Targeted therapy for small cell lung cancer

Abstract

To provide methods for treatment of lung cancers, particularly small cell lung cancer with targeted therapy.SOLUTION: A method for treatment of a lung cancer in a patient comprises: administering to the patient an effective dose of a targeted therapeutic agent that specifically binds to one or more cell surface antigens including CD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165, and EpCAM.SELECTED DRAWING: None

Description

Background Art

[0001] Background Targeted therapies such as antibodies and specific ligands have been found to be effective in fighting cancer, especially in cases where conventional therapies have failed. Even more promising is the fact that antibodies against cancer generally act through a mechanism distinct from traditional chemotherapy or radiation therapy and can therefore often produce additive or synergistic effects when combined with these traditional therapies.

[0002] Antibodies can achieve their therapeutic effects through a variety of mechanisms. Antibodies can have a direct effect in bringing about apoptosis or programmed cell death. Antibodies can block growth factor receptors and effectively halt the proliferation of tumor cells. In cells expressing monoclonal antibodies, they can trigger the formation of anti-idiotype antibodies. Indirect effects include mobilizing cytotoxic cells such as monocytes and macrophages. This type of antibody-mediated cell killing is called antibody-dependent cell-mediated cytotoxicity (ADCC). Monoclonal antibodies also bind to complement, leading to direct cytotoxicity known as complement-dependent cytotoxicity (CDC).

[0003] CD47 is a valuable target for anti-cancer therapy due to its function as an inhibitor of macrophage phagocytosis and its widespread expression on a variety of human neoplasms. CD47 can transmit an inhibitory signal that blocks phagocytosis by binding to signal regulatory protein alpha (SIRPα), a receptor expressed on the surface of macrophages. Blocking the interaction between CD47 and SIRPα with an antibody not only stimulates macrophages to engulf cancer cells in vitro but also exerts a robust anti-cancer effect in vivo. Other CD47 blocking agents include "next-generation" CD47 antagonists that bind and block human CD47 with unusually high affinity.

[0004] High-affinity SIRPα variants can lower the threshold for macrophage activation and promote the phagocytic response promoted by tumor-specific antibodies by disabling the inhibitory signals transmitted by SIRPα. The extent to which the anti-cancer activity of a given therapeutic antibody is enhanced by CD47 blockade appears to depend on a number of factors, including the level of antigen expression on the surface of malignant cells, the isotype of its heavy chain, and its ability to bind to Fc receptors on immune effectors, including the orientation it assumes upon antigen binding. Thus, high-affinity SIRPα monomers represent a rapid, safe, and effective alternative to several other approaches, including the drug / toxin conjugation strategies being pursued in this direction.

[0005] The identification of effective targets and combinations for targeted therapies remains a high area of interest. The present invention addresses this need.

Summary of the Invention

[0006] Methods and compositions for the treatment of lung cancer by targeted therapy are provided. In some embodiments, the lung cancer is small cell lung cancer. In some embodiments, the therapy targets one or more cell surface antigens including CD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165, EpCAM, and the like. In some embodiments, the targeted therapy comprises administering a therapeutic dose of an antibody that specifically binds to a cell surface marker selected from CD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165, and EpCAM to an individual suffering from lung cancer.

[0007] In some embodiments, the targeted therapy is combined with a CD47 blocking agent. Cancer cells evade macrophage surveillance by upregulating CD47 expression. An agent that masks the CD47 protein, such as an antibody or small molecule that binds to CD47 or SIRPα and prevents the interaction between CD47 and SIRPα, is administered to a patient, thereby increasing the clearance of cancer cells via phagocytosis. The agent that blocks CD47 is combined with a monoclonal antibody directed against one or more lung cancer cell markers, and this composition can be synergistic in enhancing the phagocytosis and elimination of cancer cells compared to the use of a single agent.

[0008] Combinations of specific agents for therapy of interest include anti-CD47 and anti-CD56, anti-CD47 and anti-CD44, anti-CD47 and anti-CD99, anti-CD47 and anti-EpCam. In some such embodiments, the anti-CD47 agent is a high-affinity SIRPα polypeptide, which can be provided in monomeric or multimeric form, for example, as a fusion protein with an IgG Fc polypeptide.

[0009] In other embodiments, the therapy provides a bispecific antibody that targets CD47 and a second cancer cell marker, including bispecific antibodies that target CD47 and CD56, CD47 and CD44, CD47 and EpCam, etc. Compositions of such bispecific antibodies are also provided, and the bispecific antibodies are preferably human antibodies or humanized antibodies and can be modified, for example, by pegylation, to extend their half-life in the blood. [Invention 1001] A method for the treatment of lung cancer in a patient, the method comprising administering to the patient an effective dose of a targeted therapeutic agent that specifically binds to one or more cell surface antigens including CD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165, and EpCAM. [Invention 1002] The method of the present invention 1001, further comprising the step of administering a second targeted agent that selectively blocks the binding of CD47 to SIRPα. [The present invention 1003] The method of the present invention 1002, wherein the second targeted agent comprises a soluble SIRPα polypeptide. [The present invention 1004] The method of the present invention 1003, wherein the first agent is an antibody selective for a cell surface marker. [The present invention 1005] The method of the present invention 1001, wherein the lung cancer is small cell lung cancer. [The present invention 1006] The method of the present invention 1005, wherein the marker is selected from CD56, CD99, CD44, and EpCam. [The present invention 1007] The method of the present invention 1002, wherein the first and second agents are included in the form of a bispecific antibody selective for CD47 and a second cancer-associated cell marker. [Brief Description of the Drawings]

[0010]

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DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description of the Embodiment Methods and compositions for the treatment of lung cancer are provided by a therapeutic agent, such as an antibody targeted to a marker of lung cancer, such as an antibody targeted to one or more cell surface antigens including CD_{24}, CD_{166}, CD_{56}, CD_{326}, CD_{298}, CD_{29}, CD_{63}, CD_{9}, CD_{164}, CD_{99}, CD_{46}, CD_{59}, CD_{57}, CD_{165}, EpCAM, etc. In some embodiments, combinations of agents, such as synergistic combinations, are provided, where one agent is an anti-CD47 blocking agent and a second agent is targeted to a lung cancer marker, such as CD_{24}, CD_{166}, CD_{56}, CD_{326}, CD_{298}, CD_{29}, CD_{63}, CD_{9}, CD_{164}, CD_{99}, CD_{46}, CD_{59}, CD_{57}, CD_{165}, EpCAM, etc.

[0012] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments to be described, and can naturally be changed by itself. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the technical terms used in this specification are for the purpose of describing only specific embodiments and are not intended to be limiting.

[0013] When a range of values is provided, unless clearly stated otherwise in context, each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, as well as any other stated value or intervening value in the recited range, is understood to be included in the present invention. The upper and lower limits of these smaller ranges may independently be included within those smaller ranges, and are also included in the present invention in accordance with any specifically excluded limit values in the recited range. Where the recited range includes one or both of these limit values, ranges excluding either or both of such included limit values are also included in the present invention.

[0014] The methods described herein can be performed in any logically possible order of the recited events, as well as in the order of the recited events.

[0015] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0016] All publications mentioned herein are hereby incorporated by reference into this specification for the purpose of disclosing and describing the methods and / or materials related to which these publications are cited.

[0017] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude any optional elements. Accordingly, the specification is intended to serve as a basis for antecedent basis for using exclusive terms such as "solely", "only", etc. in connection with the recitation of claim elements, or for using "negative" limitations.

[0018] The publications discussed herein are provided solely to show the state of the art as of their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of the publications provided may be different from the actual publication dates, which may need to be independently confirmed.

[0019] Definitions Synergistic combination. A synergistic combination may provide a therapeutic effect comparable to the effectiveness of the individual components of the combination, i.e., monotherapy, while reducing adverse side effects, such as damage to non-target tissues, immune status, and other clinical signs. Alternatively, a synergistic combination may provide enhanced effectiveness compared to the effectiveness of the individual components of the combination, i.e., monotherapy, the effect of which may be measured by total tumor cell count; length of time to recurrence; and other signs of the patient's health status.

[0020] The synergistic combination of the present invention combines an agent targeted to inhibit or block the function of CD47 with an agent targeted to inhibit or block a second lung cancer cell marker, usually a cell surface marker. The combination can be provided by a combination of agents, for example a combination of two distinct proteins each specific for a different marker, or as a multispecific agent, for example an antibody combining specificities for two or more different markers.

[0021] Combination therapy: As used herein, the term "combination therapy" refers to a situation where a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more agents can be administered simultaneously; in some embodiments, such agents can be administered sequentially; in some embodiments, such agents are administered in a repeated dosing regimen.

[0022] Dosage form: As used herein, the term "dosage form" refers to physically distinct units of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Each unit contains a predetermined amount of the active agent. In some embodiments, such amount is an appropriate unit dosage (or a fraction thereof) according to a dosing regimen (i.e., a therapeutic dosing regimen) that has been found to correlate with a desirable or beneficial outcome when administered to the relevant population. One of ordinary skill in the art will understand that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0023] Dosing regimen: As used herein, the term "dosing regimen" typically refers to a set of unit doses (typically two or more) that are individually administered to a subject and separated by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more administrations. In some embodiments, the dosing regimen includes multiple administrations that are each separated from one another by a period of the same length; in some embodiments, the dosing regimen includes multiple administrations and includes at least two different periods separating the individual administrations. In some embodiments, all of the administrations within a dosing regimen are in the same amount of unit dose. In some embodiments, the different administrations within a dosing regimen are in different amounts. In some embodiments, the dosing regimen includes a first administration at a first dosage amount, followed by one or more additional administrations at a second dosage amount that is different from the first dosage amount. In some embodiments, the dosing regimen includes a first administration at a first dosage amount, followed by one or more additional administrations at a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desirable or beneficial outcome when administered to an entire relevant population (i.e., is a therapeutic dosing regimen).

[0024] CD47 polypeptide. Three transcript variants of human CD47 (variant 1, NM 001777; variant 2, NM 198793; and variant 3, NM 001025079) encode three isoforms of the CD47 polypeptide. The longest of the three isoforms, CD47 isoform 1 (NP 001768), is 323 amino acids in length. CD47 isoform 2 (NP 942088) is 305 amino acids in length. CD47 isoform 3 is 312 amino acids in length. The three isoforms have identical sequences in the first 303 amino acids. Amino acids 1-8 contain a signal sequence, amino acids 9-142 contain the CD47 immunoglobulin-like domain, which is a soluble fragment, and amino acids 143-300 are a transmembrane domain.

[0025] A "functional derivative" of a native sequence polypeptide is a compound having qualitative biological properties in common with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of the native sequence, as well as derivatives and fragments of the native sequence polypeptide, provided that they have biological activity in common with the corresponding native sequence polypeptide. The term "derivative" encompasses both amino acid sequence variants of the polypeptide and its covalent modifications. Derivatives and fusions of soluble CD47 find use as CD47 mimetic molecules.

[0026] The first 142 amino acids of the CD47 polypeptide include the extracellular region of CD47 (SEQ ID NO: 1). The three isoforms have the same amino acid sequence in the extracellular region and thus soluble CD47 can be made using any of the isoforms. "Soluble CD47" is a CD47 protein lacking a transmembrane domain. Soluble CD47 is not localized to the cell surface but is secreted from the cells expressing it.

[0027] In vitro assays for the biological activity of CD47 include, for example, inhibition of phagocytosis of porcine cells by human macrophages, binding to the SIRPα receptor, SIRPα tyrosine phosphorylation, etc. Exemplary assays for the biological activity of CD47 involve contacting a human macrophage composition in the presence of a candidate agent. The cells are incubated with the candidate agent for about 30 minutes and lysed. The cell lysate is mixed with an anti-human SIRPα antibody to immunoprecipitate SIRPα. The precipitated protein is separated by SDS PAGE, then transferred to nitrocellulose and probed with an antibody specific for phosphotyrosine. A candidate agent useful as a CD47 mimetic increases SIRPα tyrosine phosphorylation by at least 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90%, compared to the level of phosphorylation observed in the absence of the candidate agent. Another exemplary assay for the biological activity of CD47 measures phagocytosis of hematopoietic cells by human macrophages. A candidate agent useful as a CD47 mimetic results in downregulation of phagocytosis by at least about 10%, at least about 20%, at least about 50%, at least about 70%, at least about 80%, or up to about 90%, compared to the level of phagocytosis observed in the absence of the candidate agent.

[0028] "Manipulating phagocytosis" means upregulation or downregulation in phagocytosis by at least about 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90%, compared to the level of phagocytosis observed in the absence of the intervention. Thus, in the context of reducing phagocytosis of circulating hematopoietic cells, particularly in the context of transplantation, manipulating phagocytosis means downregulation in phagocytosis by at least about 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90%, compared to the level of phagocytosis observed in the absence of the intervention.

[0029] Anti-CD47 agents. As used herein, the term "anti-CD47 agent" refers to any agent that reduces the binding of CD47 (e.g., on a target cell) to SIRPα (e.g., on a phagocyte). Non-limiting examples of suitable anti-CD47 agents include, but are not limited to, SIRPα agents, including high affinity SIRPα polypeptides, anti-SIRPα antibodies, soluble CD47 polypeptides, and anti-CD47 antibodies or antibody fragments. In some embodiments, a suitable anti-CD47 agent (e.g., an anti-CD47 antibody, a SIRPα agent, etc.) specifically binds to CD47 and reduces the binding of CD47 to SIRPα. In some embodiments, a suitable anti-CD47 agent (e.g., an anti-SIRPα antibody, a soluble CD47 polypeptide, etc.) specifically binds to SIRPα and reduces the binding of CD47 to SIRPα. A suitable anti-CD47 agent that binds to SIRPα does not activate SIRPα (e.g., in phagocytes that express SIRPα).

[0030] The efficacy of a suitable anti-CD47 agent can be evaluated by assaying the agent (further described below). In an exemplary assay, target cells are incubated in the presence or absence of a candidate agent. An agent for use in the methods of the invention is considered to upregulate phagocytosis by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, or at least 200%) compared to phagocytosis in the absence of the agent. Similarly, an in vitro assay for the level of tyrosine phosphorylation of SIRPα is considered to show a decrease in phosphorylation of at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) compared to phosphorylation observed in the absence of the candidate agent.

[0031] In some embodiments, the anti-CD47 agent does not activate CD47 upon binding. If CD47 is activated, a process similar to apoptosis (i.e., programmed cell death) can occur (Manna and Frazier, Cancer Research, 64, 1026-1036, Feb. 1 2004). Thus, in some embodiments, the anti-CD47 agent does not directly induce cell death of CD47-expressing cells by apoptosis.

[0032] SIRPα agents. SIRPα agents include a portion of SIRPα or a fragment thereof that retains binding activity that is sufficient to bind to CD47 with a recognizable affinity that is normally present between the signal sequence and the transmembrane domain. Suitable SIRPα agents reduce (e.g., block, impede, etc.) the interaction between the native protein SIRPα and CD47. SIRPα agents are generally thought to include at least the d1 domain of SIRPα. In some embodiments, the SIRPα agent is a fusion protein, for example, fused in-frame with a second polypeptide. In some embodiments, the second polypeptide can increase the size of the fusion protein, for example, so that the fusion protein is not rapidly removed from the bloodstream. In some embodiments, the second polypeptide is a part or the whole of the immunoglobulin Fc region. The Fc region helps phagocytosis by providing an "eat me" signal that enhances the blockade of the "don't eat me" signal provided by the high-affinity SIRPα agent. In other embodiments, the second polypeptide is any suitable polypeptide that is substantially similar to Fc and provides, for example, an increase in size, a multimerization domain, and / or additional binding or interaction with Ig molecules.

[0033] In some embodiments, the subject anti-CD47 agent is a "high affinity SIRPα agent", which includes an SIRPα-derived polypeptide and analogs thereof. The high affinity SIRPα agent is described in International Application PCT / US13 / 21937, which is specifically incorporated herein by reference. The high affinity SIRPα agent is a variant of the native SIRPα protein. In some embodiments, the high affinity SIRPα agent is soluble, the polypeptide lacks the SIRPα transmembrane domain, and includes at least one amino acid change compared to the wild-type SIRPα sequence, and the amino acid change increases the affinity of the SIRPα polypeptide for CD47, for example, by decreasing the dissociation rate (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.

[0034] The high affinity SIRPα agent includes a portion of SIRPα that is capable of binding to CD47 with a recognizable affinity, such as a high affinity, which is normally present between the signal sequence and the transmembrane domain, or a fragment thereof that retains the binding activity. The high affinity SIRPα agent is generally thought to include at least the d1 domain of SIRPα having modifications of amino acid residues that increase the affinity.

[0035] The SIRPα agent can be used as a "monomer", and the binding domain of SIRPα is used, and the binding domain is provided as a soluble monomeric protein. In other embodiments, the SIRPα variant of the present invention is, for example, a fusion protein fused in-frame with a second polypeptide, and in particular, the second polypeptide provides multimerization. In some embodiments, the second polypeptide is part or all of the immunoglobulin Fc region. The Fc region helps phagocytosis by providing an "eat me" signal that enhances the blockade of the "don't eat me" signal provided by the high-affinity SIRPα agent. In other embodiments, the second polypeptide is any suitable polypeptide that is substantially similar to Fc and provides, for example, an increase in size, a multimerization domain, and / or additional binding or interaction with Ig molecules.

[0036] Amino acid changes that provide an increase in affinity are located in the d1 domain, and thus the high-affinity SIRPα agent contains the d1 domain of human SIRPα having at least one amino acid change in the d1 domain compared to the wild-type sequence. Such high-affinity SIRPα agents optionally include additional amino acid sequences, such as antibody Fc sequences; a portion of the wild-type human SIRPα protein other than the d1 domain, including but not limited to residues 150-374 of the native protein or fragments thereof, usually a fragment adjacent to the d1 domain. The high-affinity SIRPα agent can be a monomer or a multimer, i.e., a dimer, trimer, tetramer, etc.

[0037] Anti-CD47 antibodies. In some embodiments, the subject anti-CD47 agent specifically binds to CD47 (i.e., an anti-CD47 antibody) and is an antibody that reduces the interaction between CD47 on one cell (e.g., an infected cell) and SIRPα on another cell (e.g., a phagocyte). In some embodiments, a suitable anti-CD47 antibody is one that does not activate CD47 upon binding, e.g., an antibody that does not induce apoptosis upon binding. Non-limiting examples of suitable antibodies include clone B6H12, 5F9, 8B6, and C3 (e.g., those described in International Patent Publication WO 2011 / 143624, which is specifically incorporated herein by reference). Suitable anti-CD47 antibodies include fully human, humanized, or chimeric forms of such antibodies. Humanized antibodies (e.g., hu5F9-G4) are particularly useful for in vivo applications in humans due to their low antigenicity. Similarly, caninized antibodies, felinized antibodies, etc. are particularly useful for applications in dogs, cats, and other species, respectively. Antibodies of interest include humanized antibodies, or caninized antibodies, felinized antibodies, equinized antibodies, bovinized antibodies, porcinized antibodies, etc., and variants thereof.

[0038] Anti-SIRPα antibodies. In some embodiments, the subject anti-CD47 agent is an antibody that specifically binds to SIRPα (i.e., an anti-SIRPα antibody) and reduces the interaction between CD47 on one cell (e.g., an infected cell) and SIRPα on another cell (e.g., a phagocytic cell). Since activation of SIRPα is thought to inhibit phagocytosis, a suitable anti-SIRPα antibody can bind to SIRPα without activating or stimulating signal transduction via SIRPα. Rather, a suitable anti-SIRPα antibody promotes preferential phagocytosis of damaged cells over normal cells. Such cells (e.g., infected cells) that express higher levels of CD47 compared to other cells (non-infected cells) are thought to be preferentially phagocytosed. Thus, a suitable anti-SIRPα antibody specifically binds to SIRPα (without activating / stimulating a signal transduction response sufficient to inhibit phagocytosis) and blocks the interaction between SIRPα and CD47. Suitable anti-SIRPα antibodies include fully human, humanized, or chimeric forms of such antibodies. Humanized antibodies are particularly useful for in vivo applications in humans due to their low antigenicity. Similarly, antibodies such as canineized antibodies, felineized antibodies, etc. are particularly useful for applications in dogs, cats, and other species, respectively. Antibodies of interest include humanized antibodies, or antibodies such as canineized antibodies, felineized antibodies, equinized antibodies, bovineized antibodies, porcineized antibodies, etc., and variants thereof.

[0039] Soluble CD47 polypeptide. In some embodiments, the subject anti-CD47 agent is a soluble CD47 polypeptide that specifically binds to SIRPα and reduces the interaction between CD47 on one cell (e.g., an infected cell) and SIRPα on another cell (e.g., a phagocytic cell). Since activation of SIRPα is thought to inhibit phagocytosis, a suitable soluble CD47 polypeptide can bind to SIRPα without activating or stimulating signaling through SIRPα. On the contrary, a suitable soluble CD47 polypeptide promotes preferential phagocytosis of infected cells over non-infected cells. Such cells (e.g., infected cells) that express a higher level of CD47 compared to normal non-target cells (normal cells) are thought to be preferentially phagocytosed. Thus, a suitable soluble CD47 polypeptide specifically binds to SIRPα without activating / stimulating a signaling response sufficient to inhibit phagocytosis.

[0040] In some cases, a suitable soluble CD47 polypeptide can be a fusion protein (e.g., those structurally described in U.S. Patent Publication US20100239579, which is specifically incorporated herein by reference). However, only fusion proteins that do not activate / stimulate SIRPα are suitable for the methods provided herein. Suitable soluble CD47 polypeptides include any peptide or peptide fragment that can specifically bind to SIRPα and inhibit the interaction between CD47 and SIRPα without stimulating SIRPα activity sufficient to inhibit phagocytosis, including mutant or native CD47 sequences (e.g., extracellular domain sequences or extracellular domain variants).

[0041] In certain embodiments, the soluble CD47 polypeptide comprises the extracellular domain of CD47, including a signal peptide, such that the extracellular portion of CD47 is typically 142 amino acids in length and has the amino acid sequence set forth in SEQ ID NO: 3. Also included within the soluble CD47 polypeptides described herein are CD47 extracellular domain variants that include at least 65% - 75%, 75% - 80%, 80 - 85%, 85% - 90%, or 95% - 99% (or any percent identity not specifically recited between 65% - 100%) of the amino acid sequence, and the variants retain the ability to bind to SIRPα without stimulating SIRPα signaling.

[0042] In certain embodiments, the signal peptide amino acid sequence can be replaced with a signal peptide amino acid sequence derived from another polypeptide (e.g., an immunoglobulin or CTLA4). For example, unlike full-length CD47, which is a cell surface polypeptide that traverses the cell membrane, soluble CD47 polypeptides are secreted; thus, the polynucleotide encoding a soluble CD47 polypeptide can include a nucleotide sequence encoding a signal peptide associated with a polypeptide that is normally secreted from the cell.

[0043] In other embodiments, the soluble CD47 polypeptide comprises the extracellular domain of CD47 lacking a signal peptide. In an exemplary embodiment, the CD47 extracellular domain lacking a signal peptide has the amino acid sequence set forth in SEQ ID NO: 1 (124 amino acids). As described herein, the signal peptide is either cleaved during protein trafficking or remains anchored to the cell membrane (such peptides are also referred to as signal anchors), such that the signal peptide of a secreted or transmembrane protein is not exposed on the cell surface. The signal peptide sequence of CD47 is thought to be cleaved from the precursor CD47 polypeptide in vivo.

[0044] In other aspects, soluble CD47 polypeptides include CD47 extracellular domain variants. Such soluble CD47 polypeptides retain the ability to bind to SIRPα without stimulating SIRPα signaling. CD47 extracellular domain variants can have an amino acid sequence that is at least 65% - 75%, 75% - 80%, 80 - 85%, 85% - 90%, or 95% - 99% identical to the reference human CD47 sequence (including any percent identity within any one of the recited ranges).

[0045] The term "antibody" or "antibody component" is intended to include any molecular structure that includes polypeptide chains and has a specific shape that fits and recognizes an epitope, and the complex of the molecular structure and the epitope is stabilized by one or more non-covalent binding interactions. The antibodies utilized in the present invention may be polyclonal antibodies, but monoclonal antibodies are preferred because they can be replicated by cell culture or recombination and can be modified to reduce their antigenicity.

[0046] Polyclonal antibodies can be made by standard protocols by injecting an antigenic composition into a production animal. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. When using the whole protein or a large protein portion, the antibodies can be made by immunizing the production animal with the protein and an appropriate adjuvant (e.g., Freund's, Freund's complete, water-in-oil emulsion, etc.). When smaller peptides are used, it is advantageous to conjugate the peptide with a larger molecule to create an immunogenic conjugate. Commercially available and commonly used conjugate proteins for such use include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). To make antibodies against a specific epitope, peptides derived from the full sequence can be used. Alternatively, excellent immune responses can be elicited when a polypeptide is conjugated to a carrier protein such as ovalbumin, BSA, or KLH to make antibodies against a relatively short peptide portion of a protein target. Alternatively, in the case of monoclonal antibodies, hybridomas can be formed by isolating stimulated immune cells such as those derived from the spleen of an inoculated animal. These cells are then fused to immortalized cells such as myeloma cells or transformed cells that can be infinitely replicated under cell culture, thereby producing an immortal immunoglobulin-secreting cell line. In addition, antibodies or antigen-binding fragments can be produced by genetic engineering. Humanized antibodies, chimeric antibodies, or xenogeneic human antibodies that do not elicit much of an immune response when administered to humans are preferred for use in the present invention.

[0047] In addition to intact immunoglobulins (or their recombinant counterparts), immunoglobulin fragments containing epitope binding sites (e.g., Fab', F(ab')2, or other fragments) are useful as antibody components in the present invention. Such antibody fragments can be made from intact immunoglobulins by lysine, pepsin, papain, or other protease cleavage. "Fragments", i.e., minimal immunoglobulins, can be designed using recombinant immunoglobulin techniques. For example, "Fv" immunoglobulins for use in the present invention can be produced by linking the variable light chain region to the variable heavy chain region via a peptide linker (e.g., polyglycine, or another sequence that forms neither an α helix nor a β sheet motif).

[0048] Antibodies include free antibodies and antigen-binding fragments derived therefrom, as well as conjugates, such as conjugates of pegylated antibodies, drugs, radioisotopes, or toxins. Monoclonal antibodies directed against specific epitopes or combinations of epitopes enable the targeting and / or depletion of cell populations expressing a marker. Using various techniques with monoclonal antibodies, cell populations expressing a marker can be screened, which can include magnetic separation using magnetic beads coated with the antibody, "panning" with the antibody adhered to a solid phase matrix (i.e., plate), and flow cytometry (see, e.g., U.S. Patent No. 5,985,660; and Morrison et al. Cell, 96:737-49 (1999)). These techniques enable screening for specific populations of cells in immunohistochemistry of biopsy samples; detection of the presence of markers shed into the blood and other biological fluids by cancer cells, etc.

[0049] Humanized forms of such antibodies are also within the scope of the present invention. Humanized antibodies are particularly useful for in vivo applications in humans because of their low antigenicity.

[0050] The term "bispecific or dual - specificity antibody" refers to synthetic or recombinant antibodies that recognize two or more proteins. Bispecific antibodies directed against combinations of epitopes enable the targeting and / or depletion of cell populations expressing such combinations of epitopes. Exemplary bispecific antibodies include those that target combinations of CD47 and SCLC cancer cell markers. The production of bispecific antibodies is described in the literature, for example, in U.S. Patent No. 5,989,830 and U.S. Patent No. 5,798,229, which are incorporated herein by reference. Higher - order specificities, such as trispecific antibodies, are described by Holliger and Hudson (2005) Nature Biotechnology 23:1126 - 1136.

[0051] The efficacy of a CD47 inhibitor can be evaluated by assaying CD47 activity. The above - described assay or a modified version thereof is used. In an exemplary assay, SCLC is incubated with bone - marrow - derived macrophages in the presence or absence of a candidate agent. An inhibitor of cell - surface CD47 is thought to up - regulate phagocytosis by at least about 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90% compared to phagocytosis in the absence of the candidate agent. Similarly, an in vitro assay for the level of tyrosine phosphorylation of SIRPα is thought to show a decrease in phosphorylation by at least about 10%, or up to 20%, or 50%, or 70%, or 80%, or up to about 90% compared to phosphorylation observed in the absence of the candidate agent.

[0052] In one aspect of the present invention, an agent, or a pharmaceutical composition comprising the agent, is provided in an amount effective to inhibit detectably the binding of CD47 to the SIRPα receptor present on the surface of phagocytic cells. The effective amount is determined by empirical routine testing in the art. The effective amount can vary depending on the number of cells transplanted, the site of transplantation, and factors specific to the transplant recipient.

[0053] The terms "phagocytic cell" and "phagocyte" are used interchangeably herein to refer to cells capable of phagocytosis. There are four main categories of phagocytes: macrophages, monocytes (histiocytes and monocytes), polymorphonuclear leukocytes (neutrophils), and dendritic cells.

[0054] The term "biological sample" encompasses a variety of sample types obtained from organisms and can be used in diagnostic or monitoring assays. The term includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. The term includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components. The term includes clinical samples and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0055] The terms "treatment", "treating", "treat" etc. are generally used herein to refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in that it completely or partially prevents a disease or its symptoms from occurring, and / or therapeutic in that it partially or completely stabilizes or cures a disease and / or adverse effects resulting from the disease. "Treatment" as used herein encompasses any treatment of a disease in mammals, such as mice, rats, rabbits, pigs, humans and other primates including apes, particularly humans, and includes (a) preventing a disease or symptoms from occurring in a subject that may be predisposed to having the disease or symptoms but has not yet been diagnosed as having it; (b) inhibiting the disease symptoms, i.e., stopping its progression; or (c) alleviating the disease symptoms, i.e., causing regression of the disease or symptoms.

[0056] The terms "recipient", "individual", "subject", "host", and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for which a diagnosis, treatment, or therapy is desired.

[0057] As used herein, "host cell" refers to a microorganism, or a eukaryotic cell, or a cell line cultured as a single cell entity that can be or has been used as a recipient for a recombinant vector or other transport polynucleotide, and includes the progeny of the original transfected cell. It is understood that due to natural, accidental, or intentional mutations, the form or the genomic DNA or the complementary strand of the total DNA of the progeny of a single cell may not necessarily be identical to that of the original parent.

[0058] The terms "cancer", "neoplasm", "tumor", and "carcinoma" are used interchangeably herein and refer to cells that exhibit relatively autonomous growth and as a result exhibit an abnormal growth phenotype characterized by a significant loss of cell growth control. Generally, the cells of interest for detection or treatment in the present application include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. The detection of cancerous cells is of particular interest. The term "normal" as used in the context of "normal cells" is intended to refer to cells that have not been transformed or that exhibit the morphology of non-transformed cells of the tissue type being examined. "Cancerous phenotype" generally refers to any of a variety of biological phenomena characteristic of cancerous cells, and the phenomena can vary depending on the type of cancer. A cancerous phenotype is generally identified by, for example, abnormalities in cell growth or proliferation (e.g., uncontrolled growth or proliferation), abnormalities in the regulation of the cell cycle, abnormalities in cell motility, abnormalities in cell-cell interactions, or abnormalities such as metastasis.

[0059] "Therapeutic target" refers to a gene or gene product whose activity, when modulated (e.g., by modulating expression, biological activity, etc.), can provide modulation of the cancer phenotype. As used throughout, "modulation" is intended to refer to an increase or decrease in the specified phenomenon (e.g., modulation of biological activity refers to an increase or decrease in biological activity).

[0060] Lung cancer Lung cancer is the leading cause of cancer-related death worldwide. Approximately 85% of cases are associated with smoking. Symptoms can include cough, chest discomfort or pain, weight loss, and, less commonly, hemoptysis; however, many patients present with metastatic disease without any clinical symptoms. Diagnosis is typically made by chest X-ray or CT and confirmed by biopsy. Treatment includes surgery, chemotherapy, radiation therapy, or a combination, depending on the stage of the disease. Over the past several decades, the prognosis for lung cancer patients, particularly those with stage IV (metastatic) disease, has been poor.

[0061] Respiratory epithelial cells require long-term exposure to carcinogenic agents and the accumulation of numerous genetic mutations (an effect called field carcinogenesis) before becoming neoplastic. In some patients with lung cancer, secondary or additional mutations in genes that stimulate cell growth (K-ras, MYC) cause abnormalities in growth factor receptor signaling (EGFR, HER2 / neu) and inhibit apoptosis, which contributes to the proliferation of abnormal cells. In addition, mutations that inhibit tumor suppressor genes (p53, APC) can lead to cancer. Other mutations that may be involved include the EML-4-ALK translocation, as well as mutations in ROS-1, BRAF, and PI3KCA. Genes such as these that primarily contribute to lung cancer are called driver mutations. Driver mutations can cause or contribute to lung cancer among smokers, but these mutations may particularly be the cause of lung cancer among non-smokers.

[0062] Chest X-ray is often the first imaging test. It can show well-defined abnormalities such as a single mass or multiple masses, or solitary pulmonary nodules, enlarged hilar regions, widened mediastinum, tracheobronchial stenosis, atelectasis, non-resolving parenchymal infiltrates, cavitary lesions, or pleural thickening or pleural effusion of unknown cause. These findings are suggestive but not diagnostic of lung cancer and require follow-up with CT scan or combined PET-CT scan and cytopathological confirmation.

[0063] CT shows many characteristic anatomical patterns and appearances that can strongly suggest a diagnosis. CT can also guide core needle biopsy of accessible lesions and is useful for staging. If a lesion found on plain X-ray is highly likely to be lung cancer, PET-CT can be performed. This test combines anatomical images from CT and functional images from PET. PET images can help distinguish between inflammatory and malignant processes.

[0064] SCLC has two stages: limited stage and extensive stage. Limited-stage SCLC disease is cancer that is confined to one hemithorax (including ipsilateral lymph nodes) and can be encompassed within one tolerable radiotherapy port, except when there is a pleural effusion or pericardial effusion. Extensive-stage disease is cancer outside of a single hemithorax or the presence of malignant cells detected in a pleural effusion or pericardial effusion. Less than one-third of patients with SCLC present with limited-stage disease; the remaining patients often have distant metastases of the extensive type. The overall prognosis of SCLC is poor. The median survival of limited-stage SCLC is 20 months, with a 5-year survival rate of 20%. Patients with extensive-stage SCLC are particularly poor, with a 5-year survival rate of <1%.

[0065] NSCLC has four stages, I to IV (using the TNM system). The TNM staging is based on tumor size, the location of the tumor and lymph nodes, and the presence or absence of distant metastases. The 5-year survival rate of patients with NSCLC varies by stage, from 60 - 70% for patients with stage I disease to <1% for patients with stage IV disease.

[0066] Conventional treatments vary by cell type and stage of the disease. Many factors in the patient, unrelated to the tumor, influence the choice of treatment. Even if a cure by aggressive therapy might be technically possible, comorbidities including poor cardiorespiratory reserve, malnutrition, debilitation or poor physical performance, cytopenia, and psychiatric or cognitive disorders can all lead to a decision for palliative treatment rather than curative treatment, or no treatment at all.

[0067] SCLC at any stage is typically initially responsive to treatment, but the response is usually temporary. Depending on the stage of the disease, chemotherapy with or without radiotherapy is provided. In many patients, chemotherapy prolongs survival and improves quality of life enough to justify its use. Surgery generally plays no role in the treatment of SCLC, but surgery can be curative in rare patients with a small, non-spreading, local tumor (such as a solitary pulmonary nodule) who have had surgical resection before the tumor was identified as SCLC. Chemotherapy regimens of etoposide and a platinum compound (either cisplatin or carboplatin) are commonly used, and the same is true for other drugs such as irinotecan, topotecan, vinca alkaloids (vinblastine, vincristine, vinorelbine), alkylating agents (cyclophosphamide, ifosfamide), doxorubicin, taxanes (docetaxel, paclitaxel), and gemcitabine. When the disease is limited to one half of the chest, radiotherapy further improves clinical outcomes; such a response to radiotherapy was the basis for the definition of limited-stage disease. The use of cranial irradiation to prevent brain metastases is also advocated in certain cases; micrometastases are common in SCLC, and chemotherapy has a low ability to cross the blood-brain barrier.

[0068] In extensive-stage disease, treatment is based on chemotherapy rather than radiation therapy, although radiation therapy is often used as palliative treatment for metastases to the bone or brain. In patients who have a good response to chemotherapy, prophylactic brain irradiation may be used, as in limited-stage SCLC, to prevent the growth of SCLC in the brain.

[0069] Treatment for NSCLC typically involves assessment for surgical eligibility, followed by selection of surgery, chemotherapy, radiation therapy, or a combination of modalities as appropriate, depending on tumor type and stage.

[0070] Cancer treatment The present invention provides a method for reducing the growth of lung cancer cells by introducing an effective dose of a targeted therapeutic agent directed against a lung cancer cell surface marker, including but not limited to CD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165, EpCAM, etc. In some embodiments, the marker is one of CD56, CD44, CD99, and EpCAM. In preferred embodiments, the targeted therapeutic agent is combined with a CD47 blocking agent, such as a soluble SIRPα monomer or multimer, an anti-CD47 antibody, a small molecule, etc. In certain embodiments, the cancer is SCLC. Blocking the activity of CD47 prevents the downregulation of phagocytosis found in certain tumor cells.

[0071] "Reducing the growth of cancer cells" includes, but is not limited to, reducing the proliferation of cancer cells and reducing the incidence of non-cancerous cells becoming cancerous cells. Whether a reduction in cancer cell growth has been achieved is determined by [ 3 H]-thymidine incorporation; counting cell numbers over a period of time; detecting and / or measuring markers associated with SCLC; and the like.

[0072] Whether a substance, i.e., a specific amount of the substance, is effective in treating cancer can be evaluated using any of a variety of known diagnostic assays for cancer, including but not limited to biopsies, radiological examinations with contrast agents, CAT scans, and detection of tumor markers associated with cancer in the blood of an individual. The substance can be administered systemically or locally, usually systemically.

[0073] As an alternative aspect, an agent that reduces cancer cell growth, such as a chemotherapeutic drug, can be targeted to cancer cells by conjugation to a CD47-specific antibody. Thus, in some aspects, the present invention provides a method of delivering a drug to cancer cells, comprising the step of administering a drug-antibody conjugate to a subject, wherein the antibody is specific for a cancer-associated polypeptide and the drug is one that reduces cancer cell growth, and various types of which are known in the art. Targeting can be achieved by binding a drug to an antibody specific for a cancer-associated polypeptide (e.g., by linking directly or via a linker molecule, either covalently or non-covalently, to form a drug-antibody conjugate). Methods of binding a drug to an antibody are well known in the art and need not be detailed herein.

[0074] In certain aspects, a bispecific antibody can be used. For example, a bispecific antibody can be used in which one antigen-binding domain is directed against CD47 and the other antigen-binding domain is directed against a cancer cell marker such as CD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165, EpCAM, etc.

[0075] Generally, when terms are used in this specification, "antibody" or "antibody component" is intended to include any molecular structure that includes polypeptide chains and has a specific shape that fits and recognizes an epitope, and the complex of the molecular structure and the epitope is stabilized by one or more non-covalent binding interactions. In the case of monoclonal antibodies, hybridomas can be formed by isolating stimulated immune cells, such as those derived from the spleen of an immunized animal. These cells are then fused to immortalized cells, such as myeloma cells or transformed cells, that can be replicated infinitely under cell culture, thereby producing an immortal immunoglobulin-secreting cell line. The immortal cell line used is preferably selected such that it is deficient in an enzyme required for the utilization of a particular nutrient. Many such cell lines (such as myelomas) are known to those skilled in the art and include, for example, thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyl transferase (HGPRT). These deficiencies allow for the selection of fused cells according to their ability to grow, for example, on hypoxanthine-aminopterin-thymidine medium (HAT).

[0076] Antibodies that have a reduced tendency to induce a severe or harmful immune response (such as anaphylactic shock) in humans and also have a reduced tendency to prime an immune response (such as the human anti-mouse antibody "HAMA" response) that would interfere with repeated dosing with a therapeutic or imaging antibody substance are preferred for use in the present invention. These antibodies are preferred for all routes of administration. Thus, humanized antibodies, chimeric antibodies, or xenogeneic human antibodies that do not elicit much of an immune response when administered to humans are preferred for use in the present invention.

[0077] Chimeric antibodies can be produced by recombinant means by combining mouse variable light and heavy chain regions (VK and VH) obtained from mouse hybridoma clones (or from other animals) with human constant light and heavy chain regions, thereby producing antibodies having mainly human domains. The production of such chimeric antibodies is well known in the art and can be achieved by standard means (e.g., those described in U.S. Patent No. 5,624,659, which is hereby incorporated by reference in its entirety). Humanized antibodies contain even more human-like immunoglobulin domains and are engineered to incorporate only the complementarity-determining regions of the animal-derived antibody. This is achieved by carefully examining the sequences of the hypervariable loops of the variable regions of monoclonal antibodies and adapting them to the structure of the human antibody chains. Although seemingly complex on the surface, in practice, the process is straightforward. See, for example, U.S. Patent No. 6,187,287, which is hereby incorporated by reference in its entirety.

[0078] Alternatively, polyclonal or monoclonal antibodies can be produced from animals that have been genetically modified to produce human immunoglobulins. Transgenic animals can be produced by first producing "knockout" animals that do not produce the animal's natural antibodies and then stably transforming the animals with a human antibody locus (e.g., by using a human artificial chromosome). Then, only human antibodies are produced by the animals. Techniques for producing such animals and obtaining antibodies therefrom are described in U.S. Patents Nos. 6,162,963 and 6,150,584, which are hereby incorporated by reference in their entirety. Such fully human xenogeneic antibodies are preferred antibodies for use in the methods and compositions of the present invention. Alternatively, single-chain antibodies can be produced from phage libraries containing human variable regions. See U.S. Patent No. 6,174,708, which is hereby incorporated by reference in its entirety.

[0079] In addition to intact immunoglobulins (or their recombinant counterparts), immunoglobulin fragments containing an epitope-binding site (e.g., Fab', F(ab')2, or other fragments) are useful as antibody components in the present invention. Such antibody fragments can be made from intact immunoglobulins by lysine, pepsin, papain, or other protease cleavage. "Fragments", i.e., minimal immunoglobulins, can be designed using recombinant immunoglobulin techniques. For example, an "Fv" immunoglobulin for use in the present invention can be produced by linking the variable light chain region to the variable heavy chain region via a peptide linker (e.g., polyglycine, or another sequence that forms neither an α helix nor a β sheet motif).

[0080] An Fv fragment is a heterodimer of a variable heavy chain domain (V H ) and a variable light chain domain (V L ). The heterodimers of heavy and light chain domains that occur in intact IgG are connected by disulfide bonds. Recombinant Fvs in which V H and V L are connected by a peptide linker are typically stable; see, for example, Huston et al., Proc. Natl. Acad, Sci. USA 85:5879 5883 (1988) and Bird et al., Science 242:423 426 (1988), both of which are hereby incorporated by reference in their entirety. These have been found to retain specificity and affinity, are useful for imaging tumors, and are single-chain Fvs that have been shown to be useful for making recombinant immunotoxins for tumor therapy. However, researchers have found that some of the single-chain Fvs have a reduced affinity for the antigen and that the peptide linker can interfere with binding. As described in U.S. Patent No. 6,147,203, which is hereby incorporated by reference in its entirety, the V H region and the V LImproved Fvs containing stabilizing disulfide bonds between domains have also been produced. Any of these minimal antibodies may be utilized in the present invention, and those that have been humanized to avoid the HAMA reaction are preferred for use in the context of the present invention.

[0081] Derivatized polypeptides having attached chemical linkers, detectable components such as fluorescent dyes, enzymes, substrates, chemiluminescent components, specific binding components such as streptavidin, avidin, or biotin, or drug conjugates may be utilized in the methods and compositions of the present invention.

[0082] In some embodiments of the present invention, the polypeptide agent of the present invention is conjugated or conjugated to one or more therapeutic components, cytotoxic components, or imaging components. As used herein, "cytotoxic component" (C) simply means a component that inhibits cell growth or promotes cell death when it approaches or is taken up by cells. Suitable cytotoxic components in this regard include radioisotopes (radionuclides), differentiation-inducing factors and chemotoxic agents such as small chemotoxic drugs, toxin proteins, and derivatives thereof. Taking into account the need for pharmacokinetic stability and reduced overall toxicity to patients, the agent can be conjugated to the polypeptide agent of the present invention by any suitable technique. The therapeutic agent can be bound directly or indirectly (e.g., via a linker group) to a suitable component. A direct reaction is possible when each has a functional group capable of reacting with the other. For example, a nucleophilic group such as an amino group or a sulfhydryl group may have the ability to react with a carbonyl-containing group such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide). Alternatively, a suitable chemical linker group can be used. The linker group can function as a spacer that distances the polypeptide agent of the present invention from the agent to avoid interference with binding ability. The linker group can also serve to increase the chemical reactivity of substituents on the component or on the polypeptide agent of the present invention, and thus increase the binding efficiency. The increased chemical reactivity can facilitate the use of components or the use of functional groups on the components that would otherwise not be possible.

[0083] Suitable linking chemistries include maleimidyl linkers (which react with sulfhydryls on the antibody component) and alkyl halide linkers, as well as succinimidyl linkers (which react with primary amines on the antibody component). A number of primary amines and sulfhydryl groups are present on immunoglobulins, and additional groups can be engineered into recombinant immunoglobulin molecules. It will be apparent to those skilled in the art that a variety of bifunctional or polyfunctional reagents (such as those described in the catalog of Pierce Chemical Co., Rockford, Ill.), both homofunctional and heterofunctional, can be employed as linker groups. The linkage can be effected, for example, via amino, carboxyl, sulfhydryl, or oxidized carbohydrate residues. A number of references describe such methodologies, for example, U.S. Patent No. 4,671,958. As an alternative method of conjugation, as described in U.S. Patents Nos. 5,057,313 and 5,156,840, a cytotoxic component can be conjugated to the polypeptide agent of the invention via an oxidized carbohydrate residue at a glycosylation site. Yet another alternative method of conjugating the polypeptide agent of the invention to a cytotoxic or therapeutic component is by use of a non-covalent binding pair such as streptavidin / biotin or avidin / biotin. In these embodiments, one member of the pair is covalently bound to a component such as anti-CD47, CV1, etc., and the other member of the binding pair is covalently bound to a therapeutic component, cytotoxic component, or imaging component.

[0084] If the cytotoxic component is more potent when released from the binding moiety of the polypeptide agent of the present invention, it may be desirable to use a linker group that is cleavable either during or upon internalization into the cell, or a linker group that is gradually cleavable over time in the extracellular environment. A number of different cleavable linker groups have been described. Mechanisms for intracellular release of the cytotoxic component agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Patent No. 4,489,710), cleavage by irradiation of a photolabile bond (e.g., U.S. Patent No. 4,625,014), cleavage by hydrolysis of derivatized amino acid side chains (e.g., U.S. Patent No. 4,638,045), cleavage by serum complement-mediated hydrolysis (e.g., U.S. Patent No. 4,671,958), and cleavage by acid-catalyzed hydrolysis (e.g., U.S. Patent No. 4,569,789).

[0085] It may also be desirable to conjugate two or more components to the polypeptide agent of the present invention. By poly-derivatizing the agent, several strategies can be carried out simultaneously, for example, a therapeutic antibody can be labeled for tracking by visualization techniques. Regardless of the particular embodiment, conjugates with two or more components can be prepared in a variety of ways. For example, two or more components can be directly conjugated to the polypeptide molecule, or a linker (e.g., a dendrimer) that provides multiple sites for attachment can be used. Alternatively, a carrier having the ability to hold two or more cytotoxic components or imaging components can be used.

[0086] Carriers can carry agents in a variety of ways, including direct or linker-mediated covalent bonds and non-covalent associations. Suitable covalent carriers include proteins such as albumin (e.g., U.S. Patent No. 4,507,234), peptides, and polysaccharides such as amino dextran (e.g., U.S. Patent No. 4,699,784), each of which has multiple sites for attachment of components. Carriers can also carry agents by non-covalent associations such as non-covalent bonds or by encapsulation, such as within liposomal vesicles (e.g., U.S. Patents Nos. 4,429,008 and 4,873,088). Encapsulation carriers are particularly useful for the conjugation of imaging components to antibody components for use in the present invention, since a sufficient amount of imaging components (dyes, magnetic resonance imaging reagents, etc.) for detection can be more easily associated with the antibody component. In addition, encapsulation carriers are useful in therapeutic regimens involving chemical toxicity, since they can allow the therapeutic composition to gradually release the chemical toxic component over time while concentrating it near tumor cells.

[0087] Preferred radionuclides for use as cytotoxic components are radionuclides suitable for pharmacological administration. Such radionuclides include 123 I, 125 I, 131 I, 90 Y, 211 At, 67 Cu, 186 Re, 188 Re, 212 Pb, and 212 Bi. Iodine and astatine isotopes are more preferred radionuclides for use in the therapeutic compositions of the present invention, as a large number of references have accumulated regarding their use.

[0088] Preferred chemical toxic agents include small molecule drugs such as carboplatin, cisplatin, vincristine, taxanes such as paclitaxel and docetaxel, hydroxyurea, gemcitabine, vinorelbine, irinotecan, tirapazamine, matrilysin, methotrexate, pyrimidine and purine analogs, and other suitable small toxins known in the art. Preferred chemical toxic differentiation inducers include phorbol esters and butyric acid. The chemical toxic component can be directly conjugated to the antibody component via a chemical linker or can be encapsulated within a carrier and then bound to the antibody. Preferred toxin proteins for use as cytotoxic components include ricin A and B, abrin, diphtheria toxin, bryodin 1 and 2, momordin, trichokirin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, yamabobo antiviral protein, and other toxin proteins known in the art of pharmaceutical biochemistry. These toxin agents are preferably encapsulated within a carrier for binding to an antibody because they can induce an undesirable immune response in a patient, especially when injected intravascularly.

[0089] With respect to administration, the targeted therapeutic agent, or combination of targeted therapeutic agents, can be administered separately or together; generally, within the same normal time frame, such as within 1 week, within 3 to 4 days, within 1 day, or simultaneously with each other.

[0090] One or more active substances are mixed, prior to administration, with a non-toxic pharmaceutically acceptable carrier substance. Usually, this is an aqueous solution such as physiological saline or phosphate buffered saline (PBS), Ringer's solution, lactated Ringer's solution, or any isotonic solution physiologically acceptable for administration by the selected means. Preferably, the solution is sterile and pyrogen-free and is manufactured and packaged under current Good Manufacturing Processes (GMP) approved by the FDA. A clinician of ordinary skill is familiar with the appropriate range of pH, tonicity, and additives or preservatives in formulating a pharmaceutical composition for administration by intravascular injection, intranodal, intraperitoneal direct injection, or other routes. In addition to additives for adjusting pH or tonicity, the active substance can be stabilized against aggregation and polymerization by amino acids and nonionic surfactants, polysorbates, and polyethylene glycol. Optionally, additional stabilizers can include various physiologically acceptable sugars and salts. Also, in addition to amino acids, polyvinylpyrrolidone can be added. Suitable therapeutic immunoglobulin solutions stabilized for storage and administration to humans are described in U.S. Patent No. 5,945,098, which is hereby incorporated by reference in its entirety. Other active substances, such as human serum albumin (HSA), can be added to the therapeutic or imaging composition to stabilize the antibody conjugate.

[0091] The compositions of the present invention can be administered using any medically appropriate procedure, such as intravascular (intravenous, intra-arterial, intracapillary) administration, injection into a tumor, etc. Intravascular injection can be by intravenous or intra-arterial injection. The effective amount of the therapeutic composition administered to a particular patient depends on a variety of factors, some of which vary from patient to patient. A qualified clinician will be able to determine the effective amount of the therapeutic composition to administer to a patient to slow the growth of tumor cells and promote the death of tumor cells. The dosage of the active substance depends on the treatment of the tumor, the route of administration, the nature of the treatment method, the sensitivity of the tumor to the treatment method, etc. LD 50Using animal data and other information available regarding conjugated cytotoxic or imaging components, a clinician can determine the maximum safe dose for an individual, depending on the route of administration. For example, considering that the amount of fluid in which the therapeutic composition is administered is greater, the dose administered intravenously may be higher than the dose administered locally. Similarly, a composition that is rapidly removed from the body can be administered at a higher dose or by repeated dosing to maintain a therapeutic concentration. Using ordinary skill, a qualified clinician will be able to optimize the dosage of a particular therapeutic or imaging composition during the course of routine clinical trials.

[0092] Typically, the effective dosage is from 0.001 to 100 milligrams per kilogram of the subject's body weight. The ratio of anti-CD47 to a second agent can range from 1:100, 1:50, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 50:1, 100:1. The agent can be administered to the subject in a series of two or more administrations. For therapeutic compositions, regular periodic administration (e.g., every 2 - 3 days) may be required, or it may be desirable to reduce toxicity. For therapeutic compositions utilized in a repeated dosing regimen, an antibody component that does not induce HAMA or other immune responses is preferred.

Example

[0093] Example 1 High-affinity SIRPα variants enhance macrophage-mediated destruction of small cell lung cancer CD47 enables cancer cells to evade the immune system through signaling via the inhibitory receptor SIRPα on macrophages. The inventors have recently developed next-generation CD47 antagonists by engineering the N-terminal immunoglobulin domain of SIRPα. These "high-affinity SIRPα variants" have a low affinity (K) for human CD47 of 11.1 pM, approximately 50,000-fold improved compared to wild-type SIRPα. D) When combined with tumor - specific antibodies, the high - affinity SIRPα variants act as adjuvants for immunotherapy, maximizing the macrophage - mediated destruction of cancer cells.

[0094] The inventors herein applied these agents to small - cell lung cancer (SCLC), a cancer with a poor prognosis and in which clinically approved antibodies do not exist. The inventors found SCLC cell lines and primary samples in which high levels of CD47 were expressed on their surfaces. Using human macrophages, the inventors found that CD47 blockade therapy can induce macrophage phagocytosis of SCLC cells. Treatment of mice bearing primary human SCLC tumors with a CD47 - blocking antibody could inhibit tumor growth and significantly prolong survival. To identify novel SCLC antigens that can be targeted in combination with high - affinity SIRPα variants, SCLC samples were screened by flow cytometry using a comprehensive antibody array.

[0095] The inventors verified tumor - specific antigens on the surface of SCLC cells and identified antibodies against these antigens that can stimulate phagocytosis in vitro. When combined with high - affinity SIRPα monomers, the ability of these antibodies to stimulate phagocytosis was dramatically enhanced.

[0096] Example 2 CD47 blockade therapy stimulates macrophage - mediated destruction of small - cell lung cancer Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with a poor prognosis. There are no clinically approved antibodies, targeted therapies, or immunotherapies for this disease. The inventors have found that SCLC samples express high levels of CD47, a cell surface molecule that enables cancer cells to evade the immune system. In particular, CD47 promotes immune evasion through signaling via SIRPα, an inhibitory receptor on macrophages. The inventors hypothesized that CD47 blockade therapy could be applied to the treatment of SCLC. The inventors have found that CD47 blockade therapy can induce macrophage phagocytosis of SCLC samples in vitro. Also, CD47 blockade therapy inhibited tumor growth and significantly prolonged survival in mice bearing SCLC tumors. Furthermore, using an inclusive antibody array, the inventors have identified several new and established therapeutic targets on the surface of SCLC cells. Antibodies against these targets can induce macrophage phagocytosis and are enhanced when combined with CD47 blockade therapy. These findings suggest that therapies that interfere with the CD47-SIRPa system can benefit patients with SCLC, especially when combined with tumor-specific antibodies.

[0097] Small cell lung cancer (SCLC), which originates from neuroendocrine cells of the lung, is one of the most lethal cancer subtypes in humans. Each year, more than 25,000 patients in the United States alone are diagnosed with SCLC, and patients typically survive only 6 - 12 months after diagnosis. The 5-year survival rate remains poor and has been around 5% since the 1970s. Except for the combination of radiation and chemotherapy, there have been no new treatment approaches implemented in the past 30 years. Despite numerous clinical trials, targeted therapies for SCLC have not been approved. SCLC is strongly associated with heavy smoking, and the increasing smoking rates in developing countries are expected to continue to increase the global prevalence of SCLC in the future. For these reasons, there is a need to identify novel therapeutic targets and develop new treatments for patients with SCLC.

[0098] One of the most promising advances in the field of oncology is immunotherapy, which aims to stimulate the patient's own immune system to attack and eliminate cancer. As tumors progress, they acquire mechanisms to avoid destruction by the immune system. By understanding these mechanisms, the inventors were able to develop a new strategy to make the immune system recognize cancer cells as foreign. Previous studies have identified the cell surface molecule CD47 as a "self marker" that prevents cells of the innate immune system from attacking hematological malignancies and certain types of solid tumors. CD47 acts by sending inhibitory signals via SIRPα, a receptor expressed on the surface of macrophages and other myeloid cells. In this sense, the CD47-SIRPα interaction serves as a myeloid-specific immune checkpoint. Several agents have been generated that interfere with signaling by the CD47-SIRPα system, including anti-CD47 antibodies and engineered variants of its receptor SIRPα. Recent studies have shown that CD47 blockade lowers the threshold for macrophage phagocytosis of cancer. The inventors hypothesized that SCLC cells also express CD47, and that CD47 blockade therapy could stimulate macrophage phagocytosis of SCLC cells and inhibit the growth of SCLC tumors in vivo.

[0099] Furthermore, CD47 blockade therapy has been shown to enhance the macrophage response to monoclonal antibodies. Rituximab or Her2 against lymphoma +Monoclonal antibodies such as trastuzumab against breast cancer have demonstrated a great success in cancer treatment. Monoclonal antibodies have not been clinically approved for the treatment of SCLC. Therefore, the inventors aimed to identify new SCLC surface antigens that can be targeted by monoclonal antibodies. Although treatment with monoclonal antibodies can result in a robust antitumor effect, they often cannot bring about a cure when used as a single agent, highlighting the need to improve the efficacy of these approaches. Therefore, the inventors aimed to combine CD47 blockade therapy with other antibodies to achieve the maximum antitumor response against SCLC.

[0100] As the first step in the inventors' approach, the inventors examined whether CD47 is expressed on the surface of SCLC samples. Next, the inventors investigated whether CD47 blockade therapy can stimulate macrophage phagocytosis of SCLC in vitro. Using a mouse model of human cancer, the response of SCLC samples to CD47 blockade therapy in vivo was evaluated. To identify new therapeutic targets on the surface of SCLC samples, the inventors performed high-throughput flow cytometry using an antibody array. Finally, the inventors aimed to demonstrate that combining an antibody against the identified antigen with CD47 blockade therapy can further increase phagocytosis. The overall goal of this study was to demonstrate the rationale for CD47 blockade therapy against SCLC and to identify additional antibodies that can be used to target SCLC. In this way, the inventors aimed to identify new combinations of immunotherapies that can be used for the benefit of patients with SCLC.

[0101] Results CD47 is expressed on the surface of SCLC. To evaluate whether CD47 blockade therapy can be applied to SCLC, the inventors first examined the expression of CD47 on the surface of SCLC cells. The inventors obtained six SCLC cell lines and subjected them to flow cytometry to evaluate CD47 expression on the cell surface. All six cell lines exhibited high CD47 expression (Figure 5A). The inventors also evaluated CD47 surface expression on SCLC patient-derived xenografts obtained from primary SCLC patient samples. Similar to the cell lines, the H29 patient sample also expressed high levels of CD47 on its surface (Figure 5B). These findings suggested that CD47 is an immunotherapy target on SCLC.

[0102] CD47 blocking antibodies induce phagocytosis of SCLC by human macrophages. To demonstrate CD47 as a true therapeutic target on SCLC, the inventors performed in vitro phagocytosis assays using human macrophages and SCLC samples. Macrophages were co-cultured with SCLC cells in the presence of vehicle control or anti-CD47 antibody. The inventors tested the anti-CD47 antibody clone Hu5F9-G4, which is a humanized anti-CD47 antibody that blocks the interaction between CD47 and SIRPa and is under investigation in a Phase I clinical trial for solid tumors (ClinicalTrials.gov identifier: NCT02216409). Phagocytosis was measured using high-throughput flow cytometry and was evaluated by the percentage of macrophages that had ingested calcein AM-labeled SCLC cells (Figures 5C and D). Fluorescence-activated cell sorting was used to identify the double-positive population containing macrophages with ingested tumor cells (Figure 5E). Four SCLC samples were subjected to evaluation in the phagocytosis assay. Three cell lines (NCI-H524, NCI-1688, and NCI-H82) showed a significant increase in phagocytosis when treated with the CD47 blocking antibody (Figure 5F). One cell line, NCI-H196, appeared to be resistant to phagocytosis, suggesting that additional mechanisms modify the sensitivity of this cell line to macrophage attack. The patient-derived xenograft H29 was also subjected to the phagocytosis assay by human macrophages. Treatment of this sample with the anti-CD47 antibody also resulted in a significant increase in phagocytosis (Figure 5G).

[0103] CD47 blocking antibodies inhibit the growth of SCLC tumors in vivo. To evaluate the potential of CD47 blocking agents when administered as a therapy for human SCLC, the inventors established a xenograft model of human SCLC. The inventors implanted NCI-H82 cells into the left lower flank of NSG mice that lack functional T cells, B cells, and NK cells but retain functional macrophages. Approximately one week after implantation, the mice were randomly assigned to treatment with a vehicle control administered every other day or 250 μg of the anti-CD47 antibody clone Hu5F9-G4. Tumor volume measurements were used to evaluate the mice for response to the therapy. Two weeks after treatment, a significant difference in the median tumor volume that persisted throughout the remainder of the experiment was observed (Figure 6A). Approximately one month after treatment, the median tumor volume for the vehicle control cohort was 837.8 mm 3 compared to 160.2 mm 3 for the cohort treated with the anti-CD47 antibody (P = 0.0281). Thus, CD47 blocking antibodies could result in a significant inhibition of tumor growth.

[0104] The inventors generated a GFP-luciferase+ NCI-H82 cell line and monitored growth and metastasis in vivo. As an orthotopic model of human SCLC, the inventors implanted GFP-luciferase+ NCI-H82 cells into the left intrathoracic space. Four days after injection, engraftment was confirmed by bioluminescence imaging. The inventors then randomly assigned the mice to two cohorts treated with a vehicle control administered every other day or 250 μg of the anti-CD47 antibody clone Hu5F9-G4. The inventors monitored tumor growth over time by bioluminescence imaging. Again, the CD47 blocking antibody resulted in a significant inhibition of tumor growth. In addition, the inventors observed a significant survival benefit for the cohort treated with the CD47 blocking antibody. Postmortem analysis revealed tumors formed in the intrathoracic or perithoracic regions. Mice in the vehicle control group also exhibited substantial metastases to the liver, which was not observed in the cohort treated with the anti-CD47 antibody.

[0105] Since cell lines typically represent a clonal population of cells, the inventors next tested the in vivo efficacy of a CD47-blocking antibody against patient-derived xenografts that more closely model treatment in patients, as it maintains the heterogeneity of the cancer cell population within the tumor. The primary SCLC sample H29 was transduced to express GFP-luciferase, enabling dynamic measurement of tumor growth in vivo. The tumor was then implanted into the left lower flank of the mouse and allowed to establish for approximately two weeks. The mice were then randomly assigned to two treatment cohorts: a vehicle control administered every other day or 250 μg of the anti-CD47 antibody clone Hu5F9-G4. The inventors found that the anti-CD47 antibody significantly inhibited tumor growth, as evaluated by tumor volume measurements and bioluminescence imaging (Figures 6B–D). Treatment with CD47 blockade therapy also resulted in a significant survival benefit. By 125 days after implantation, all mice in the control group had died, while most mice in the anti-CD47 antibody group had only small tumors that could not progress, even at 225 days after implantation (Figure 6E). These models demonstrate that CD47 blockade therapy can be effective in patients with SCLC.

[0106] Serum MCP-3 is a biomarker for response to CD47 blockade therapy. To identify potential biomarkers for response to CD47 blockade therapy, the inventors again implanted NCI-H82 cells into mice. The inventors allowed the tumors to grow to approximately 1.5 cm in diameter and then treated the mice with a single dose of vehicle control or the anti-CD47 antibody clone Hu5F9-G4. The inventors collected serum samples immediately before treatment and 24 hours after treatment. The inventors subjected the serum samples to multiplex analysis of 38 cytokines. From this analysis, the inventors found that macrophage chemoattractant protein 3 (MCP-3) increased systemically after treatment with the anti-CD47 antibody clone Hu5F9-G4 (Figure 7A). No significant increase in MCP-3 was observed in mice without tumors treated with the anti-CD47 antibody clone Hu5F9-G4 (Figure 7A). The inventors also performed a similar experiment using the patient-derived xenograft H29. Similarly, mice bearing tumors were subjected to a single dose of the anti-CD47 antibody clone Hu5F9-G4. Serum cytokine analysis again revealed that MCP-3 increased significantly after treatment with the CD47-blocking antibody (Figure 7B). Thus, MCP-3 may serve as a biomarker for response to CD47 blockade therapy in patients. The secretion of MCP-3 may be a positive feedback mechanism that recruits more macrophages to the tumor and may partially explain the robust effect of CD47 blockade therapy in vivo.

[0107] Therapeutic targets on SCLC are identified by an inclusive antibody array. Monoclonal antibodies have proven to be part of the most effective treatments against cancer. However, there are few known antibody targets on the surface of SCLC. For this reason, the inventors aimed to characterize the surface antigen profile of SCLC cells using an inclusive antibody array. The inventors subjected four SCLC cell lines and the primary SCLC sample H29 to analysis using the BioLegend LEGENDScreen array, an inclusive collection of 332 antibodies against human cell surface antigens. ***Considerations regarding histograms defining negative, low, and high antigens (Figure 8A). The inventors identified 39 antigens highly expressed on the surface of SCLC samples and targeted them as potential therapeutic antibody targets. When the inventors ranked these antigens by the median of their staining intensity, they found that CD47 was the most strongly stained surface antigen (Figure 8B). Another antigen highly expressed across all samples was CD56 (NCAM), a known marker for neuroendocrine tumors and a therapeutic target currently under evaluation for SCLC, thus validating the inventors' approach. Several other highly expressed surface antigens that could potentially be targeted by monoclonal antibody therapy, including CD24, CD29, and CD99, were also identified (Figure 8B). Interestingly, other immune checkpoint ligands such as CD80, CD86, PD-L1, or PD-L2 were not expressed at a detectable level on the surface of SCLC samples.

[0108] By combining an antibody with CD47 blockade, the phagocytosis of SCLC is enhanced. To evaluate the therapeutic potential of the antigens identified by the LEGENDScreen array, the inventors next evaluated their ability to be targeted by an antibody and induce phagocytosis in vitro. The inventors obtained antibodies against several highly expressed surface antigens, including CD56 (clones HCD56 and MEM-188), CD24, CD29, and CD99. In addition, the inventors obtained the sequence for lorbuvizumab, an anti-CD56 antibody being evaluated in clinical trials as an antibody-drug conjugate, and they recombinantly produced it as a naked antibody. The inventors tested these antibodies alone and in combination with the high-affinity CD47 antagonist CV1, which blocks CD47 but does not contribute to additional Fc stimulation (Figures 9A and B). The inventors tested the ability of these antibodies to induce phagocytosis by human macrophages of two different SCLC cell lines, NCI-H82 (Figure 9A) and NCI-H524 (Figure 9B). Of the three anti-CD56 antibodies tested, the inventors found that lorbuvizumab could bring about the greatest increase in phagocytosis and that this effect was significantly enhanced by combination with CV1. Antibodies against CD24 or CD99 also could induce phagocytosis comparable to or exceeding that by treatment with the anti-CD47 clone Hu5F9-G4. As expected, phagocytosis by Hu5F9-G4 was completely blocked when combined with CV1, because CV1 competes for the same binding surface and binds with extremely high affinity. Interestingly, the anti-CD29 antibody could not induce phagocytosis even in combination with CV1, providing important evidence that additional factors, such as surface binding conformation or the ability to bind to Fc receptors, can modify the macrophage response to therapeutic antibodies.

[0109] Since ralbotuzumab is in the evaluation stage as a therapeutic agent for SCLC, the inventors examined its ability to induce phagocytosis over a varying range of concentrations. Treatment with ralbotuzumab alone resulted in a dose-response relationship with respect to inducing macrophage phagocytosis. Importantly, the inventors found that the addition of CV1 resulted in a greater degree of phagocytosis over each ralbotuzumab concentration tested (Figure 9C). These findings demonstrate that CV1 can increase both the maximum efficacy and potency of ralbotuzumab, as previously observed when CV1 was combined with rituximab, trastuzumab, and cetuximab.

[0110] Due to its poor prognosis and lack of effective treatment options, there is an urgent need to identify new treatments for SCLC. Immunotherapy is emerging as part of the most promising new therapies for cancer, and here the inventors show that the myeloid-specific immune checkpoint CD47 is a true immunotherapy target for SCLC. CD47 is highly expressed on the surface of all SCLC samples tested, and the inventors found that blocking CD47 enables macrophage phagocytosis of SCLC samples in vitro. Using a number of xenograft models, the CD47-blocking antibody Hu5F9-G4 was able to inhibit tumor growth and extend survival in mice bearing SCLC tumors. Importantly, the inventors observed antitumor efficacy in a patient-derived xenograft model of SCLC that maintains the complexity of the tumor-forming cell population and thus serves as a more accurate model for treatment in humans. In addition, the inventors identified MCP-3 as a serum biomarker that correlates with response to CD47 blockade therapy. Since the anti-CD47 antibody Hu5F9-G4 is under investigation in a Phase I clinical trial for human solid malignancies (ClinicalTrials.gov identifier: NCT02216409), the inventors' findings provide the scientific rationale for further evaluation of anti-CD47 antibodies in a subset of patients with SCLC.

[0111] Furthermore, using an inclusive antibody array, the inventors identified several antigens on the surface of SCLC samples that could be targeted by monoclonal antibody therapy. Using the high-affinity SIRPα variant CV1, a next-generation CD47 antagonist, the inventors found that CD47 blockade increased the efficacy of anti-tumor antibodies against SCLC, as demonstrated for other cancers. The combination of the high-affinity SIRPα variant and an independent tumor-binding antibody provided an optimal strategy for targeting CD47 in SCLC. Blockade of CD47 on the surface of SCLC was not sufficient to induce macrophage phagocytosis, but instead, CD47 blockade increased macrophage phagocytosis in the presence of SCLC-binding antibodies. Antibodies against CD56, CD24, and CD99 were shown to be effective in inducing phagocytosis of SCLC, particularly when combined with CV1.

[0112] In addition, the inventors found that CD47 blockade could enhance the efficacy of lorvotuzumab, an antibody that has passed clinical trials against SCLC, as an antibody-drug conjugate (ADC) with the cytotoxic agent mertansine. Combining a therapeutic antibody with CD47 blockade therapy is an alternative way to enhance the efficacy of therapeutic antibodies. One benefit of CV1 over ADCs is that it can be combined with any antibody without further manipulation. ADCs often rely on internalization to deliver their cytotoxic payloads, and this dependence can limit efficacy and increase side effects. CD47 blockade can add additional specificity provided by cell-cell interactions, as it stimulates macrophages to identify cells for removal, compared to what is achieved by ADCs. Nevertheless, lorvotuzumab-mertansine may benefit from combination with CV1 if its ability to bind to Fc receptors is preserved.

[0113] The inventors' approach to identifying novel SCLC surface antigens can be applied to other types of cancer and may be used in the future to construct oligoclonal cocktails of antibodies that can be used to mimic the natural humoral immune response to foreign pathogens or cells. Combining these cocktails with CD47 blockade therapy and other immunotherapies can elicit an effective immune response against SCLC cells. These studies indicate that SCLC is responsive to CD47 blockade therapy.

[0114] Materials and Methods Cell lines and culture: NCI-H82, NCI-524, NCI-H69, and NCI-1688 were obtained from ATCC. Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Hyclone), 1× Glutamax (Invitrogen), and 100 U / mL penicillin and 100 μg / mL streptomycin (Invitrogen). Cell lines were grown in suspension (NCI-H82, NCI-524, NCI-H69) and dissociated by gentle pipetting or short incubation with 1× TrypLE (Invitrogen). NCI-1688 cells were grown in an adherent monolayer and removed by short incubation with 1× TrypLE. Cell lines were cultured at 37°C in a humidified incubator containing 5% carbon dioxide.

[0115] Human macrophage differentiation: The leukapheresis system chamber was obtained from an anonymous blood donor at the Stanford Blood Center. Monocytes were purified using AutoMACS (Miltenyi) with CD14+ microbeads or CD14+ whole blood microbeads (Miltenyi) according to the manufacturer's instructions. The purified CD14+ monocytes were seeded onto 15 cm tissue culture dishes at a density of 10 million monocytes per plate. Monocytes were differentiated into macrophages by culturing for approximately 7 - 10 days in IMDM supplemented with 10% human AB serum (Invitrogen), 1× GlutaMax (Invitrogen), as well as 100 U / mL penicillin and 100 μg / mL streptomycin.

[0116] In vitro phagocytosis assay: The in vitro phagocytosis assay was performed as previously described. Briefly, SCLC cancer cells were removed from plates and washed with serum-free IMDM. GFP-luciferase+ cells or cells labeled with Calcein AM (Invitrogen) were used as target cells. Macrophages were washed twice with HBSS and then incubated with 1× TrypLE in a humidified incubator at 37 °C for approximately 20 minutes. Macrophages were removed from plates using a cell lifter (Corning) and then washed twice with serum-free IMDM. Phagocytosis reactions were performed using 50,000 macrophages and 100,000 tumor cells. Co-cultures were performed with antibody therapy for 2 hours at 37 °C. After co-culture, cells were washed with autoMACS Running Buffer (Miltenyi) and prepared for analysis by flow cytometry. Macrophages were stained with fluorophore-conjugated antibodies (BioLegend) against CD45 in the presence of 100 μg / mL mouse IgG (Lampire). Dead cells were excluded from the analysis by staining with DAPI (Sigma). Samples were analyzed by flow cytometry using an LSR Fortessa (BD Biosciences) equipped with a high-speed sampler. Phagocytosis was evaluated as the percentage of macrophages and normalized to the maximum response by each independent donor when indicated. Statistical significance was determined and data were fitted to sigmoidal dose-response curves using Prism 5 (Graphpad). + evaluated as the percentage of macrophages and normalized to the maximum response by each independent donor when indicated. Statistical significance was determined and data were fitted to sigmoidal dose-response curves using Prism 5 (Graphpad).

[0117] Additional agents used in the phagocytosis assay included the high affinity SIRPα variant CV1 monomer, which was produced as previously described and used at a concentration of 1 μM for blocking. Antibodies against the identified SCLC antigens, including anti-CD56 (NCAM) clone HCD56 (BioLegend), anti-CD56 (NCAM) clone MEM-188 (BioLegend), anti-CD24 clone ML5 (Biolegend), anti-CD29 clone TS2 / 16 (BioLegend), anti-CD99 clone 12E7 (Abcam), were used in the phagocytosis assay at a concentration of 10 μg / mL. In addition, lorvotuzumab was recombinantly produced using the heavy and light chain variable region sequences available in the KEGG database (Drug: D09927). The lorvotuzumab variable regions were cloned into pFUSE-CHIg-hG1 and pFUSE2-CLIg-hK (Invivogen) for expression. Lorvotuzumab was recombinantly produced by transient transfection of 293F cells (Invitrogen) using 293fectin (Invitrogen), followed by purification through a HiTrap Protein A column (GE Healthcare). The purified antibody was eluted with 100 mM citrate buffer (pH 3.0) and neutralized with 1 / 10 volume of Tris buffer (pH 8.0). The antibody was desalted using a PD-10 column (GE Healthcare).

[0118] Sorting of macrophage populations after phagocytosis: 2.5 million human macrophages and 5 million GFP + NCI-H82 cells were combined with 10 μg / mL of anti-CD47 antibody (clone Hu5F9-G4) in serum-free medium and incubated for 2 hours. Macrophages were identified by staining with anti-CD45, and the macrophage population was sorted using a FACSAria II cell sorter (BD Biosciences). Cells from the sorted population were centrifuged onto microscope slides and then stained with Modified Wright-Giemsa stain (Sigma-Aldrich) according to the manufacturer's instructions and imaged using a DM5500 B upright optical microscope (Leica).

[0119] Mouse: Nod.Cg-Prkdc scid IL2rg tm1Wjl All in vivo experiments were performed using NOD.Cg-Prkdc scid IL2rg tm1Wjl / SzJ (NSG) mice. Tumors were implanted into mice at approximately 6 - 10 weeks of age, and experiments were conducted using age- and sex-matched cohorts. Mice were maintained in a barrier facility under the care of the Stanford Veterinary Services Center and were handled according to a protocol approved by the Stanford University Administrative Panel on Laboratory Animal Care.

[0120] In vivo SCLC treatment model: 1.25×10 6 Individual NCI-H82 cells were subcutaneously implanted into the flank of NSG mice. Tumors were allowed to grow for 8 days and then the mice were randomly assigned to treatment groups with PBS or 250 μg of anti-CD47 antibody (clone Hu5F9-G4). Treatments were administered every other day by intraperitoneal injection. Tumor growth was monitored by measuring tumor dimensions used to calculate tumor volume according to the ellipsoid formula (π / 6 × length × width 2 ). For the patient-derived xenograft model of SCLC, 3×10 6 Individual GFP-luciferase + H29 cells were subcutaneously implanted into the flank of NSG mice together with 25% Matrigel (BD Biosciences). Tumors were allowed to grow for 15 days and then the mice were randomly assigned to treatment groups with PBS or 250 μg of anti-CD47 antibody (clone Hu5F9-G4). Treatments were administered every other day by intraperitoneal injection. Tumor growth was monitored by bioluminescence imaging and by measuring tumor volume as described above. Statistical significance of tumor growth was determined by the Mann-Whitney test. Survival was analyzed by the Mantel-Cox test. Pilot in vivo experiments using H82 and H29 cells were performed using a smaller cohort of mice with similar results.

[0121] GFP fluorescence from tumor nodules was visualized using an M205 FA fluorescence dissection microscope (Leica) equipped with a DFC 500 camera (Leica).

[0122] Bioluminescence imaging: Mice bearing GFP-luciferase+ tumors were imaged as previously described. Briefly, anesthetized mice were injected with 200 μL of D-luciferin (firefly) potassium salt (Biosynth) reconstituted to 16.67 mg / mL in sterile PBS. Bioluminescence imaging was performed for 20 minutes using an IVIS Spectrum (Caliper Life Sciences) to record the maximum luminance. The total light flux value at the peak was evaluated from the anatomical region of interest using Living Image 4.0 (Caliper Life Sciences) and used for analysis.

[0123] Antibody screening based on comprehensive FACS: The antigens on the surface of SCLC samples were analyzed using LEGENDScreen Human Cell Screening Kits (BioLegend) according to the manufacturer's protocol, including the following modifications. Briefly, lyophilized antibodies were reconstituted in molecular biology grade water and added to cell samples at a 1:8 dilution. Approximately 20 - 40×10 6 total cells per SCLC sample were used for analysis. NCI-H82 was labeled with calcein-AM and analyzed simultaneously with NCI-H524. NCI-H69 was labeled with calcein-AM and analyzed simultaneously with NCI-H1688. The primary patient sample H69 was analyzed independently. It was freshly dissociated from low passage xenografts and mouse lineage cells were excluded from the analysis by staining with Pacific Blue anti-mouse H-2k d (BioLegend). Samples were incubated with antibodies for 30 minutes on ice protected from light. For all samples, dead cells were excluded from the analysis by staining with DAPI.

[0124] TIFF0007716299000001.tif100167TIFF0007716299000002.tif99167

Claims

Use of a combination in the manufacture of a medicament for treating an individual having small cell lung cancer, wherein the combination is an amount effective to increase depletion of small cell lung cancer cells of (i) an antibody that specifically binds to SIRPα and blocks the binding of CD47 to SIRPα, and (ii) a taxane selected from docetaxel and paclitaxel, said use.

2. The use according to claim 1, wherein the cancer is metastatic.

3. Use of a combination in the manufacture of a medicament for treating an individual having small cell lung cancer, wherein the combination is an amount effective to increase depletion of small cell lung cancer cells of (i) a substance that selectively blocks the binding of CD47 to SIRPα, (ii) a targeted therapeutic substance that specifically binds to one or more cell surface antigens on lung cancer cells, and (iii) a taxane; the substance that selectively blocks the binding of CD47 to SIRPα is (a) an antibody that specifically binds to CD47, (b) an antibody that specifically binds to SIRPα, or (c) a soluble SIRPα polypeptide ; the targeted therapeutic substance that specifically binds to one or more cell surface antigens on lung cancer cells is an antibody; and the one or more cell surface antigens on lung cancer cells are selected from CD24, CD56, CD166, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, and CD165, said use.

4. The use according to claim 1, wherein the combination of substances is administered simultaneously.

5. The use according to claim 1, wherein the combination of substances is administered sequentially.

6. The use according to claim 1, wherein the individual is human.

7. The use according to claim 3, wherein the substance that selectively blocks the binding of CD47 to SIRPα is an antibody.

8. The use according to claim 7, wherein the antibody specifically binds to CD47.

9. The use according to claim 7, wherein the antibody specifically binds to SIRPα. 【Claim 1)】 The use according to claim 3, wherein the substance that selectively blocks the binding of CD47 to SIRPα is a soluble SIRPα polypeptide.

11. The use according to claim 3, wherein the combination of substances is administered simultaneously. The use according to claim 3, wherein the combination of substances is administered sequentially.

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