is immunoglobulin constant domain(s).
- the immunoglobulin moiety in such chimeras may be obtained from any species, usually human, and includes lgG1 , lgG2, lgG3 or lgG4 subtypes, IgA, IgE, IgD or IgM.
- the immunoglobulin moiety may comprise one or more domains, e.g. CH1 , CH2, CH3, etc.
- Chimeras constructed from a sequence linked to an appropriate immunoglobulin constant domain sequence are known in the art.
- the encoded chimeric polypeptide may retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CH1 of the heavy chain or the corresponding region of the light chain.
- the precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion or binding characteristics of the SIRPa polypeptide-immunoglobulin chimeras.
- the SIRPa polypeptide-immunoglobulin chimeras are assembled as monomers, or hetero- or homo-multimers, and particularly as dimers or tetramers.
- an immunoglobulin light chain may be included, either covalently associated to an SIRPa polypeptide-immunoglobulin heavy chain fusion polypeptide, or directly fused to the SIRPa polypeptide.
- a single chain construct may be used to provide both heavy and light chain constant regions.
- the second polypeptide is a marker sequence, such as a peptide that facilitates purification of the fused polypeptide.
- the marker amino acid sequence can be a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 9131 1 ), among others, many of which are commercially available.
- a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 9131 1
- hexa-histidine provides for convenient purification of the fusion protein.
- HA hemagglutinin protein
- Wilson et al., Cell 37: 767, 1984 The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
- the high affinity SIRPa binding domain is provided as a monomeric protein, which may be an isolated d1 domain, or a d1 domain fused to SIRPa or chimeric sequences.
- Monomeric SIRPa binding domains are useful, for example, as an adjuvant to increase the phagocytosis of cells expressing CD47, when combined with a cell-specific binding agent, e.g. an antibody, particularly a tumor cell specific antibody as defined herein.
- Monomeric SIRPa binding domains are also useful as adjuvants for increasing phagocytosis, as well as other immune functions, e.g.
- ADCC uptake of antigens for antigen presentation, and the like by a number of immune cells, such as macrophages, dendritic cells, neutrophils, granulocytes and the like, which express SIRPa and respond to blockade with the SIRPa reagents of the invention.
- Monomeric high affinity SIRPa binding domains are also useful as imaging agents, e.g. when conjugated to a detectable label.
- high affinity SIRPa reagents of the invention include reagents further modified to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
- variants of the present invention further include analogs containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.
- the high affinity SIRPa reagent is coupled or conjugated to one or more imaging moieties, i.e. a detectable label.
- imaging moieties i.e. a detectable label.
- cytotoxic moiety refers to a moiety that inhibits cell growth or promotes cell death when proximate to or absorbed by the cell. Suitable cytotoxic moieties in this regard include radioactive isotopes (radionuclides), chemotoxic agents such as differentiation inducers and small chemotoxic drugs, toxin proteins, and derivatives thereof.
- imaging moiety refers to a moiety that can be utilized to increase contrast between a tumor and the surrounding healthy tissue in a visualization technique, e.g., radiography, positron-emission tomography, magnetic resonance imaging, direct or indirect visual inspection.
- suitable imaging moieties include radiography moieties, e.g. heavy metals 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. It will be appreciated by one of ordinary skill that some overlap exists between what is a therapeutic moiety and what is an imaging moiety.
- therapeutic or imaging agents can be conjugated to the high affinity SIRPa reagent moiety by any suitable technique, with appropriate consideration of the need for pharmacokinetic stability and reduced overall toxicity to the patient.
- a direct reaction between an agent and SIRPa is possible when each possesses a functional group capable of reacting with the other.
- a nucleophilic group such as an amino or sulfhydryl group
- a carbonyl-containing group such as an anhydride or an acid halide
- an alkyl group containing a good leaving group e.g., a halide
- a suitable chemical linker group may be used.
- a linker group can function as a spacer in order to avoid interference with binding capabilities.
- bifunctional or polyfunctional reagents both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, III.), may be employed as a linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology.
- the SIRPa is linked to the cytotoxic or imaging moiety by the use of a non-covalent binding pair, such as streptavidin/biotin, or avidin/biotin.
- one member of the pair is covalently coupled to SIRPa and the other member of the binding pair is covalently coupled to the cytotoxic or imaging moiety. It may be desirable to couple more than one cytotoxic and/or imaging moiety.
- an antibody may be made useful as a contrasting agent for several visualization techniques, or a therapeutic antibody may be labeled for tracking by a visualization technique.
- a carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations.
- Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties.
- a carrier may also bear an agent by non-covalent associations, such as non-covalent bonding or by encapsulation
- Carriers and linkers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds.
- a radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
- Radiographic moieties for use as imaging moieties in the present invention include compounds and chelates with relatively large atoms, such as gold, iridium, technetium, barium, thallium, iodine, and their isotopes. It is preferred that less toxic radiographic imaging moieties, such as iodine or iodine isotopes, be utilized in the compositions and methods of the invention. Such moieties may be conjugated to the high affinity SIRPa reagent antibody moiety through an acceptable chemical linker or chelation carrier.
- Positron emitting moieties for use in the present invention include 18 F, which can be easily conjugated by a fluorination reaction with the high affinity SIRPa reagent.
- Magnetic resonance contrast moieties include chelates of chromium(lll), manganese(ll), iron(ll), nickel(ll), copper(ll), praseodymium(lll), neodymium(lll), samarium(lll) and ytterbium(lll) ion. Because of their very strong magnetic moment, the gadolinium(lll), terbium(lll), dysprosium(lll), holmium(lll), erbium(lll), and iron(lll) ions are especially preferred.
- Optically visible moieties for use as imaging moieties include fluorescent dyes, or visible-spectrum dyes, visible particles, and other visible labeling moieties.
- 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 inspected visually. Endoscopic visualization procedures may be more compatible with the use of such labels.
- Acceptable dyes include FDA-approved food dyes and colors, which are non-toxic, although pharmaceutically acceptable dyes which have been approved for internal administration are preferred.
- the effective amount of an imaging conjugate compositions to be given to a particular patient will depend on a variety of factors, several of which will be different from patient to patient.
- a competent clinician will be able to determine an effective amount to facilitate the visualization of a tumor. Dosage will depend on the treatment of the tumor, route of administration, the nature of the therapeutics, sensitivity of the tumor to the therapeutics, etc. Utilizing ordinary skill, the competent clinician will be able to optimize the dosage of a particular therapeutic or imaging composition in the course of routine clinical trials.
- a typical dose may be from 0.001 to 100 milligrams of conjugate per kilogram subject body weight. Relatively large doses, in the range of 0.1 to 10 mg per kilogram of patient body weight may be used for imaging conjugates with a relatively non-toxic imaging moiety. The amount utilized will depend on the sensitivity of the imaging method, and the relative toxicity of the imaging moiety.
- SIRPa variants of the present invention can be produced by any suitable means known or later discovered in the field, e.g., produced from eukaryotic or prokaryotic cells, synthesized in vitro, etc. Where the protein is produced by prokaryotic cells, 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.
- polypeptides may be prepared by cell-free translation systems, or synthetic in vitro synthesis, using conventional methods as known in the art.
- Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, CA, Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
- the polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis.
- a lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
- the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.
- Methods which are 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/genetic recombination. Alternatively, RNA capable of encoding the polypeptides of interest may be chemically synthesized. One of skill in the art can readily utilize well-known codon usage tables and synthetic methods to provide a suitable coding sequence for any of the polypeptides of the invention. The nucleic acids may be isolated and obtained in substantial purity.
- the nucleic acids will be obtained substantially free of other naturally-occurring nucleic acid sequences, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant," e.g., flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.
- the nucleic acids of the invention can be provided as a linear molecule or within a circular molecule, and can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. Expression of the nucleic acids can be regulated by their own or by other regulatory sequences known in the art.
- the nucleic acids of the invention can be introduced into suitable host cells using a variety of techniques available in the art.
- SIRPa variants can be provided in pharmaceutical compositions suitable for therapeutic use, e.g. for human treatment.
- pharmaceutical compositions of the present invention include one or more therapeutic entities of the present invention or pharmaceutically acceptable salts, esters or solvates thereof.
- pharmaceutical compositions of the present invention include one or more therapeutic entities of the present invention in combination with another therapeutic agent, e.g., another anti-tumor agent.
- compositions comprising an active therapeutic agent and a other pharmaceutically acceptable excipient.
- the preferred form depends on the intended mode of administration and therapeutic application.
- the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles 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 solutions, dextrose solution, and Hank's solution.
- the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
- compositions of the present invention can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
- macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
- Methods are provided for treating, reducing or preventing cancer, including without limitation lymphomas, leukemias, carcinomas, melanomas, glioblastomas, sarcomas, myelomas, etc. as primary or metastatic cancers, by inhibiting the interaction between SIRPa and CD47, thereby increasing in vivo phagocytosis of the tumor cells.
- Such methods include administering to a subject in need of treatment a therapeutically effective amount or an effective dose of a high affinity SIRPa reagent of the invention, including without limitation combinations of the reagent with a chemotherapeutic drug, a tumor-specific antibody, or an ESA.
- Effective doses of the therapeutic entity of the present invention vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
- the patient is a human, but nonhuman mammals may also be treated, e.g. companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., and the like. Treatment dosages can be titrated to optimize safety and efficacy.
- the therapeutic dosage may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
- dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1 -10 mg/kg.
- An exemplary treatment regime entails administration once every two weeks or once a month or once every 3 to 6 months.
- Therapeutic entities of the present invention are usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the therapeutic entity in the patient.
- therapeutic entities of the present invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.
- a relatively low dosage may be administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In other therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
- methods of the present invention include treating, reducing or preventing tumor growth, tumor metastasis or tumor invasion of cancers including lymphomas, leukemias, carcinomas, melanomas, glioblastomas, sarcomas, myelomas, etc.
- cancers including lymphomas, leukemias, carcinomas, melanomas, glioblastomas, sarcomas, myelomas, etc.
- pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of disease in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
- the high affinity SIRPa polypeptides of the invention can be used in vitro and in vivo to monitor the course of disease therapy, for example, by measuring the increase or decrease in the number of cells expressing CD47, particularly cancer cells expressing CD47, it can be determined whether a particular therapeutic regimen aimed at ameliorating disease is effective.
- the SIRPa polypeptide may be detectably labeled.
- the high affinity SIRPa polypeptides of the invention may be used in vitro in binding assays in which they can be utilized in liquid phase or bound to a solid phase carrier.
- the polypeptides in these immunoassays can be detectably labeled in various ways.
- types of assays which can utilize high affinity SIRPa polypeptides of the invention are flow cytometry, e.g. FACS, MACS, histochemistry, competitive and non-competitive immunoassays in either a direct or indirect format; and the like.
- Detection of CD47 using the high affinity SIRPa polypeptides can be done with assays which are run in either the forward, reverse, or simultaneous modes, including histochemical assays on physiological samples.
- Those of skill in the art will know, or can readily discern, other assay formats without undue experimentation.
- the high affinity SIRPa polypeptides can be bound to many different carriers and used to detect the presence of CD47 expressing cells.
- carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
- the nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding proteins, or will be able to ascertain such, using routine experimentation.
- [001 08] There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bio-luminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the polypeptides of the invention, or will be able to ascertain such, using routine experimentation. Furthermore, the binding of these labels to the polypeptides of the invention can be done using standard techniques common to those of ordinary skill in the art.
- CD47 may be detected by the high affinity SIRPa polypeptides of the invention when present in biological fluids and tissues. Any sample containing a detectable amount of CD47 can be used.
- a sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or a solid or semi-solid such as tissues, feces, and the like, or, alternatively, a solid tissue such as those commonly used in histological diagnosis.
- [001 1 0] Another labeling technique which may result in greater sensitivity consists of coupling the polypeptides to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.
- haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.
- the imaging conjugates of a high affinity SIRPa reagent can be administered to the subject in a series of more than one administration. The imaging conjugate compositions may be administered at an appropriate time before the visualization technique.
- administration within an hour before direct visual inspection may be appropriate, or administration within twelve hours before an MRI scan may be appropriate. Care should be taken, however, to not allow too much time to pass between administration and visualization, as the imaging compound may eventually be cleared from the patient's system.
- compositions for the treatment of cancer can be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means.
- a typical route of administration is intravenous or intratumoral, although other routes can be equally effective.
- compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
- the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997.
- the agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
- the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
- GMP Good Manufacturing Practice
- Toxicity of the high affinity SIRPa reagents described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 (the dose lethal to 50% of the population) or the LD 100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
- the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
- the dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
- kits comprising the compositions (e.g., high affinity SI RPa reagents and formulations thereof) of the invention and instructions for use.
- the kit can further contain a least one additional reagent, e.g. a chemotherapeutic drug, anti-tumor antibody, ESA, etc.
- Kits typically include a label indicating the intended use of the contents of the kit.
- the term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
- a C-terminal biotin acceptor peptide (BAP)-LNDI FEAQKIEWHE tag was added and proteins were co-expressed with BirA ligase with excess biotin (100 ⁇ ).
- SIRPa variant FD6 was expressed in the E. coli periplasm with an N-terminal maltose-binding protein (MBP) tag, which was removed by treatment with 3C protease.
- MBP maltose-binding protein
- the CD47 IgSF domain was co-expressed with EndoF in Hi5 cells in the presence of kifunensine to remove glycosylation.
- SIRPa variant proteins were cloned into pFUSE-hlgG4-Fc2 and pFUSE-hlgG2-Fc2 vectors (Invivogen) in frame with the IL2 signal sequence.
- SIRPa variant fusion proteins were expressed in Freestyle 293-F cells (Invitrogen) following transfection with 293fectin (Invitrogen). Supernatants were harvested after 96 hours of protein expression and purified over HiTrap Protein A columns (GE Healthcare).
- Jurkat and DLD-1 cells were transduced with a Iuciferase2 (Promega)-eGFP expressing lentivirus derived from the pCDH-CMV-MCS-EF1 -puro HIV-based lentiviral vector (Systems Bioscience). Cells were sorted for GFP+ cells using a BD FACSAria II flow cytometer and maintained in culture in the same manner as the parental lines.
- Iuciferase2 Promega
- Macrophage derivation and culture For derivation of human macrophages, leukocyte reduction system chambers were obtained from the Stanford Blood Center from anonymous donors. Peripheral blood mononuclear cells were obtained by centrifugation over a Ficoll Paque Premium (GE Healthcare) density gradient. CD14+ monocytes were purified using CD14 microbeads (Miltenyi) and an AutoMACS Pro Separator (Miltenyi). Monocytes were differentiated to macrophages by culturing in IMDM+GlutaMax (lnvitrogen)+10% AB human serum (lnvitrogen)+1 % penicillin/streptomycin for 7 days at which time they were used for phagocytosis assays.
- IMDM+GlutaMax lanvitrogen
- AB human serum lanvitrogen
- penicillin/streptomycin penicillin/streptomycin
- Mouse macrophages were derived by isolating bone marrow from C57BI/Ka Rosa26- mRFP1 transgenic mice and culturing in RPMI (Invitrogen) +10% FBS +1 % GlutaMax + 1 % penicillin/streptomycin supplemented with ⁇ g/mL murine M-CSF (Peprotech). After 7 days of differentiation, macrophages were harvested and used for phagocytosis assays.
- In vitro phagocytosis assays were performed using mouse and human macrophages. For evaluation by flow cytometry, approximately 50,000 macrophages were added per well in 96-well tissue culture plates. 200,000 GFP+ tumor cells were pre-incubated for 30 minutes with antibody or SIRPa variant therapies in serum-free medium then added to the macrophages. Anti-CD47 clone 2D3 (eBioscience) and cetuximab (Bristol-Myers Squibb) were used as described for opsonization. Macrophages and target cells were incubated for 2 hours in a humidified 37° incubator containing 5% carbon dioxide.
- phagocytosis index was scored as the number of target cells per macrophage multiplied by 100.
- High-affinity SIRPa lowers the threshold for macrophage phagocytosis of cancer cells
- the ability of tumors to evade the immune system is an emerging hallmark of cancer, and new therapeutic strategies that direct immune responses against cancer cells show promise in experimental and clinical settings.
- Macrophages commonly infiltrate tumors, and recent studies have identified CD47 as an anti-phagocytic "don't eat me" signal that is highly expressed on many types of cancer to avoid macrophage-mediated destruction.
- Antibodies that block binding of CD47 to SIRPa, an inhibitory receptor on macrophages, greatly increase phagocytosis of cancer cells— identifying an exciting new axis to manipulate with anti-tumor immunotherapies.
- Directed evolution and protein engineering were used to modify the binding domain of SIRPa, whose wild-type affinity is too weak to be therapeutically useful, as a high- affinity competitive antagonist of CD47.
- SIRPa variants that bind to CD47 with an approximately 50,000-fold increase in affinity relative to wild-type SIRPa.
- the variants act as single agents to induce phagocytosis in vitro and reduce growth of human tumors in vivo.
- single domain high-affinity SIRPa monomers are not sufficient to induce maximal phagocytosis on their own, they greatly enhance the efficacy of established therapeutic monoclonal antibodies when given in combination therapies.
- CD47 is a pervasive mechanism that tumor cells use to evade the immune system, the molecules generated in this study benefit a large number of cancer patients, both as monotherapies and as adjuvants to other targeted biologies.
- the resulting first generation SIRPa variants bound CD47 with 20-100-fold higher affinity than wild type SI RPa, as measured by surface plasmon resonance.
- we performed a second generation of directed evolution by constructing a library that achieved full-coverage of thirteen residues mutated in the first generation selectants.
- KD dissociation constants
- t 1 2 decay half- lives
- CV1 has the following amino acid changes, relative to the wild-type allele: V6I ; V27I ; 131 F; E47V; K53R; E54Q; H56P; S66T; V92I.
- CV1 may comprise, for example, a d1 domain amino acid sequence as follows:
- the CD loop of FD6 contains three of the four contact mutations present in the consensus sequence ( Figure 1 E). We speculate these mutations stabilize the CD loop, which positions the positive charge of Arg53 into a cluster of glutamic acids on CD47 ( Figure 1 E). The remainder of the binding interface between FD6 and CD47 highly resembles the wild-type SIRPa:CD47 interface, with the most notable exception being the mutation of Ile31 to Phe. These structural studies indicate the high-affinity SIRPa variants can serve as efficacious CD47 antagonists. [00136] To test the functional properties of the high-affinity SIRPa variants, we first examined their ability to bind and antagonize CD47 on the surface of cancer cells.
- SIRPa variants with increased CD47 affinity exhibited greater potency in binding (Figs. 8a, c) and blocking cell-surface CD47 (Fig. 2a and Fig. 8b).
- both FD6 and CV1 variants exhibited potent antagonism relative to wild-type SIRPa.
- both high- affinity variants were more potent CD47 antagonists than Fab fragments produced from anti- CD47 antibody clone B6H12, a well-characterized CD47 antagonist that demonstrates therapeutic efficacy in vitro and in vivo (Fig. 8a).
- phagocytosis assays were performed using DLD-1 colon cancer cells treated with the anti-EGFR antibody cetuximab. Phagocytosis was evaluated in response to titrating concentrations of cetuximab alone, in combination with wild-type SIRPa monomer, or in combination with high-affinity SIRPa monomers. Relative to both cetuximab alone or in combination with wild-type SIRPa monomer, the combination of cetuximab plus high-affinity SIRPa monomer produced a significant increase in both the maximal efficacy and potency of cetuximab (Fig. 2g).
- CV1 -hlgG4 exhibited anti-tumor efficacy as a single agent, we next evaluated its efficacy in a model of human bladder cancer, a type of cancer for which no targeted biologies currently exist.
- GFP-luciferase + 639-V bladder cancer cells were injected into the dorsal subcutaneous tissue of NSG mice. Engraftment was confirmed by bioluminescence imaging, and mice were randomized into groups for daily treatment with vehicle control or CV1 -hlgG4. Treatment with CV1 -hlgG4 substantially reduced tumor growth rates as evaluated by bioluminescence imaging (Figs. 3e, f).
- CV1 -hlgG4 resultsed in decreases in red blood cell indices (Fig. 10a).
- CV1 -hlgG4 treated mice also developed palpable stromal tissue surrounding the sites of tumor engraftment. Histopathological examination of this tissue revealed small tumor nodules embedded in an extensive inflammatory infiltrate containing macrophages (Figs. 10b, c).
- mice were randomized into groups for a three-week course of daily treatment with either vehicle, CV1 monomer alone, rituximab alone, or a combination of rituximab plus CV1 monomer. Treatment with CV1 monomer or rituximab alone only reduced tumor growth, while treatment with the combination therapy dramatically eradicated tumors in the majority of mice (Figs. 4a, b, c).
- High-affinity SIRPa-Fc fusion proteins and anti- CD47 antibodies combine a CD47 blocking component and a pro-phagocytic antibody Fc into a single molecule; hence they exhibit efficacy as single agents but have greater potential for off target toxicity.
- the combination of high-affinity SIRPa monomers with a separate anti-tumor monoclonal antibody, such as rituximab specifically enhances anti-tumor responses. While this strategy offers clear benefits, particularly a lack of discernible toxicity, it is dependent on the availability and efficacy of clinically-approved monoclonal antibodies to achieve maximal responses.
- the high-affinity SIRPa reagents constitute a novel class of anti-tumor biologies that are amenable to further engineering. Modifications can be designed to alter efficacy, specificity, tissue penetrance, clearance, and toxicity. Furthermore, since many tumors overexpress CD47 and expression levels correlate with poor patient outcomes, high-affinity SIRPa variants can be adapted as non-invasive imaging agents for cancer. CD47 is commonly used by tumor cells to evade the immune system, thus high-affinity SIRPa variants could be valuable therapeutics for a variety of human cancers. High-affinity SIRPa-Fc fusion proteins demonstrate efficacy as single agents, and thus can be particularly useful as treatments for cancers for which no targeted therapies currently exist.
- high-affinity SIRPa monomers can be used as universal adjuvants to conventional monoclonal antibody therapies.
- this study deepens our knowledge of macrophage responses to malignant cells and supports use of the high- affinity SIRPa reagents as immune-based therapies for cancer.
- CD47 IgSF domain (residues 1 -1 17), with a C15G mutation and 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 a Superdex- 75 column.
- CD47 was co-expressed with endoglycosidase-H (endoH) in the presence of kifunensine.
- Monomeric SIRPa variants (residues 1 -1 18) were expressed as MBP-fusions in the periplasm of BL-21 (DE3) E.
- coli using a modified pMal-p2X expression vector (New England Biolabs) containing a rhinovirus 3C protease cleavage site after the MBP tag and a C-terminal 8 ⁇ histidine tag.
- Cells were induced at an OD 600 of 0.8 with 1 mM IPTG and incubated with shaking at 22 °C for 24 hours.
- Periplasmic protein was obtained by osmotic shock and the MBP-fusion proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) chromatography.
- Ni-NTA nickel-nitrilotriacetic acid
- Eluted proteins were digested with 3C protease at 4 °C for 12 hours to remove MBP and further purified by an additional Ni-NTA chromatography step, followed by size exclusion chromatography with a Superdex-S75 column.
- endotoxin was removed using Triton X-1 14 as previously described and endotoxin removal confirmed using the ToxinSensor Chromogenic LAL Endotoxin Assay Kit (Genscript).
- SIRPoFc fusions were produced by cloning SIRPa variants into a modified pFUSE-hlgG4-Fc vector (Invivogen) with an IL-2 signal sequence and engineered Ser228 Pro mutation. Proteins were expressed by transient transfection in Freestyle 293-F cells (Invitrogen) and purified over HiTrap Protein A columns (GE Healthcare). Chimeric anti-CD47 clone B6H12-hlgG4 was recombinantly produced by stable expression in CHO cells (Lonza).
- proteins were expressed with a carboxy terminal biotin acceptor peptide tag (GLNDIFEAQKIEWHE) and purified as described above.
- the purified proteins were biotinylated in vitro with BirA ligase and then repurified from the reaction mixture by size exclusion chromatography.
- B6H12 antibody was desalted into 20 mM sodium citrate pH 6.0, 25 mM cysteine, 5 mM EDTA and diluted to a concentration of 4 mg/mL. The antibody was then mixed with 250 ⁇ _ immobilized ficin resin (Thermo Scientific) per ml_ of antibody and incubated with rotation at 37 °C for five hours. The digested fragments were purified by passing the reaction mixture over a monoQ column (B6H12 Fab resided in the flow through), followed by gel filtration with a Superdex-200 column.
- Yeast display and library generation of SIRPa variants The N-terminal V-set domain of SIRPa (residues 1 -1 18) was displayed on the surface of S. cerevisiae strain EBY100 as a C terminal fusion to Aga2 using the pCT302 vector as previously described.
- GGNTTACAACTGTTTCAGAGTCCACAAAGAGAGAAAAC3 ' (SEQ ID NO:25) 5 ' GCCGGCATCTG CTGGGGTGATGTTACTGATGCTAANGGAAANGTCAANGTTTTCTCTCTTTGTGGA3 ' ; (SEQ ID NO: 26) 5 'ACCCCAGCAGATGCCGGCACCTACTACTGTNTTAAGNTTCGGAAAGGGAGCCCTGACACGGAG3 ' , (SEQ ID NO : 27 ) 5 'AGAGGGTTTGGCACGCACAGACAGCTCAGTGCCTGCTCCAGACTTAANCTCCGTGTCAGGGCTCCC3 ' .
- the PCR products were 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 4.0-10 8 transformants.
- the resulting library contained 2 x 10 8 transformants.
- yeast were pelleted by centrifugation at 5,000g for five minutes and washed twice with 10 mL PBE. Magnetically-labeled yeast were resuspended in 5 mL PBE and separated with an LS MACS column according to the manufacturer's instructions (Miltenyi). Eluted yeast were pelleted, resuspended in SDCAA medium, and expanded for the next round of selection. Four additional rounds of selection were performed similarly to the first round with the following modifications: 1 x-10 8 yeast were resuspended in 500 ⁇ _ PBE containing FITC-labeled anti-c-Myc antibody (Miltenyi) or successively decreasing concentrations of biotinylated CD47 protein, from 1 ⁇ to 100 nM.
- yeast were washed with PBE and for selections with CD47, labeled with streptavidin-PE (Invitrogen) or streptavidin-Alexa Fluor 647 (produced in house) for 15 minutes.
- Yeast were washed twice more with PBE and magnetically labeled with 50 ⁇ _ of the appropriate anti-fluorophore microbeads (anti-FITC, anti-PE, or anti-Alexa Fluor 647; Miltenyi) for 15 minutes.
- Yeast were washed once, resuspended in 3 ml_ PBE, and separated with an LS column as in the first round.
- yeast were selected with monomeric, biotinylated CD47 protein, as in rounds two through five of the first generation selections.
- the first round was selected with 20 nM biotinylated CD47 and the second round with 1 nM biotinylated CD47, using a larger staining volume (10 ml_ PBE) to avoid ligand depletion.
- kinetic selection was performed. Briefly, yeast were stained with 20 nM biotinylated CD47 for one hour, washed with PBE, and then resuspended in 500 ⁇ _ PBE containing 1 ⁇ non- biotinylated CD47.
- yeast were incubated at 25 °C for 90 minutes (round three) or 300 minutes (rounds four and five), after which they were washed with ice-cold PBE and stained with fluorescently labeled streptavidin.
- yeast were separated using MACS, as described for the first generation library.
- yeast were co-labeled with FITC-labeled anti-c-Myc and streptavidin-Alexa Fluor 647 and selected with a FACSAria cell sorter (BD Biosciences).
- Crystals were obtained by addition of 0.1 ⁇ _ protein to an equal volume of 2.0 M ammonium sulfate and 0.1 M Tris pH 7.3, and were cryoprotected in paraffin oil. Diffraction studies were performed at beamline 8-2 at the Advanced Light Source (Berkeley, CA, USA). An anisotropic 1 .9 A dataset was obtained and processed with HKL-3000. The FD6:CD47 complex was solved by molecular replacement with the individual models of CD47 and SIRPa from Protein Data Bank accession code 2JJS. Refinement was carried out using PHENIX and model adjustment performed with COOT. Bulk solvent flattening was used for solvent correction. Initial refinement used rigid body, coordinate, and real-space refinement, along with individual atomic displacement parameter refinement. TLS refinement was added in later refinement iterations.
- DLD-1 cells ATCC
- HT-29 cells ATCC
- Raji cells ATCC
- Jurkat cells ATCC
- 639-V cells DSMZ
- RPMI+GlutaMax Invitrogen
- 10% fetal bovine serum Omega Scientific
- GFP-luciferase + lines were generated by transduction using a pCDH-CMV-MCS-EF1 puro HIV-based lentiviral vector (Systems Biosciences) engineered to express an eGFP-luciferase2 (pgl4) fusion protein. Stable lines were created by sorting for GFP expression on FACSAria II cell sorters (BD Biosciences).
- Leukocyte reduction system (LRS) chambers were obtained from the Stanford Blood Center from anonymous donors, and peripheral blood mononuclear cells were enriched by density gradient centrifugation over Ficoll- Paque Premium (GE Healthcare).
- Monocytes were purified on an AutoMACS (Miltenyi) using anti-CD14 microbeads (Miltenyi) and differentiated to macrophages by culture for 7-10 days in IMDM+GlutaMax (Invitrogen) supplemented with 10% AB-Human Serum (Invitrogen) and 100 U/mL penicillin and 100 ⁇ g/mL streptomycin (Invitrogen).
- Phagocytosis assays were performed by co-culture of 50,000 macrophages with 100,000 GFP+ tumor cells for 2 hours, then analyzed using an LSRFortessa cell analyzer with high throughput sampler (BD Biosciences).
- Antibodies used for treatment included: mouse lgG1 isotype control (eBioscience), anti-CD47 clone 2D3 (eBioscience), anti-EpCam (BioLegend), cetuximab (Bristoll-Myers Squibb), and rituximab (Genentech). Macrophages were identified by flow cytometry using anti-CD14, anti-CD45, or anti-CD206 antibodies (BioLegend).
- Dead cells were excluded from the analysis by staining with DAPI (Sigma). Phagocytosis was evaluated as the percentage of GFP + macrophages using FlowJo v9.4.10 (Tree Star) and was normalized to the maximal response by each independent donor against each cell line. Statistical significance was determined by 2-way ANOVA with Bonferroni post-tests, and, when indicated, data were fit to sigmoidal dose- response curves using Prism 5 (Graphpad).
- mice Nod.Cg-Prkdc scld IL2rgtmiwji/SzJ (NSG) mice were used for all in vivo experiments.
- mice were engrafted with tumors at approximately 6-10 weeks of age, and experiments were performed with age and sex-matched cohorts of 8-15 mice. Mice were maintained in a barrier facility under the care of the Stanford Veterinary Services Center and handled according to protocols approved by the Stanford University Administrative Panel on Laboratory Animal Care.
- Bioluminescence imaging Anesthetized mice were injected with 200 ⁇ _ D-luciferin (firefly) potassium salt (Biosynth) reconstituted at 16.67 mg/mL in sterile PBS. Bioluminescence imaging was performed using an S Spectrum (Caliper Life Sciences) over 20 minutes to record maximal radiance. Peak total flux values were assessed from the anatomical region of interest using Living Image 4.0 (Caliper Life Sciences) and used for analysis.
- FD6-hlgG4 (FD6 underlined, human lgG4 S228P in bold), which includes the CH2, CH3 and hinge regions of human lgG4, and the d1 domain of high affinity SIRPa FD6:
- CV1 -hlgG4 (CV-1 underlined, human lgG4 S228P in bold), which includes the CH2, CH3 and hinge regions of human lgG4, and the d1 domain of high affinity SIRPa CV1 . Note that the CV1 amino acid substitutions are "built" on the human wild-type allele 2:
- FD6-hlgG2 (FD6 underlined, human lgG2 in bold), which includes the CH2, CH3 and hinge regions of human lgG2, and the d1 domain of high affinity SIRPa FD6: (SEQ ID NO:42)
- CV1 -hlgG2 (CV-1 underlined, human lgG2 in bold), which includes the CH2, CH3 and hinge regions of human lgG2, and the d1 domain of high affinity SIRPa CV-1 :
- FD6-zipper (FD6 underlined, GCN4 leucine-zipper in bold), which includes the d1 domain of FD6 fused to GCN4, and which utilizes the leucine zipper function to dimerize:
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WO2014094122A1 (en) * | 2012-12-17 | 2014-06-26 | Trillium Therapeutics Inc. | Treatment of cd47+ disease cells with sirp alpha-fc fusions |
WO2014149477A1 (en) | 2013-03-15 | 2014-09-25 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for achieving therapeutically effective doses of anti-cd47 agents |
WO2014179132A1 (en) | 2013-04-29 | 2014-11-06 | The Board Of Trustees Of The Leland Stanford Junior University | Use of anti-cd47 agents to enhance immunization |
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